Structure and function of retroviral integrase

Intasomes from different retroviral genera display unexpected architectural diversity.

“The structures revealed unexpected architectural diversity among intasomes from different retroviral genera.”

• cites [25] Hare, S., Gupta, S. S., Valkov, E., Engelman, A. & Cherepanov, P. Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464, 232–236 (2010). The first high-resolution structure of a retroviral intasome reveals the IN–viral DNA architecture underlying DNA recombination as well as the mechanism of clinical INSTI action. sourceDisputed

  The Abstract reports a crystal structure of the prototype foamy virus intasome, not structures from different retroviral genera. In “Overall architecture of the PFV intasome” (page unavailable), the authors instead suggest that the HIV-1 intasome could have a similar overall architecture and topology. The paper does not report the claimed cross-genera architectural diversity. (LLM, conf=0.78)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [29] Maertens, G. N., Hare, S. & Cherepanov, P. The mechanism of retroviral integration from X-ray structures of its key intermediates. Nature 468, 326–329 (2010). The first high-resolution structures of retroviral target capture and strand transfer intasome complexes reveal the requirement for target DNA distortion during integration. sourceCould not verify

  The Abstract (page not available) reports structures of prototype foamy virus intasomes and says they may help model HIV integration intermediates. A search for architectural diversity and comparisons across retroviral genera found no passage reporting such a comparison or its result. (LLM, conf=0.77)

  Experimental — accuracy not yet measured
• cites [30] Yin, Z. et al. Crystal structure of the Rous sarcoma virus intasome. Nature 530, 362–366 (2016). Together with Ballandras-Colas et al. (2016), these intasome structures first reveal the requirement for more than four IN protomers for functional integration. sourceSupported
• cites [31] Ballandras-Colas, A. et al. Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function. Nature 530, 358–361 (2016). sourceSupported
• cites [32] Ballandras-Colas, A. et al. A supramolecular assembly mediates lentiviral DNA integration. Science 355, 93–95 (2017). Cryo-EM structures of intasomes derived from the sheep lentivirus MVV reveal a higher-order macromolecular machine composed of 16 IN protomers and define the conserved intasome core shared by all retroviral intasome structures. sourceSupported
• cites [33] Passos, D. O. et al. Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome. Science 355, 89–92 (2017). Initial cryo-EM structures of HIV-1 strand transfer complex intasomes reveal a higher-order IN dodecamer architecture. sourceNot checked — no full text of the source
• cites [34] Li, M. et al. A peptide derived from lens epithelium-derived growth factor stimulates HIV-1 DNA integration and facilitates intasome structural studies. J. Mol. Biol. 432, 2055–2066 (2020). sourceSupported
• cites [35] Cook, N. J. et al. Structural basis of second-generation HIV integrase inhibitor action and viral resistance. Science 367, 806 (2020). High-resolution cryo-EM structures of simian immunodeficiency virus intasomes reveal the mechanism of INSTI resistance. sourcePartly supported

  The passage comparing SIVrcm and maedi-visna virus intasomes (labeled Abstract; page unavailable) describes different assembly patterns, supporting architectural diversity. But those examples are both lentiviruses. The paper’s comparison with a different retroviral genus, prototype foamy virus, identifies differences in the environment of bound drugs rather than establishing the claimed architectural diversity across genera. (LLM, conf=0.79)

  Experimental — accuracy not yet measured
• cites [36] Passos, D. O. et al. Structural basis for strand-transfer inhibitor binding to HIV intasomes. Science 367, 810 (2020). This study provides high-resolution cryo-EM structures of HIV-1 intasomes with several advanced INSTIs. sourceNot checked — no full text of the source
• cites [37] Bhatt, V. et al. Structural basis of host protein hijacking in human T-cell leukemia virus integration. Nat. Commun. 11, 3121 (2020). Together with Barski et al. (2020), cryo-EM studies reveal that deltaretrovirus intasomes are composed of IN tetramers and visualize details of the IN–PP2A interface. sourceSupported

ALLINIs do not act by inhibiting integration.

“Counterintuitively, the underlying mode of ALLINI action is not via inhibition of integration140,142,174–177,179–181.”

• cites [140] Jurado, K. A. et al. Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation. Proc. Natl Acad. Sci. USA 110, 8690–8695 (2013). This is the first study to reveal that ALLINIs potently inhibit HIV-1 maturation. sourceNot checked — no full text of the source
• cites [142] Balakrishnan, M. et al. Non-catalytic site HIV-1 integrase inhibitors disrupt core maturation and induce a reverse transcription block in target cells. PLoS ONE 8, e74163 (2013). sourcePartly supported

  The Introduction and Discussion say that NCINIs’ primary antiviral effect occurs during virus production, through disrupted core maturation, rather than through inhibition of integration. But the Results also show that GS-B blocks integration when applied to target cells at high concentrations. The categorical claim that ALLINIs do not act by inhibiting integration therefore overstates the paper’s finding. (LLM, conf=0.91)

  Experimental — accuracy not yet measured
• cites [174] Le Rouzic, E. et al. Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage. Retrovirology 10, 144 (2013). sourceDisputed

  The paper describes a dual mode of action at integration and post-integration, rather than saying ALLINIs do not act by inhibiting integration. In the “A B” section (page not supplied), it explicitly attributes the integration block to inhibition of integrase strand transfer and the integrase–LEDGF interaction; it reports that the post-integration effect is stronger. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [175] Gupta, K. et al. Allosteric inhibition of human immunodeficiency virus integrase: late block during viral replication and abnormal multimerization involving specific protein domains. J. Biol. Chem. 289, 20477–20488 (2014). sourceSupported
• cites [176] Desimmie, B. A. et al. LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions. Retrovirology 10, 57 (2013). sourceDisputed

  The Discussion says LEDGINs block HIV-1 integration and that this study confirms inhibition at the integration step while identifying an additional late-stage effect. The source therefore contradicts the claim that their action is not via inhibition of integration. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [177] Sharma, A. et al. A new class of multimerization selective inhibitors of HIV-1 integrase. PLoS Pathog. 10, e1004171 (2014). sourceDisputed

  The Introduction says ALLINIs inhibit both early and late stages of HIV-1 replication and that, in target cells, they impair integration. The comparative-analysis section likewise reports that the ALLINI GS-B blocks integration in target cells. The paper distinguishes this from KF116, a MINI that does not detectably affect integration at therapeutically relevant concentrations. (LLM, conf=0.89)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [179] Gupta, K. et al. Structural basis for inhibitor-induced aggregation of HIV Integrase. PLoS Biol. 14, e1002584 (2016). sourceSupported
• cites [180] Koneru, P. C. et al. HIV-1 integrase tetramers are the antiviral target of pyridine-based allosteric integrase inhibitors. eLife 8, e46344 (2019). sourceSupported
• cites [181] Gupta, K. et al. Allosteric HIV integrase inhibitors promote formation of inactive branched polymers via homomeric carboxy-terminal domain interactions. Structure 29, 213–225.e5 (2021). sourceNot checked — no full text of the source

Intasomes from several retroviral species have been assembled from purified components and visualized by X-ray crystallography or cryo-EM.

“The intasomes from several retroviral species were assembled from purified components and visualised by X-ray crystallography or single-particle cryogenic electron microscopy (cryo-EM)25,29–39 (Figs. 2A, 3).”

• cites [25] Hare, S., Gupta, S. S., Valkov, E., Engelman, A. & Cherepanov, P. Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464, 232–236 (2010). The first high-resolution structure of a retroviral intasome reveals the IN–viral DNA architecture underlying DNA recombination as well as the mechanism of clinical INSTI action. sourceSupported
• cites [29] Maertens, G. N., Hare, S. & Cherepanov, P. The mechanism of retroviral integration from X-ray structures of its key intermediates. Nature 468, 326–329 (2010). The first high-resolution structures of retroviral target capture and strand transfer intasome complexes reveal the requirement for target DNA distortion during integration. sourceSupported
• cites [30] Yin, Z. et al. Crystal structure of the Rous sarcoma virus intasome. Nature 530, 362–366 (2016). Together with Ballandras-Colas et al. (2016), these intasome structures first reveal the requirement for more than four IN protomers for functional integration. sourceSupported
• cites [31] Ballandras-Colas, A. et al. Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function. Nature 530, 358–361 (2016). sourceSupported
• cites [32] Ballandras-Colas, A. et al. A supramolecular assembly mediates lentiviral DNA integration. Science 355, 93–95 (2017). Cryo-EM structures of intasomes derived from the sheep lentivirus MVV reveal a higher-order macromolecular machine composed of 16 IN protomers and define the conserved intasome core shared by all retroviral intasome structures. sourceSupported
• cites [33] Passos, D. O. et al. Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome. Science 355, 89–92 (2017). Initial cryo-EM structures of HIV-1 strand transfer complex intasomes reveal a higher-order IN dodecamer architecture. sourceNot checked — no full text of the source
• cites [34] Li, M. et al. A peptide derived from lens epithelium-derived growth factor stimulates HIV-1 DNA integration and facilitates intasome structural studies. J. Mol. Biol. 432, 2055–2066 (2020). sourceSupported
• cites [35] Cook, N. J. et al. Structural basis of second-generation HIV integrase inhibitor action and viral resistance. Science 367, 806 (2020). High-resolution cryo-EM structures of simian immunodeficiency virus intasomes reveal the mechanism of INSTI resistance. sourceSupported
• cites [36] Passos, D. O. et al. Structural basis for strand-transfer inhibitor binding to HIV intasomes. Science 367, 810 (2020). This study provides high-resolution cryo-EM structures of HIV-1 intasomes with several advanced INSTIs. sourceNot checked — no full text of the source
• cites [37] Bhatt, V. et al. Structural basis of host protein hijacking in human T-cell leukemia virus integration. Nat. Commun. 11, 3121 (2020). Together with Barski et al. (2020), cryo-EM studies reveal that deltaretrovirus intasomes are composed of IN tetramers and visualize details of the IN–PP2A interface. sourceSupported

• AssumptionRests on an assumption: Intasome structures assembled in vitro from purified components represent functional states relevant to integration in vivo.

The stoichiometry of HIV-1 and SIV intasomes remains debated, with complexes containing four and eight to sixteen integrase molecules observed.

“The stoichiometric state of primate lentiviral intasomes from HIV-1 and the simian immunodeficiency virus (SIV) remain a subject of debate, with complexes containing four and eight to sixteen IN molecules observed by cryo-EM and negative stain electron microscopy33–36.”

• cites [33] Passos, D. O. et al. Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome. Science 355, 89–92 (2017). Initial cryo-EM structures of HIV-1 strand transfer complex intasomes reveal a higher-order IN dodecamer architecture. sourceNot checked — no full text of the source
• cites [34] Li, M. et al. A peptide derived from lens epithelium-derived growth factor stimulates HIV-1 DNA integration and facilitates intasome structural studies. J. Mol. Biol. 432, 2055–2066 (2020). sourcePartly supported

  The Introduction and ‘Structure of the HIV-1 CSC intasome assembled with P5-IN’ section report heterogeneous HIV-1 intasomes, including tetramers and dodecamers, and describe a cryo-EM reconstruction. Our copy does not substantiate the claim about SIV intasomes, sixteen-integrase complexes, or observations by negative-stain electron microscopy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [35] Cook, N. J. et al. Structural basis of second-generation HIV integrase inhibitor action and viral resistance. Science 367, 806 (2020). High-resolution cryo-EM structures of simian immunodeficiency virus intasomes reveal the mechanism of INSTI resistance. sourcePartly supported

  The paper reports dodecamer-like particles in both HIV-1 and SIVrcm preparations and a cryo-EM model of an SIVrcm repeat unit containing two integrase tetramers. It does not report a four-integrase HIV-1 or SIV intasome; its explicit observation of predominantly sixteen-integrase complexes concerns maedi-visna virus instead. The cited passages are labeled Abstract in our copy, with no page number provided. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [36] Passos, D. O. et al. Structural basis for strand-transfer inhibitor binding to HIV intasomes. Science 367, 810 (2020). This study provides high-resolution cryo-EM structures of HIV-1 intasomes with several advanced INSTIs. sourceNot checked — no full text of the source

PFV, HTLV-1, and STLV intasomes contain four integrase subunits arranged as a dimer-of-dimers.

“While the intasomes from the prototype foamy virus (PFV, a simiispumavirus) as well as the δ-retroviruses HTLV-1 and simian T-cell lymphotropic virus (STLV) contain four IN subunits (arranged as a dimer-of-dimers)25,29,37,38”

• cites [25] Hare, S., Gupta, S. S., Valkov, E., Engelman, A. & Cherepanov, P. Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464, 232–236 (2010). The first high-resolution structure of a retroviral intasome reveals the IN–viral DNA architecture underlying DNA recombination as well as the mechanism of clinical INSTI action. sourcePartly supported

  The “Crystallization of the PFV intasome” section (page unavailable) reports that two PFV integrase dimers form a tetramer, supporting the PFV portion of the claim. The paper does not establish the stated arrangement for HTLV-1 or STLV intasomes. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [29] Maertens, G. N., Hare, S. & Cherepanov, P. The mechanism of retroviral integration from X-ray structures of its key intermediates. Nature 468, 326–329 (2010). The first high-resolution structures of retroviral target capture and strand transfer intasome complexes reveal the requirement for target DNA distortion during integration. sourcePartly supported

  The Abstract describes the PFV intasome as a tetramer of integrase with a cleft between IN dimers, supporting the four-subunit, dimer-of-dimers arrangement for PFV. The paper does not address HTLV-1 or STLV intasomes, so it does not support that part of the claim. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [37] Bhatt, V. et al. Structural basis of host protein hijacking in human T-cell leukemia virus integration. Nat. Commun. 11, 3121 (2020). Together with Barski et al. (2020), cryo-EM studies reveal that deltaretrovirus intasomes are composed of IN tetramers and visualize details of the IN–PP2A interface. sourcePartly supported

  The Introduction describes the PFV intasome as tetrameric, and the Results (IN tetramer in the HTLV-1 intasome; page not provided) describe HTLV-1 as four integrase subunits forming two inner–outer pairs. Our copy does not establish the claimed four-subunit arrangement for STLV. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [38] Barski, M. S. et al. Cryo-EM structure of the deltaretroviral intasome in complex with the PP2A regulatory subunit B56γ. Nat. Commun. 11, 5043 (2020). sourcePartly supported

  The Architecture section reports four integrase subunits in the STLV-1 intasome, and the Fig. 2 caption describes them as a dimer of dimers. The Discussion says PFV also has a tetrameric intasome. The paper does not establish the corresponding intasome arrangement for HTLV-1; its structural analysis of the assembled intasome uses STLV-1. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

ASLV and MMTV intasomes contain eight integrase chains arranged as a tetramer-of-dimers.

“those from ASLV and mouse mammary tumour virus (MMTV, β-retrovirus) harbour eight IN chains (a tetramer-of-dimers)30,31,39.”

• cites [30] Yin, Z. et al. Crystal structure of the Rous sarcoma virus intasome. Nature 530, 362–366 (2016). Together with Ballandras-Colas et al. (2016), these intasome structures first reveal the requirement for more than four IN protomers for functional integration. sourceSupported
• cites [31] Ballandras-Colas, A. et al. Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function. Nature 530, 358–361 (2016). sourceSupported
• cites [39] Pandey, K. K. et al. Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome. Commun. Biol. 4, 330 (2021). sourceSupported

The viral DNA ends make extensive interactions with all three types of integrase domains within the conserved intasome core.

“Inserted into this assembly, the vDNA ends make extensive interactions with all three types of IN domains within the CIC.”

• cites [25] Hare, S., Gupta, S. S., Valkov, E., Engelman, A. & Cherepanov, P. Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464, 232–236 (2010). The first high-resolution structure of a retroviral intasome reveals the IN–viral DNA architecture underlying DNA recombination as well as the mechanism of clinical INSTI action. sourceSupported

The conserved intasome core contains two integrase catalytic core domain dimers, each contributing one active site for catalysis.

“The CIC contains two IN CCD dimers (painted cyan and green in Figs. 2–4), each of which contributes one active site for catalysis.”

• cites [25] Hare, S., Gupta, S. S., Valkov, E., Engelman, A. & Cherepanov, P. Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464, 232–236 (2010). The first high-resolution structure of a retroviral intasome reveals the IN–viral DNA architecture underlying DNA recombination as well as the mechanism of clinical INSTI action. sourceSupported

The observed integration target site preference supports the large target DNA distortion required to accommodate scissile phosphodiester bonds at opposing integrase active sites.

“The observed integration target site preference accordingly supports the large target DNA distortion required to accommodate scissile phosphodiester bonds at opposing IN active sites, as observed in the PFV structure29.”

• cites [29] Maertens, G. N., Hare, S. & Cherepanov, P. The mechanism of retroviral integration from X-ray structures of its key intermediates. Nature 468, 326–329 (2010). The first high-resolution structures of retroviral target capture and strand transfer intasome complexes reveal the requirement for target DNA distortion during integration. sourceSupported
  Experimental — accuracy not yet measured

The retroviral integrase active site becomes fully structured only when the viral DNA end engages within the intasome.

“The active site of retroviral IN is fully structured only upon engagement of the vDNA end in the context of the intasome25.”

• cites [25] Hare, S., Gupta, S. S., Valkov, E., Engelman, A. & Cherepanov, P. Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464, 232–236 (2010). The first high-resolution structure of a retroviral intasome reveals the IN–viral DNA architecture underlying DNA recombination as well as the mechanism of clinical INSTI action. sourcePartly supported

  The introduction (page unavailable in our copy) says the active site is *believed* to adopt its functional state only upon viral-DNA binding. The section “The active site of the functional intasome” describes an assembled active site engaged with viral DNA, but does not establish the unqualified claim that it becomes fully structured only then. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Brief incubation of PFV intasome crystals with Mn2+ before snap-freezing trapped the active site prior to strand transfer.

“Brief incubation of the crystals in the presence of Mn2+ ions prior to snap-freezing in liquid nitrogen trapped the active site prior to strand transfer.”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourceSupported

Sharp bending of the target DNA duplex substantially widens the major groove at the integration site, allowing opposing strand phosphodiester bonds to insert into the intasome active sites.

“Sharp bending of the target DNA duplex substantially widened the major groove at the site of integration, permitting the insertion of opposing strand phosphodiester bonds into the intasome active sites (Fig. 2A).”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourcePartly supported

  The Introduction (page unavailable) says target DNA binds in a severely bent conformation that forces its phosphodiesters toward the integrase active sites. Our copy does not say that the bending substantially widens the DNA major groove at the integration site; the groove it mentions is between integrase subunits. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Footprinting studies suggest that as much as 200-250 bp at each viral DNA end are protected by a stable protein complex within retroviral pre-integration complexes.

“Furthermore, while the intasome structure covers only ~20 bp of vDNA, footprinting studies suggested that as much as 200–250 bp at each vDNA end are protected by a stable protein complex within retroviral PICs45,46.”

• cites [45] Wei, S. Q., Mizuuchi, K. & Craigie, R. A large nucleoprotein assembly at the ends of the viral DNA mediates retroviral DNA integration. EMBO J. 16, 7511–7520 (1997). sourcePartly supported

  The Footprinting by Mu-mediated PCR section reports protection at each viral DNA end in pre-integration complexes and associates it with a nucleoprotein structure. But it says protection extends approximately 500 bp, with no sharply defined border, rather than only as much as 200–250 bp. No page number is available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [46] Chen, H., Wei, S. Q. & Engelman, A. Multiple integrase functions are required to form the native structure of the human immunodeficiency virus type I intasome. J. Biol. Chem. 274, 17358–17364 (1999). sourceSupported

Relocation of the new phosphodiester bond joining viral DNA to target DNA protects it from nucleophilic attack by the target DNA 3' hydroxyl.

“Relocation of the new phosphodiester bond that joins vDNA to the target protects it from a nucleophilic attack by the 3’ hydroxyl of the target DNA28,29.”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourcePartly supported

  The “Target DNA binding and strand transfer” section reports that the newly formed viral–target DNA phosphodiester bond moves out of the active site and says this was postulated to make strand transfer irreversible. It does not specifically identify prevention of attack by the target DNA 3′ hydroxyl as the mechanism, and the claim states the proposed protective effect as fact. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [29] Maertens, G. N., Hare, S. & Cherepanov, P. The mechanism of retroviral integration from X-ray structures of its key intermediates. Nature 468, 326–329 (2010). The first high-resolution structures of retroviral target capture and strand transfer intasome complexes reveal the requirement for target DNA distortion during integration. sourceSupported

Within the conserved intasome core, the viral DNA duplex is split at the invariant dCdA dinucleotide, leaving the 3' dA and the scissile dinucleotide unpaired.

“Within the CIC, the vDNA duplex is split at the invariant dCdA dinucleotide: while dC remains paired to its dG mate from the opposing vDNA strand, 3’ dA and the scissile dinucleotide are unpaired.”

• cites [25] Hare, S., Gupta, S. S., Valkov, E., Engelman, A. & Cherepanov, P. Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464, 232–236 (2010). The first high-resolution structure of a retroviral intasome reveals the IN–viral DNA architecture underlying DNA recombination as well as the mechanism of clinical INSTI action. sourcePartly supported

  The 'IN-DNA interactions within the PFV intasome' section says the cytosine of the invariant CA dinucleotide remains in a distorted base pair, while the terminal adenine is isolated from its complement. It describes separation of the viral DNA strands, but does not say that a scissile dinucleotide is unpaired. No page number is provided in our copy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

The PFV intasome crystallized with blunt-ended viral DNA underwent catalysis when incubated with Mg2+ or Mn2+ salts.

“The PFV intasome crystallised with blunt-ended vDNA underwent catalysis upon incubation of the crystals in the presence of Mg2+ or Mn2+ salts.”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourceSupported
  Experimental — accuracy not yet measured

Target DNA binds a groove between the intasome active sites, nestling on the synaptic C-terminal domains.

“The target DNA bound a groove between the active sites of the intasome, nestling on the synaptic CTDs.”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourcePartly supported

  The Introduction (page unavailable) says target DNA binds in a groove between the inner integrase subunits and presents its phosphodiesters to the active sites. It does not specify that the DNA nestles on the synaptic C-terminal domains, so that part of the claim is unsupported by our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

The metal ions in the integrase active site swap their roles between 3'-processing and strand transfer.

“Thus, the metal ions in the IN active site swap their roles during 3’-processing versus strand transfer.”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourceSupported

Intasomes from several retroviral species and prokaryotic and eukaryotic transpososomes have been visualized as post-catalytic strand transfer complexes, all showing sharp deformations of target DNA.

“Intasomes from several retroviral species as well as prokaryotic and eukaryotic transpososomes have been visualised as post-catalytic strand transfer complexes, all of which display sharp deformations of target DNA29,30,32,33,37,51–54.”

• cites [29] Maertens, G. N., Hare, S. & Cherepanov, P. The mechanism of retroviral integration from X-ray structures of its key intermediates. Nature 468, 326–329 (2010). The first high-resolution structures of retroviral target capture and strand transfer intasome complexes reveal the requirement for target DNA distortion during integration. sourcePartly supported

  The paper reports a post-catalytic strand transfer structure of the prototype foamy virus intasome and severe bending of its target DNA (Abstract; page unavailable). It does not establish the sentence’s broader claim about structures from several retroviral species and prokaryotic and eukaryotic transpososomes all displaying sharp target-DNA deformations. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [30] Yin, Z. et al. Crystal structure of the Rous sarcoma virus intasome. Nature 530, 362–366 (2016). Together with Ballandras-Colas et al. (2016), these intasome structures first reveal the requirement for more than four IN protomers for functional integration. sourceSupported
• cites [32] Ballandras-Colas, A. et al. A supramolecular assembly mediates lentiviral DNA integration. Science 355, 93–95 (2017). Cryo-EM structures of intasomes derived from the sheep lentivirus MVV reveal a higher-order macromolecular machine composed of 16 IN protomers and define the conserved intasome core shared by all retroviral intasome structures. sourceSupported
• cites [33] Passos, D. O. et al. Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome. Science 355, 89–92 (2017). Initial cryo-EM structures of HIV-1 strand transfer complex intasomes reveal a higher-order IN dodecamer architecture. sourceNot checked — no full text of the source
• cites [37] Bhatt, V. et al. Structural basis of host protein hijacking in human T-cell leukemia virus integration. Nat. Commun. 11, 3121 (2020). Together with Barski et al. (2020), cryo-EM studies reveal that deltaretrovirus intasomes are composed of IN tetramers and visualize details of the IN–PP2A interface. sourcePartly supported

  The Results section ('IN tetramer in the HTLV-1 intasome'; page unavailable) reports sharp target-DNA kinks in the HTLV-1 strand-transfer complex and describes a similar target-DNA trajectory in the RSV intasome. The paper does not establish the claim about prokaryotic or eukaryotic transpososomes, or that all the complexes listed display sharp deformations. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [51] Richardson, J. M., Colloms, S. D., Finnegan, D. J. & Walkinshaw, M. D. Molecular architecture of the Mos1 paired-end complex: the structural basis of DNA transposition in a eukaryote. Cell 138, 1096–1108 (2009). sourcePartly supported

  The Results and Discussion (‘The PEC contains a dimeric transposase’; page not available) describe a visualized eukaryotic Mos1 transpososome, supporting that part of the citation. But the structure contains transposon ends positioned as if just cleaved, not a post-catalytic strand-transfer complex. In ‘A model for the target capture complex,’ target DNA is modeled rather than observed, so this paper does not show a sharp deformation of target DNA. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [52] Montano, S. P., Pigli, Y. Z. & Rice, P. A. The mu transpososome structure sheds light on DDE recombinase evolution. Nature 491, 413–417 (2012). sourcePartly supported

  The Methods (Overview; page not specified) describe a final strand-transfer complex of Mu, and Overall Architecture (Target DNA; page not specified) reports that its target DNA bends by about 140°. Convergent and divergent evolution also describes bent target DNA in the PFV intasome, but says Mos1 was crystallized without target DNA and bending in Tn5 was inferred by modeling. The paper therefore does not support the claim that all the complexes mentioned were visualized as post-catalytic complexes displaying sharply deformed target DNA, or its reference to several retroviral species. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [53] Morris, E. R., Grey, H., McKenzie, G., Jones, A. C. & Richardson, J. M. A bend, flip and trap mechanism for transposon integration. eLife 5, e15537 (2016). sourceSupported
• cites [54] Ghanim, G. E., Kellogg, E. H., Nogales, E. & Rio, D. C. Structure of a P element transposase-DNA complex reveals unusual DNA structures and GTP-DNA contacts. Nat. Struct. Mol. Biol. 26, 1013–1022 (2019). sourceSupported

The side chains of the D,D-35-E residues coordinate a pair of Mg2+ ions that facilitate 3'-processing and strand transfer.

“The side chains of these residues serve to coordinate the essential pair of Mg2+ ions, which facilitate 3’-processing and strand transfer reactions.”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourceSupported
  Experimental — accuracy not yet measured
• cites [47] Yang, W., Lee, J. Y. & Nowotny, M. Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity. Mol. Cell 22, 5–13 (2006). sourcePartly supported

  The Background section says that two metal ions in a transposase active site support consecutive reactions: cleavage to generate a 3′ OH, followed by strand transfer. It also describes a conserved Asp coordinating both ions. It does not identify a D,D-35-E motif or establish that the side chains of all three specified residues coordinate the Mg2+ pair. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [48] Nowotny, M. & Yang, W. Stepwise analyses of metal ions in RNase H catalysis from substrate destabilization to product release. EMBO J. 25, 1924–1933 (2006). sourcePartly supported

  The Introduction (page unavailable) describes active-site carboxylates coordinating two magnesium ions in RNase H catalysis, and the concluding remarks suggest that a two-metal mechanism may extend to recombinases. The paper does not identify a D,D-35-E motif or establish that its residues coordinate the ions for 3′-processing and strand transfer; those reaction-specific parts of the attributed claim are unsupported. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

The 3' dA hydroxyl acts as the nucleophile during strand transfer, attacking chromosomal DNA and causing transesterification and covalent joining of viral DNA to the chromosome.

“The latter hydroxyl takes the role of the nucleophile during the strand transfer reaction to attack the phosphodiester backbone of chromosomal DNA, leading to transesterification and covalent joining of vDNA to the chromosome.”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourceSupported

Both 3'-processing and strand transfer reactions have been visualized in PFV intasome crystals.

“Both of these reactions were visualised in PFV intasome crystals28.”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourceSupported

In the intasome context, strand transfer causes reconfiguration of the active site followed by dissociation of metal ion B.

“However, in the context of the intasome, strand transfer results in reconfiguration of the active site followed by dissociation of metal ion B28.”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourcePartly supported

  In “Target DNA binding and strand transfer,” the paper reports that the metal ions move closer and the newly formed phosphodiester is ejected from the active site after strand transfer. It reports reduced occupancy at metal position B in later structures, but describes dissociation of metal B as a suggestion rather than an established sequence of events. The claim overstates that inference. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Placement of the 3' viral DNA end in the active site is enabled by fraying of the viral DNA termini through insertion of a catalytic core domain 310 helix between the DNA strands.

“The placement of the 3’ vDNA end in the active site is afforded by fraying of the vDNA termini by insertion of a CCD 310 helix between vDNA strands.”

• cites [25] Hare, S., Gupta, S. S., Valkov, E., Engelman, A. & Cherepanov, P. Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464, 232–236 (2010). The first high-resolution structure of a retroviral intasome reveals the IN–viral DNA architecture underlying DNA recombination as well as the mechanism of clinical INSTI action. sourcePartly supported

  In “IN-DNA interactions within the PFV intasome” (page unavailable), the paper describes separation of the viral DNA strands at the reactive terminus, and “The active site of the functional intasome” places the 3′ end near the active-site residues. But it attributes strand separation to the active-site loop, not insertion of a CCD 3₁₀ helix; the CCD α4 helix is described as packing into the DNA minor groove. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Brief soaking of PFV intasome crystals in Mn2+ allowed visualization of the active site in the Michaelis complex just before 3'-processing.

“Briefly soaking the crystals in Mn2+ allowed visualisation of the active site in the Michaelis complex form just prior to 3’-processing28.”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourceSupported

• AssumptionRests on an assumption: Conformational changes observed in crystallized PFV intasomes after metal soaking reflect the actual catalytic pathway.

Integrase residues that interact with target DNA, when altered, significantly change base preferences at integration sites.

“IN residues that interact with target DNA which, when altered, significantly changed base preferences at integration sites29,55–57.”

• cites [29] Maertens, G. N., Hare, S. & Cherepanov, P. The mechanism of retroviral integration from X-ray structures of its key intermediates. Nature 468, 326–329 (2010). The first high-resolution structures of retroviral target capture and strand transfer intasome complexes reveal the requirement for target DNA distortion during integration. sourceSupported
• cites [55] Aiyer, S. et al. Structural and sequencing analysis of local target DNA recognition by MLV integrase. Nucleic Acids Res. 43, 5647–5663 (2015). sourceSupported
• cites [56] Serrao, E. et al. Integrase residues that determine nucleotide preferences at sites of HIV-1 integration: implications for the mechanism of target DNA binding. Nucleic Acids Res. 42, 5164–5176 (2014). sourceSupported
• cites [57] Demeulemeester, J. et al. HIV-1 integrase variants retarget viral integration and are associated with disease progression in a chronic infection cohort. Cell Host Microbe 16, 651–662 (2014). sourceNot checked — no full text of the source

During 3'-processing, a water molecule acts as the nucleophile, cleaving a phosphodiester bond to reveal a 3' dA hydroxyl group.

“During 3’-processing, a water molecule is used as a nucleophile, and the reaction results in cleavage of a phosphodiester bond to reveal a 3’ dA hydroxyl group.”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourceSupported

The PFV intasome assembled with pre-processed viral DNA was co-crystallized with a double-stranded DNA oligonucleotide in the target capture complex.

“The PFV intasome assembled using pre-processed vDNA was co-crystallized with a double-stranded DNA oligonucleotide in the form of the target capture complex28.”

• cites [28] Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012). This study visualizes the 3′-processing and strand transfer reactions in crystals of PFV intasomes, which defined the roles of chemical nucleophiles and divalent metal ions in catalysis, and reveals substrate mimicry by INSTIs. sourceSupported

Cryo-EM structures of the PFV intasome bound to a mononucleosome show that the nucleosome adapts to present DNA in a conformation optimal for strand transfer.

“Cryo-EM structures of the PFV intasome engaged with a mononucleosome revealed that the latter adapts to present the DNA in a conformation optimal for strand transfer64,65.”

• cites [64] Maskell, D. P. et al. Structural basis for retroviral integration into nucleosomes. Nature 523, 366–369 (2015). Together with Wilson et al. (2019), cryo-EM studies elucidate how the PFV intasome engages and locally remodels a nucleosome for integration. sourceSupported
• cites [65] Wilson, M. D. et al. Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamer. Nat. Commun. 10, 4189 (2019). sourceSupported

• AssumptionRests on an assumption: Nucleosome interactions observed in cryo-EM structures reflect physiological chromatin integration.

During nuclear import, CPSF6 is thought to compete with Nup153 for capsid binding to release the viral core from the nuclear pore complex.

“During nuclear import, CPSF6 is thought to compete with Nup153 for capsid binding to release the viral core from the nuclear pore complex81.”

• cites [81] Bejarano, D. A. et al. HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid interactions at the nuclear pore complex. eLife 8, e41800 (2019). sourceSupported

C-terminal proteolytic processing of spumaviral Gag proteins, which is required for infection, occurs adjacent to the chromatin binding sequence.

“C-terminal proteolytic processing of spumaviral Gag proteins, which is required for infection, occurs adjacent to the chromatin binding sequence (CBS)90.”

• cites [90] Müllers, E. The foamy virus Gag proteins: what makes them different? Viruses 5, 1023–1041 (2013). sourcePartly supported

  Section 2 states that FV Gag undergoes C-terminal proteolytic processing and that Gag processing is essential for viral infectivity. Section 2.3 places the chromatin-binding sequence in the C-terminal GRII region, and the Figure 3 caption identifies the primary cleavage site at amino acid 621. The text provided does not establish that the cleavage site is adjacent to the chromatin-binding sequence. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

The PFV Gag chromatin binding sequence binds nucleosomes via an acidic patch predominantly composed of core histone H2A/H2B heterodimers.

“The PFV Gag CBS binds nucleosomes via an acidic patch predominantly composed of core histone H2A/H2B heterodimers91,92”

• cites [91] Tobaly-Tapiero, J. et al. Chromatin tethering of incoming foamy virus by the structural Gag protein. Traffic 9, 1717–1727 (2008). sourcePartly supported

  The section “The CBS of PFV Gag interacts with core histones H2A/H2B” (p. 6) reports that the PFV Gag CBS associates with H2A and H2B and binds host chromosomes through them. The Discussion (p. 7) cautions that additional factors or other nucleosomal components may be involved. The paper does not identify an acidic patch as the binding site or establish direct binding to an H2A/H2B heterodimer. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [92] Lesbats, P. et al. Structural basis for spumavirus GAG tethering to chromatin. Proc. Natl Acad. Sci. USA 114, 5509–5514 (2017). sourceSupported

This is consistent with predicted variations in target DNA conformations required to integrate across 4-6 bp spacings.

“which is consistent with predicted variations in target DNA conformations required to integrate across 4–6 bp spacings67.”

• cites [67] Serrao, E., Ballandras-Colas, A., Cherepanov, P., Maertens, G. N. & Engelman, A. N. Key determinants of target DNA recognition by retroviral intasomes. Retrovirology 12, 39 (2015). sourceSupported

LEDGF/p75 markedly increased integrase strand transfer activity in vitro.

“The lentiviral IN binding protein LEDGF/p75 significantly stimulated IN strand transfer activity in vitro17,43,96,97.”

• cites [17] Llano, M. et al. LEDGF/p75 determines cellular trafficking of diverse lentiviral but not murine oncoretroviral integrase proteins and is a component of functional lentiviral preintegration complexes. J. Virol. 78, 9524–9537 (2004). sourceCould not verify

  The Results describe LEDGF/p75 binding to lentiviral integrase and show that LEDGF/p75-associated preintegration complexes can integrate DNA in vitro. They do not compare integrase strand-transfer activity with and without LEDGF/p75 or report stimulation of that activity (searched: strand transfer, stimulation, and in vitro integration activity). (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [43] Cherepanov, P. et al. HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in human cells. J. Biol. Chem. 278, 372–381 (2003). sourceNot checked — no full text of the source
• cites [96] Busschots, K. et al. The interaction of LEDGF/p75 with integrase is lentivirus-specific and promotes DNA binding. J. Biol. Chem. 280, 17841–17847 (2005). sourceNot checked — no full text of the source
• cites [97] Cherepanov, P. LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro. Nucleic Acids Res. 35, 113–124 (2007). sourceSupported
  Experimental — accuracy not yet measured

CPSF6 plays a critical role in regulating nuclear pre-integration complex incursion.

“In addition to its role in nuclear import, CPSF6 plays a critical role in regulating nuclear PIC incursion.”

• cites [84] Francis, A. C. et al. HIV-1 replication complexes accumulate in nuclear speckles and integrate into speckle-associated genomic domains. Nat. Commun. 11, 3505 (2020). This study demonstrates that HIV-1 integration favours regions of chromatin that are located close to nuclear speckles. sourcePartly supported

  The Results section (“Relocation outside of NSs diminishes nuclear VRC stability”; page unavailable) suggests that CPSF6 is involved in transporting HIV-1 viral replication complexes to nuclear speckles. The Discussion (page unavailable) is more cautious: it says CPSF6 and/or other factors may tether the complexes there. These passages support a role in intranuclear positioning, but not the stronger assertion that CPSF6 itself plays a critical role in regulating nuclear PIC incursion. (LLM, conf=0.81)

  Experimental — accuracy not yet measured
• cites [85] Achuthan, V. et al. Capsid-CPSF6 interaction licenses nuclear HIV-1 trafficking to sites of viral DNA integration. Cell Host Microbe 24, 392–404.e398 (2018). sourceSupported

Chromatinized DNA has been shown to be a suitable or even preferred target for retroviral integration in vitro and in vivo.

“Yet, chromatinized DNA was shown to be a suitable, or even preferred target for retroviral integration in vitro and in vivo59–64.”

• cites [59] Pryciak, P. M. & Varmus, H. E. Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection. Cell 69, 769–780 (1992). sourceNot checked — no full text of the source
• cites [60] Pruss, D., Bushman, F. D. & Wolffe, A. P. Human immunodeficiency virus integrase directs integration to sites of severe DNA distortion within the nucleosome core. Proc. Natl Acad. Sci. USA 91, 5913–5917 (1994). sourceNot checked — no full text of the source
• cites [61] Wang, G. P., Ciuffi, A., Leipzig, J., Berry, C. C. & Bushman, F. D. HIV integration site selection: analysis by massively parallel pyrosequencing reveals association with epigenetic modifications. Genome Res. 17, 1186–1194 (2007). sourceSupported
• cites [62] Roth, S. L., Malani, N. & Bushman, F. D. Gammaretroviral integration into nucleosomal target DNA in vivo. J. Virol. 85, 7393–7401 (2011). sourceNot checked — no full text of the source
• cites [63] Baller, J. A., Gao, J., Stamenova, R., Curcio, M. J. & Voytas, D. F. A nucleosomal surface defines an integration hotspot for the Saccharomyces cerevisiae Ty1 retrotransposon. Genome Res. 22, 704–713 (2012). sourceSupported
• cites [64] Maskell, D. P. et al. Structural basis for retroviral integration into nucleosomes. Nature 523, 366–369 (2015). Together with Wilson et al. (2019), cryo-EM studies elucidate how the PFV intasome engages and locally remodels a nucleosome for integration. sourceSupported

In MLV, chromatin tethering is accomplished by p12, a small phosphoprotein encoded within the gag gene, which acts in a manner seemingly indistinguishable from the PFV Gag chromatin binding sequence.

“In MLV, the chromatin tethering function is accomplished by p12, a small phosphoprotein that is encoded within the gag gene, which acts in a manner seemingly indistinguishable from the PFV Gag CBS93–95.”

• cites [93] Elis, E., Ehrlich, M., Prizan-Ravid, A., Laham-Karam, N. & Bacharach, E. p12 tethers the murine leukemia virus pre-integration complex to mitotic chromosomes. PLoS Pathog. 8, e1003103 (2012). sourcePartly supported

  The Introduction identifies p12 as a cleavage product of MLV Gag, and the Discussion concludes that it tethers MLV pre-integration complexes to mitotic chromosomes. The Discussion does not establish that p12 acts like the PFV Gag chromatin-binding sequence; it says the interactions underlying p12-mediated tethering remain uncertain. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [94] Schneider, W. M. et al. Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag. Proc. Natl Acad. Sci. USA 110, 9487–9492 (2013). sourcePartly supported

  The Abstract and Discussion support a chromatin-tethering role for MuLV Gag p12: a PFV Gag chromatin-binding sequence can rescue a defective p12 mutant, and the authors infer that wild-type p12 tethers to chromosomes. They do not establish that native p12 acts in a manner indistinguishable from the PFV sequence; the Discussion leaves p12’s tethering partner unresolved. The passages provide no page numbers. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [95] Wanaguru, M., Barry, D. J., Benton, D. J., O’Reilly, N. J. & Bishop, K. N. Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis. PLoS Pathog. 14, e1007117 (2018). sourcePartly supported

  The Discussion reports that MLV p12 directly tethers the pre-integration complex to nucleosomes. It also reports competition between p12 and the PFV Gag chromatin-binding sequence and describes their binding as similar, with about 90% overlap in their chromatin interactomes. That supports a similar mode of action, but not the stronger suggestion that they are indistinguishable. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

These interactions are thought to offset the energy needed to shift nucleosomal DNA along the histone octamer core to create slack for lifting target DNA at the integration site.

“These interactions are thought to offset the energy required to shift nucleosomal DNA along the histone octamer core to create the slack for lifting the target DNA at the site of integration (Fig. 4)65.”

• cites [65] Wilson, M. D. et al. Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamer. Nat. Commun. 10, 4189 (2019). sourceSupported

Genic proviruses formed when LEDGF/p75 was absent clustered near transcription start sites.

“The genic proviruses that did form under these conditions uncharacteristically congregated around transcription start sites87,99–101.”

• cites [87] Li, W. et al. CPSF6-dependent targeting of speckle-associated domains distinguishes primate from nonprimate lentiviral integration. mBio 11, e02254-20 (2020). sourceSupported
• cites [99] Shun, M. C. et al. LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration. Genes Dev. 21, 1767–1778 (2007). sourcePartly supported

  In “A critical role for Ledgf in gene-specific HIV-1 integration” (page not available), the paper reports that HIV-1 integration near transcription start sites was about 2.6-fold more frequent in Ledgf-null cells. That analysis covers all proviruses, not specifically the genic proviruses named in the claim. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [100] Singh, P. K. et al. LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes. Genes Dev. 29, 2287–2297 (2015). sourceSupported
  Experimental — accuracy not yet measured
• cites [101] Sowd, G. A. et al. A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin. Proc. Natl Acad. Sci. USA 113, E1054–E1063 (2016). sourceSupported
  Experimental — accuracy not yet measured

CPSF6 binds the same region of the HIV-1 capsid as Nup153.

“Cleavage and polyadenylation specificity factor 6 (CPSF6), which is a component of the cellular pre-mRNA cleavage and polyadenylation machinery79, binds the same region of capsid as Nup153 (REF.78,80).”

• cites [78] Matreyek, K. A., Yucel, S. S., Li, X. & Engelman, A. Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity. PLoS Pathog. 9, e1003693 (2013). sourceSupported
• cites [80] Price, A. J. et al. Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly. PLoS Pathog. 10, e1004459 (2014). sourceSupported

The integration preferences of HIV-1 for active transcription units and MLV for promoter/enhancer regions are largely dictated by specific interactions of their integrase proteins with chromatin-bound host factors.

“The well-understood integration preferences of HIV-1 for active transcription units72 and MLV for promoter/enhancer regions73–75 are in large part dictated via specific interactions of the respective IN proteins with chromatin-bound host factors.”

• cites [72] Schroder, A. R. et al. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110, 521–529 (2002). Enabled by the release of the human genome sequence, this study convincingly demonstrates that HIV-1 integration strongly favours active transcription units within gene-dense regions of chromatin. sourceNot checked — no full text of the source
• cites [73] Wu, X., Li, Y., Crise, B. & Burgess, S. M. Transcription start regions in the human genome are favored targets for MLV integration. Science 300, 1749–1751 (2003). This is the first study to reveal that the gammaretrovirus Moloney murine leukaemia virus favours gene promoter regions for integration. sourceNot checked — no full text of the source
• cites [74] De Ravin, S. S. et al. Enhancers are major targets for murine leukemia virus vector integration. J. Virol. 88, 4504–4513 (2014). sourceNot checked — no full text of the source
• cites [75] LaFave, M. C. et al. MLV integration site selection is driven by strong enhancers and active promoters. Nucleic Acids Res. 42, 4257–4269 (2014). sourcePartly supported

  The Results and Discussion report a strong MLV integration preference for particular strong enhancers and active promoters. The Results do not exclude an integrase–protein tether, and the Discussion describes BET proteins as possible tethers, but neither establishes that such interactions largely dictate MLV integration. The HIV-1 preference is separately cited in the citing sentence and is not assessed here. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

The intasome forms a multivalent interface with the nucleosome, contacting both nucleosomal DNA gyres and the core histones.

“The intasome forms a multivalent interface with the nucleosome, contacting both nucleosomal DNA gyres and the core histones (Fig. 4).”

• cites [64] Maskell, D. P. et al. Structural basis for retroviral integration into nucleosomes. Nature 523, 366–369 (2015). Together with Wilson et al. (2019), cryo-EM studies elucidate how the PFV intasome engages and locally remodels a nucleosome for integration. sourceSupported
• cites [65] Wilson, M. D. et al. Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamer. Nat. Commun. 10, 4189 (2019). sourceSupported

Depletion of cellular Nup153 or Nup358 shifts HIV-1 integration away from gene-dense regions of chromatin.

“Depletion of cellular Nup153 or Nup358 also shifted HIV-1 integration away from gene-dense regions of chromatin88,89”

• cites [88] Koh, Y. et al. Differential effects of human immunodeficiency virus type 1 capsid and cellular factors nucleoporin 153 and LEDGF/p75 on the efficiency and specificity of viral DNA integration. J. Virol. 87, 648–658 (2013). sourceSupported
  Experimental — accuracy not yet measured
• cites [89] Ocwieja, K. E. et al. HIV integration targeting: a pathway involving transportin-3 and the nuclear pore protein RanBP2. PLoS Pathog. 7, e1001313 (2011). sourcePartly supported

  The section on HIV integration site selection reports that RanBP2/Nup358 depletion shifted HIV integration toward regions of lower gene density. Our copy does not report the same result for Nup153; the corresponding experiments concern RanBP2 and Transportin-3. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

The PFV intasome shows highly preferred nucleosomal integration sites at superhelix ±3.5 locations that overlay H2A-H2B heterodimers.

“Consequently, the PFV intasome displays highly preferred nucleosomal integration sites at superhelix ±3.5 locations that overlay the H2A-H2B heterodimers64.”

• cites [64] Maskell, D. P. et al. Structural basis for retroviral integration into nucleosomes. Nature 523, 366–369 (2015). Together with Wilson et al. (2019), cryo-EM studies elucidate how the PFV intasome engages and locally remodels a nucleosome for integration. sourceSupported

In vitro studies with intasomes from several retroviruses suggest genus-specific modes of chromosomal integration.

“However, in vitro studies with intasomes of several retroviruses suggested genus-specific modes of chromosomal integration66”

• cites [66] Benleulmi, M. S. et al. Intasome architecture and chromatin density modulate retroviral integration into nucleosome. Retrovirology 12, 13 (2015). sourceSupported

The same nucleosomal positions are favored by yeast retrotransposon Ty1 in vivo.

“The same nucleosomal positions are favoured by yeast retrotransposon Ty1 in vivo63.”

• cites [63] Baller, J. A., Gao, J., Stamenova, R., Curcio, M. J. & Voytas, D. F. A nucleosomal surface defines an integration hotspot for the Saccharomyces cerevisiae Ty1 retrotransposon. Genome Res. 22, 704–713 (2012). sourceSupported

HIV-1 nuclear import is mediated by the viral capsid, which directly interacts with several components of the cellular nuclear import machinery, including Nup153 and Nup358.

“HIV-1 nuclear import is mediated via the viral capsid, which interacts directly with several components of the cellular nuclear import machinery, including the nuclear pore complex components Nup153 and Nup358 (REF.76–78).”

• cites [76] Yamashita, M. & Emerman, M. Capsid is a dominant determinant of retrovirus infectivity in nondividing cells. J. Virol. 78, 5670–5678 (2004). sourcePartly supported

  The Discussion says capsid composition influences retroviral nuclear transport, supporting a role for capsid. It does not report direct capsid interactions with Nup153 or Nup358; instead, it says HIV capsid does not appear to act as a nuclear localization signal and that the component recognized by cellular import machinery remains unclear. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [77] Schaller, T. et al. HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency. PLoS Pathog. 7, e1002439 (2011). sourcePartly supported

  The Discussion reports direct binding between HIV-1 capsid and the cyclophilin domain of Nup358, supporting a capsid-mediated nuclear-entry pathway. The Nup153-dependent nuclear-entry section shows that Nup153 affects infection, but the Discussion describes interaction with Nup153 as a possibility, not a demonstrated direct capsid–Nup153 interaction. The claim overstates what this paper establishes for Nup153. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [78] Matreyek, K. A., Yucel, S. S., Li, X. & Engelman, A. Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity. PLoS Pathog. 9, e1003693 (2013). sourcePartly supported

  The Author Summary reports direct binding between HIV-1 capsid and NUP153, and the Figure 12 caption says capsid engagement with NUP153 is required for nuclear import. The “Model of NUP153 FG engagement” section describes docking through NUP358 as likely, rather than establishing the claimed direct interaction with NUP358 as fact. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Loss of PSIP1, the gene encoding LEDGF/p75, reduced viral DNA integration in cells by several fold.

“Knockout of the PSIP1 gene, which encodes for LEDGF/p75, similarly suppressed levels of vDNA integration in cells several fold98,99.”

• cites [98] Marshall, H. M. et al. Role of PSIP1/LEDGF/p75 in lentiviral infectivity and integration targeting. PLoS ONE 2, e1340 (2007). sourceSupported
• cites [99] Shun, M. C. et al. LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration. Genes Dev. 21, 1767–1778 (2007). sourceSupported

B56 proteins served as key structural components of recent delta-retroviral intasome structures.

“Be this as it may, B56 proteins additionally served as key structural components of the recent δ-retroviral intasome structures37,38.”

• cites [37] Bhatt, V. et al. Structural basis of host protein hijacking in human T-cell leukemia virus integration. Nat. Commun. 11, 3121 (2020). Together with Barski et al. (2020), cryo-EM studies reveal that deltaretrovirus intasomes are composed of IN tetramers and visualize details of the IN–PP2A interface. sourceSupported
• cites [38] Barski, M. S. et al. Cryo-EM structure of the deltaretroviral intasome in complex with the PP2A regulatory subunit B56γ. Nat. Commun. 11, 5043 (2020). sourceSupported

LEDGF/p75 acts as a bimodal tether in lentiviral integration targeting.

“LEDGF/p75 appears to function as a bimodal tether in lentiviral integration targeting.”

• cites [103] Llano, M. et al. Identification and characterization of the chromatin-binding domains of the HIV-1 integrase interactor LEDGF/p75. J. Mol. Biol. 360, 760–773 (2006). sourceNot checked — no full text of the source
• cites [104] Turlure, F., Maertens, G., Rahman, S., Cherepanov, P. & Engelman, A. A tripartite DNA-binding element, comprised of the nuclear localization signal and two AT-hook motifs, mediates the association of LEDGF/p75 with chromatin in vivo. Nucleic Acids Res. 34, 1653–1665 (2006). sourceSupported
• cites [105] Cherepanov, P., Devroe, E., Silver, P. A. & Engelman, A. Identification of an evolutionarily conserved domain in human lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF/p75) that binds HIV-1 integrase. J. Biol. Chem. 279, 48883–48892 (2004). sourcePartly supported

  The Discussion (page unavailable) describes LEDGF/p75 as a chromosomal protein that binds lentiviral integrases, supporting the proposed tether mechanism. It calls LEDGF/p75 a candidate targeting factor, however, and says its role in targeting during infection remains to be established; the claim goes further by saying it appears to function as that tether. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

LEDGF/p75 tethers cellular proteins such as MLL1 and MML2 methyltransferases to chromatin through IBD interactions with a linear IBM sequence.

“LEDGF/p75 similarly tethers several cellular proteins to chromatin, including MLL1 and MML2 methyltransferases, via IBD interactions with a linear IBM (for IBD-binding motif) amino acid sequence113.”

• cites [113] Tesina, P. et al. Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif. Nat. Commun. 6, 7968 (2015). sourcePartly supported

  The Discussion (page not provided) says LEDGF/p75 can tether its binding partners to chromatin through an IBD interface that recognizes the IBM, and identifies MLL1 and MLL2 as partners. It establishes this interaction for MLL1 but says MLL2 most likely uses the same interface; the citing sentence presents MLL2’s mechanism as established. The source spells the protein name MLL2, not MML2. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

The integrase CCD is the primary LEDGF/p75 binding determinant, with the NTD providing secondary contacts for full affinity.

“The CCD is the main IN binding determinant, with the NTD contributing secondary interactions for full binding affinity106,107.”

• cites [106] Maertens, G. et al. LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells. J. Biol. Chem. 278, 33528–33539 (2003). sourceSupported
• cites [107] Hare, S. et al. A novel co-crystal structure affords the design of gain-of-function lentiviral integrase mutants in the presence of modified PSIP1/LEDGF/p75. PLoS Pathog. 5, e1000259 (2009). sourceSupported

LEDGF/p75 was instrumental in reconstituting lentiviral intasomes for structural characterization.

“Similarly, LEDGF/p75 was instrumental to reconstitute lentiviral intasomes for structural characterisation32–36.”

• cites [32] Ballandras-Colas, A. et al. A supramolecular assembly mediates lentiviral DNA integration. Science 355, 93–95 (2017). Cryo-EM structures of intasomes derived from the sheep lentivirus MVV reveal a higher-order macromolecular machine composed of 16 IN protomers and define the conserved intasome core shared by all retroviral intasome structures. sourceSupported
• cites [33] Passos, D. O. et al. Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome. Science 355, 89–92 (2017). Initial cryo-EM structures of HIV-1 strand transfer complex intasomes reveal a higher-order IN dodecamer architecture. sourceNot checked — no full text of the source
• cites [34] Li, M. et al. A peptide derived from lens epithelium-derived growth factor stimulates HIV-1 DNA integration and facilitates intasome structural studies. J. Mol. Biol. 432, 2055–2066 (2020). sourcePartly supported

  The Introduction (page unavailable) says that fusing a peptide derived from LEDGF to HIV-1 integrase provided a tool for structural studies of HIV-1 intasomes, and that the authors used it to determine an intasome structure. This supports the role of an LEDGF-derived peptide, but not the broader claim that full-length LEDGF/p75 was instrumental in reconstituting lentiviral intasomes generally. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [35] Cook, N. J. et al. Structural basis of second-generation HIV integrase inhibitor action and viral resistance. Science 367, 806 (2020). High-resolution cryo-EM structures of simian immunodeficiency virus intasomes reveal the mechanism of INSTI resistance. sourcePartly supported

  The passage labeled Abstract (page unavailable) reports that LEDGF/p75 stimulated strand-transfer activity and that the reaction conditions produced stable nucleoprotein complexes examined by electron microscopy. The paper does not say that LEDGF/p75 was instrumental to reconstituting those intasomes for structural characterization. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [36] Passos, D. O. et al. Structural basis for strand-transfer inhibitor binding to HIV intasomes. Science 367, 810 (2020). This study provides high-resolution cryo-EM structures of HIV-1 intasomes with several advanced INSTIs. sourceNot checked — no full text of the source

Many HIV-1 integrase point mutants retain enzymatic activity yet fail to support viral replication.

“The literature is replete with examples of HIV-1 IN point mutants that retain enzymatic activity yet fail to support viral replication (REF.132 and references therein).”

• cites [132] Elliott, J. L. et al. Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis. eLife 9, e543111 (2020). sourceSupported

Disrupting LEDGF/p75 binding to chromatin or integrase abolishes its cofactor function during HIV-1 infection.

“Alterations that disrupted LEDGF/p75 binding to chromatin or IN ablated its cofactor function during HIV-1 infection99,103,112.”

• cites [99] Shun, M. C. et al. LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration. Genes Dev. 21, 1767–1778 (2007). sourceSupported
• cites [103] Llano, M. et al. Identification and characterization of the chromatin-binding domains of the HIV-1 integrase interactor LEDGF/p75. J. Mol. Biol. 360, 760–773 (2006). sourceNot checked — no full text of the source
• cites [112] Shun, M. C. et al. Identification and characterization of PWWP domain residues critical for LEDGF/p75 chromatin binding and human immunodeficiency virus type 1 infectivity. J. Virol. 82, 11555–11567 (2008). sourceSupported

MLV and other gamma-retroviral integrases directly interact with BET proteins, including BRD2, BRD3, and BRD4.

“MLV and other γ-retroviral INs interact directly with bromo- and extra-terminal domain (BET) proteins, including bromodomain proteins 2, 3 and 4 (REF.114–116).”

• cites [114] De Rijck, J. et al. The BET family of proteins targets moloney murine leukemia virus integration near transcription start sites. Cell Rep. 5, 886–894 (2013). sourcePartly supported

  The Discussion (p. 6) states that BRD2, BRD3, and BRD4 interact with MLV integrase, and the binding assays in Figure 2 (p. 4) support a direct interaction with BET protein domains. The passages do not establish direct binding for integrases from other gammaretroviruses; that extension is unsupported by this paper's reported tests. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [115] Gupta, S. S. et al. Bromo- and extraterminal domain chromatin regulators serve as cofactors for murine leukemia virus integration. J. Virol. 87, 12721–12736 (2013). sourceSupported
• cites [116] Sharma, A. et al. BET proteins promote efficient murine leukemia virus integration at transcription start sites. Proc. Natl Acad. Sci. USA 110, 12036–12041 (2013). sourceNot checked — no full text of the source

Lentiviruses evolved a functional mimic of the cellular IBM by juxtaposing independent integrase NTDs and CCDs.

“Thus, lentiviruses have leveraged the juxtaposition of independent IN NTDs and CCDs to evolve a functional mimic of the cellular IBM.”

• cites [113] Tesina, P. et al. Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif. Nat. Commun. 6, 7968 (2015). sourceSupported

The displaced vDNA nucleotide stacks with the metal-chelating core of the inhibitor, enhancing binding strength.

“The displaced vDNA nucleotide forms a stacking interaction with the metal chelating core of the inhibitor, greatly enhancing the strength of binding35,36,163.”

• cites [35] Cook, N. J. et al. Structural basis of second-generation HIV integrase inhibitor action and viral resistance. Science 367, 806 (2020). High-resolution cryo-EM structures of simian immunodeficiency virus intasomes reveal the mechanism of INSTI resistance. sourcePartly supported

  The passage on the DTG- and BIC-bound structures (labeled Abstract; page unavailable) says the displaced 3′ viral DNA nucleotide stacks against the inhibitor’s central body. It does not say that this stacking greatly strengthens binding; a separate passage attributes extraordinarily tight binding partly to interactions with Mg2+ ions. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [36] Passos, D. O. et al. Structural basis for strand-transfer inhibitor binding to HIV intasomes. Science 367, 810 (2020). This study provides high-resolution cryo-EM structures of HIV-1 intasomes with several advanced INSTIs. sourceNot checked — no full text of the source
• cites [163] Langley, D. R. et al. The terminal (catalytic) adenosine of the HIV LTR controls the kinetics of binding and dissociation of HIV integrase strand transfer inhibitors. Biochemistry 47, 13481–13488 (2008). sourceNot checked — no full text of the source

Dolutegravir, bictegravir, and cabotegravir are second-generation INSTIs with reduced susceptibility to viral mutations that render raltegravir and elvitegravir ineffective.

“The latter three, classified as second-generation compounds, are characterized by reduced susceptibility to viral mutations that render the first-generation drugs, raltegravir and elvitegravir, ineffective151–153.”

• cites [151] Hassounah, S. A. et al. Antiviral activity of bictegravir and cabotegravir against integrase inhibitor-resistant SIVmac239 and HIV-1. Antimicrob. Agents Chemother. 61, e01695–17 (2017). sourceSupported
• cites [152] Smith, S. J., Zhao, X. Z., Burke, T. R. Jr. & Hughes, S. H. Efficacies of cabotegravir and bictegravir against drug-resistant HIV-1 integrase mutants. Retrovirology 15, 37 (2018). sourceSupported
• cites [153] Zhang, W. W. et al. Accumulation of multiple mutations in vivo confers cross-resistance to new and existing integrase inhibitors. J. Infect. Dis. 218, 1773–1776 (2018). sourcePartly supported

  The Results report that viruses with G140S/Q148H were highly resistant to raltegravir and elvitegravir but had relatively small susceptibility changes to dolutegravir, bictegravir, and cabotegravir. The claim is broader than that finding: the Results also show that additional mutations produced substantial resistance to all three newer inhibitors. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

HIV-1 integrase directly interacts with viral genomic RNA.

“Strikingly, recent cross-linking immunoprecipitation experiments demonstrated that HIV-1 IN directly interacts with viral genomic RNA132,141.”

• cites [132] Elliott, J. L. et al. Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis. eLife 9, e543111 (2020). sourceSupported
• cites [141] Kessl, J. J. et al. HIV-1 integrase binds the viral RNA genome and is essential during virion morphogenesis. Cell 166, 1257–1268 e1212 (2016). This study provides evidence that IN binding to viral RNA has a crucial role in RNA encapsidation, virus particle maturation and HIV-1 infection. sourceNot checked — no full text of the source

Some class II phenotypes are accompanied by premature proteolytic maturation, misfolding, or mislocalisation of mutant Gag-Pol.

“At least in some cases, the class II phenotype was accompanied by premature proteolytic maturation, misfolding or perhaps mislocalisation of the mutant Gag-Pol polyprotein130,135,136.”

• cites [130] Bukovsky, A. & Gottlinger, H. Lack of integrase can markedly affect human immunodeficiency virus type 1 particle production in the presence of an active viral protease. J. Virol. 70, 6820–6825 (1996). sourceNot checked — no full text of the source
• cites [135] Mohammed, K. D., Topper, M. B. & Muesing, M. A. Sequential deletion of the integrase (Gag-Pol) carboxyl terminus reveals distinct phenotypic classes of defective HIV-1. J. Virol. 85, 4654–4666 (2011). sourcePartly supported

  The Results (page unavailable) report abnormal Gag-Pol cleavage products in several class II mutants and suggest dysregulated protease activation as a cause of abnormal intracellular polyprotein processing. They do not establish misfolding or mislocalisation of mutant Gag-Pol; the misfolded-protein pathway discussed there concerns cleaved integrase, not Gag-Pol. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [136] Hoyte, A. C. et al. Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of integrase in HIV-1 Gag-Pol polyprotein proteolytic processing. J. Biol. Chem. 292, 19814–19825 (2017). sourceNot checked — no full text of the source

Many class II HIV-1 integrase mutants incorporate normal levels of mature viral proteins.

“However, there are many examples of class II HIV-1 IN mutants that incorporated normal levels of mature viral proteins132.”

• cites [132] Elliott, J. L. et al. Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis. eLife 9, e543111 (2020). sourceSupported

Integrase may organize RNA within infectious particles, consistent with a proposed cofactor function during reverse transcription.

“These data suggest that IN may have a role in organising RNA within infectious particles, which is not inconsistent with the proposed co-factor function during reverse transcription138.”

• cites [138] Tekeste, S. S. et al. Interaction between reverse transcriptase and integrase is required for reverse transcription during HIV-1 replication. J. Virol. 89, 12058–12069 (2015). sourceSupported

Two recent studies described HIV-1 integrase mutants with normal reverse transcription and integration but delayed proviral expression.

“Two recent studies described HIV-1 IN mutants with normal reverse transcription and integration, but delayed proviral expression19,21.”

• cites [19] Winans, S. & Goff, S. P. Mutations altering acetylated residues in the CTD of HIV-1 integrase cause defects in proviral transcription at early times after integration of viral DNA. PLoS Pathog. 16, e1009147 (2020). sourcePartly supported

  The Results section on the transcription defect reports that expression from the QA mutant provirus was severely reduced at two days but reached wild-type levels by four days, supporting delayed expression. However, Fig. 1 in the Results reports approximately threefold fewer reverse-transcription products and a two- to threefold reduction in integrated provirus for some mutants; these steps were functional, not normal in abundance. The paper also found no delay in integration timing. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [21] Knyazhanskaya, E. et al. NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase. Retrovirology 16, 30 (2019). sourcePartly supported

  The Results section reports that the E212A/L213A integrase mutant did not impair reverse transcription or integration and had reduced luciferase expression. It does not establish that proviral expression was merely delayed: expression remained considerably lower at 72 hours, even after the measured gap-repair efficiency matched wild type. The paper supports the finding for this mutant, but not the claim’s characterization of its expression as delayed. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Mislocalisation of viral genomic material may explain why class II mutants fail to synthesize normal vDNA levels.

“The mislocalisation of the viral genomic material provides an attractive explanation as to why class II mutants fail to synthetize normal levels of vDNA upon infection132.”

• cites [132] Elliott, J. L. et al. Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis. eLife 9, e543111 (2020). sourceSupported

Examined class II HIV-1 integrase mutant proteins fail to engage genomic RNA due to defects in virion incorporation, tetramerization, and/or RNA binding.

“Examined class II HIV-1 IN mutant proteins failed to engage genomic RNA, though for varying reasons including defects in virion incorporation, tetramerization, and/or RNA binding132.”

• cites [132] Elliott, J. L. et al. Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis. eLife 9, e543111 (2020). sourcePartly supported

  The Introduction attributes loss of IN–viral RNA interaction to reduced or absent IN in virions, impaired tetramerization, or likely direct interference with RNA binding. However, the Results section “Class II IN mutants abolish IN binding to RNA” says nearly all tested mutants failed to bind viral RNA: E96A reduced binding but did not abolish it. The claim overstates the finding by saying the examined mutants all failed to engage RNA. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

HIV-1 integrase might persist on proviral DNA after integration.

“Accordingly, a recent report indicated that HIV-1 IN might persist on proviral DNA after integration19.”

• cites [19] Winans, S. & Goff, S. P. Mutations altering acetylated residues in the CTD of HIV-1 integrase cause defects in proviral transcription at early times after integration of viral DNA. PLoS Pathog. 16, e1009147 (2020). sourceSupported

ALLINIs suppress genic integration targeting during HIV-1 ingress.

“Consistent with the known role of LEDGF/p75 in HIV-1 integration targeting, genic integration targeting during HIV-1 ingress was suppressed by ALLINIs177,184,185.”

• cites [177] Sharma, A. et al. A new class of multimerization selective inhibitors of HIV-1 integrase. PLoS Pathog. 10, e1004171 (2014). sourceSupported
• cites [184] Feng, L. et al. The competitive interplay between allosteric HIV-1 integrase inhibitor BI/D and LEDGF/p75 during the early stage of HIV-1 replication adversely affects inhibitor potency. ACS Chem. Biol. 11, 1313–1321 (2016). sourceNot checked — no full text of the source
• cites [185] Vranckx, L. S. et al. LEDGIN-mediated inhibition of integrase-LEDGF/p75 interaction reduces reactivation of residual latent HIV. EBioMedicine 8, 248–264 (2016). sourceSupported

Substitutions at Gln148 and Asn155 destabilize the coordination spheres of the Mg2+ ion pair, indirectly affecting drug binding.

“Accordingly, substitutions at these positions exert their effects by destabilizing the coordination spheres of the Mg2+ ion pair, indirectly affecting drug binding35.”

• cites [35] Cook, N. J. et al. Structural basis of second-generation HIV integrase inhibitor action and viral resistance. Science 367, 806 (2020). High-resolution cryo-EM structures of simian immunodeficiency virus intasomes reveal the mechanism of INSTI resistance. sourcePartly supported

  The passage on Q148H/G140S (labelled Abstract; no page given) says His148 disturbs the secondary coordination shell of Mg2+ ions and shifts the bound drug, supporting the mechanism for substitutions at Gln148. Our copy does not establish the same mechanism for substitutions at Asn155. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

MINI compounds are ineffective at inhibiting early HIV-1 infection.

“MINI compounds moreover were ineffective at inhibiting the early stage of HIV-1 infection177.”

• cites [177] Sharma, A. et al. A new class of multimerization selective inhibitors of HIV-1 integrase. PLoS Pathog. 10, e1004171 (2014). sourceSupported

ALLINIs engage the LEDGF/p75 binding cleft at the integrase CCD dimer interface, distal from the active site.

“ALLINIs engage the LEDGF/p75 binding cleft at the IN CCD dimer interface, which is distal from the IN active site109,171.”

• cites [109] Cherepanov, P., Ambrosio, A. L., Rahman, S., Ellenberger, T. & Engelman, A. Structural basis for the recognition between HIV-1 integrase and transcriptional coactivator p75. Proc. Natl Acad. Sci. USA 102, 17308–17313 (2005). sourcePartly supported

  The Results and Discussion (page not available) place the LEDGF-binding site at the integrase CCD dimer interface and describe it as a potential small-molecule inhibitor target. The paper does not show that ALLINIs engage that cleft; it speculates about inhibitor binding and reports other compounds at or near the site. It identifies active sites separately but does not explicitly characterize the cleft as distal from them. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [171] Christ, F. et al. Rational design of small-molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication. Nat. Chem. Biol. 6, 442–448 (2010). sourceNot checked — no full text of the source

Many, though not all, ALLINIs effectively inhibit integrase-LEDGF/p75 binding in vitro.

“Many, though not all, of the ensuing compounds effectively inhibit IN-LEDGF/p75 binding in vitro171,173–175.”

• cites [171] Christ, F. et al. Rational design of small-molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication. Nat. Chem. Biol. 6, 442–448 (2010). sourceNot checked — no full text of the source
• cites [173] Tsiang, M. et al. New class of HIV-1 integrase (IN) inhibitors with a dual mode of action. J. Biol. Chem. 287, 21189–21203 (2012). sourcePartly supported

  The section describing tBPQAs competing with LEDGF for integrase binding reports that all three tested compounds, GS-A, GS-B and GS-C, inhibited the interaction in an in-vitro assay, with IC50 values of 19–228 nM (page unavailable in our copy). This supports effective inhibition by these compounds, but the paper does not establish the claim’s “though not all” qualification for the broader group of compounds. (LLM, conf=0.79)

  Experimental — accuracy not yet measured
• cites [174] Le Rouzic, E. et al. Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage. Retrovirology 10, 144 (2013). sourceSupported
• cites [175] Gupta, K. et al. Allosteric inhibition of human immunodeficiency virus integrase: late block during viral replication and abnormal multimerization involving specific protein domains. J. Biol. Chem. 289, 20477–20488 (2014). sourcePartly supported

  The section “GSK1264 Inhibits Binding of LEDGF(IBD) to IN F185K (CCD)” reports that GSK1264 disrupts the interaction in vitro and notes other compounds with similar activity. The Discussion reports that GSK1264 did not displace LEDGF from preformed complexes in vitro, but the paper does not establish that some ALLINI compounds fail to inhibit binding in the binding assay. No page numbers were available in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Co-crystal structures show ALLINIs bind the LEDGF/p75 IBD pocket at the integrase CCD dimerization interface.

“Many ALLINIs were co-crystallised with the isolated HIV-1 IN CCD, and the structures revealed the molecules grip onto the LEDGF/p75 IBD binding pocket at the IN CCD dimerization interface171,172,174,175,180,186,187.”

• cites [171] Christ, F. et al. Rational design of small-molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication. Nat. Chem. Biol. 6, 442–448 (2010). sourceNot checked — no full text of the source
• cites [172] Fader, L. D. et al. Discovery of BI 224436, a noncatalytic site integrase inhibitor (NCINI) of HIV-1. ACS Med. Chem. Lett. 5, 422–427 (2014). sourceNot checked — no full text of the source
• cites [174] Le Rouzic, E. et al. Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage. Retrovirology 10, 144 (2013). sourceSupported
• cites [175] Gupta, K. et al. Allosteric inhibition of human immunodeficiency virus integrase: late block during viral replication and abnormal multimerization involving specific protein domains. J. Biol. Chem. 289, 20477–20488 (2014). sourceSupported
• cites [180] Koneru, P. C. et al. HIV-1 integrase tetramers are the antiviral target of pyridine-based allosteric integrase inhibitors. eLife 8, e46344 (2019). sourcePartly supported

  The Introduction describes ALLINI binding in a pocket on the IN CCD dimer, supporting the general binding-location claim. The X-ray crystallography methods describe crystallization of isolated CCD, but do not say it was co-crystallized with ALLINIs; the cited passages also do not establish that many such structures show binding at the LEDGF/p75 IBD pocket. Pages are not available in our copy. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [186] Bonnard, D. et al. Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration. J. Biol. Chem. 293, 6172–6186 (2018). sourceSupported
• cites [187] Peese, K. M. et al. 5,6,7,8-Tetrahydro-1,6-naphthyridine derivatives as potent HIV-1-integrase-allosteric-site inhibitors. J. Med. Chem. 62, 1348–1361 (2019). sourceNot checked — no full text of the source

MINI compounds do not effectively antagonize HIV-1 integrase-LEDGF/p75 binding.

“Compounds of the latter class do not effectively antagonise HIV-1 IN-LEDGF/p75 binding.”

• cites [177] Sharma, A. et al. A new class of multimerization selective inhibitors of HIV-1 integrase. PLoS Pathog. 10, e1004171 (2014). sourceSupported

The common biochemical consequence of ALLINI binding is aberrant HIV-1 integrase hyper-multimerization.

“The common biochemical consequence of ALLINI binding is aberrant HIV-1 IN hyper-multimerization140,142,174–181.”

• cites [140] Jurado, K. A. et al. Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation. Proc. Natl Acad. Sci. USA 110, 8690–8695 (2013). This is the first study to reveal that ALLINIs potently inhibit HIV-1 maturation. sourceNot checked — no full text of the source
• cites [142] Balakrishnan, M. et al. Non-catalytic site HIV-1 integrase inhibitors disrupt core maturation and induce a reverse transcription block in target cells. PLoS ONE 8, e74163 (2013). sourceSupported
• cites [174] Le Rouzic, E. et al. Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage. Retrovirology 10, 144 (2013). sourceSupported
• cites [175] Gupta, K. et al. Allosteric inhibition of human immunodeficiency virus integrase: late block during viral replication and abnormal multimerization involving specific protein domains. J. Biol. Chem. 289, 20477–20488 (2014). sourceSupported
• cites [176] Desimmie, B. A. et al. LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions. Retrovirology 10, 57 (2013). sourcePartly supported

  The Discussion reports that LEDGINs enhance HIV-1 integrase multimerization, supporting the claim for that inhibitor class. The Background distinguishes LEDGINs from the broader category of ALLINIs, which includes inhibitors with distinct mechanisms; this paper does not establish hyper-multimerization as a common consequence of ALLINI binding generally. No page numbers are available in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [177] Sharma, A. et al. A new class of multimerization selective inhibitors of HIV-1 integrase. PLoS Pathog. 10, e1004171 (2014). sourceSupported
• cites [178] Deng, N. et al. Allosteric HIV-1 integrase inhibitors promote aberrant protein multimerization by directly mediating inter-subunit interactions: structural and thermodynamic modeling studies. Protein Sci. 25, 1911–1917 (2016). sourceSupported
• cites [179] Gupta, K. et al. Structural basis for inhibitor-induced aggregation of HIV Integrase. PLoS Biol. 14, e1002584 (2016). sourceSupported
• cites [180] Koneru, P. C. et al. HIV-1 integrase tetramers are the antiviral target of pyridine-based allosteric integrase inhibitors. eLife 8, e46344 (2019). sourceSupported
• cites [181] Gupta, K. et al. Allosteric HIV integrase inhibitors promote formation of inactive branched polymers via homomeric carboxy-terminal domain interactions. Structure 29, 213–225.e5 (2021). sourceNot checked — no full text of the source

HIV-1 is more susceptible to ALLINIs when exposed during egress than during ingress.

“Under these conditions, it became clear that HIV-1 was more susceptible to ALLINIs when exposed to the compounds during egress as compared to ingress140,142,174–176.”

• cites [140] Jurado, K. A. et al. Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation. Proc. Natl Acad. Sci. USA 110, 8690–8695 (2013). This is the first study to reveal that ALLINIs potently inhibit HIV-1 maturation. sourceNot checked — no full text of the source
• cites [142] Balakrishnan, M. et al. Non-catalytic site HIV-1 integrase inhibitors disrupt core maturation and induce a reverse transcription block in target cells. PLoS ONE 8, e74163 (2013). sourceSupported
• cites [174] Le Rouzic, E. et al. Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage. Retrovirology 10, 144 (2013). sourceSupported
• cites [175] Gupta, K. et al. Allosteric inhibition of human immunodeficiency virus integrase: late block during viral replication and abnormal multimerization involving specific protein domains. J. Biol. Chem. 289, 20477–20488 (2014). sourceSupported
• cites [176] Desimmie, B. A. et al. LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions. Retrovirology 10, 57 (2013). sourcePartly supported

  The ‘LEDGINs inhibit multiple steps in HIV replication’ section (page unavailable) reports a lower EC50 for CX05045’s late effect on progeny-virion infectivity (1.46 μM) than for its early effect on integration (4.45 μM). This supports greater susceptibility during late-stage exposure for that compound, but the paper does not establish the comparison for ALLINIs as a whole; its Background explicitly distinguishes the broader ALLINI category from LEDGINs. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Primate lentiviral intasomes are unlikely to crystallize because they tend to stack artificially in solution.

“Although the α-retroviral strand transfer intasome complex structure was solved by X-ray crystallography30, it seems highly doubtful, given their propensities to artificially stack upon one another in solution35,36, that primate lentiviral intasomes would ever crystallize.”

• cites [30] Yin, Z. et al. Crystal structure of the Rous sarcoma virus intasome. Nature 530, 362–366 (2016). Together with Ballandras-Colas et al. (2016), these intasome structures first reveal the requirement for more than four IN protomers for functional integration. sourceSupported
• cites [35] Cook, N. J. et al. Structural basis of second-generation HIV integrase inhibitor action and viral resistance. Science 367, 806 (2020). High-resolution cryo-EM structures of simian immunodeficiency virus intasomes reveal the mechanism of INSTI resistance. sourcePartly supported

  The paper reports prominent stacks in SIVrcm intasome preparations and describes how neighboring intasomes form them (Abstract; page not provided). It does not say that this stacking makes primate lentiviral intasomes unlikely to crystallize; that is the citing authors’ inference. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [36] Passos, D. O. et al. Structural basis for strand-transfer inhibitor binding to HIV intasomes. Science 367, 810 (2020). This study provides high-resolution cryo-EM structures of HIV-1 intasomes with several advanced INSTIs. sourceNot checked — no full text of the source

ALLINIs have proven too toxic for in-depth clinical trials despite high potency.

“Although highly potent compounds, ALLINIs to date have proven too toxic for in-depth clinical trials183, warranting attempts to develop safer compounds that can hopefully advance to the clinic.”

• cites [183] Bruggemans, A. et al. GS-9822, a preclinical LEDGIN candidate, displays a block-and-lock phenotype in cell culture. Antimicrob. Agents Chemother. 65, e02328–20 (2021). sourcePartly supported

  The Introduction and Discussion report that potent GS-9822 had urothelial toxicity in monkeys, halting its further advancement. They do not establish that ALLINIs as a class have proven too toxic for in-depth clinical trials: the Introduction says BI224436 reached a phase Ia trial without safety concerns at the reported doses, and the Discussion describes another candidate with no significant toxicity reported in rats or dogs. Page numbers are unavailable in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Subsequent single-particle cryo-EM structures of alpha-, beta-, delta-retroviral and lentiviral intasomes revealed unexpected diversity in the number of integrase molecules needed to construct the active CIC.

“By leveraging the resolution revolution, subsequent single-particle cryo-EM structures of α-, β-, δ-retroviral and lentiviral intasomes revealed unexpected diversity among the number of IN molecules required to construct the active CIC30–33,37,38.”

• cites [30] Yin, Z. et al. Crystal structure of the Rous sarcoma virus intasome. Nature 530, 362–366 (2016). Together with Ballandras-Colas et al. (2016), these intasome structures first reveal the requirement for more than four IN protomers for functional integration. sourcePartly supported

  The paper’s Abstract reports an octameric RSV intasome and contrasts it with a tetrameric PFV intasome, supporting diversity in integrase-molecule number. But its RSV structure was determined by crystallography, not single-particle cryo-EM; the paper mentions a cryo-EM structure of a beta-retroviral intasome but does not report the claimed subsequent cryo-EM structures across all four groups. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [31] Ballandras-Colas, A. et al. Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function. Nature 530, 358–361 (2016). sourceSupported
• cites [32] Ballandras-Colas, A. et al. A supramolecular assembly mediates lentiviral DNA integration. Science 355, 93–95 (2017). Cryo-EM structures of intasomes derived from the sheep lentivirus MVV reveal a higher-order macromolecular machine composed of 16 IN protomers and define the conserved intasome core shared by all retroviral intasome structures. sourceSupported
• cites [33] Passos, D. O. et al. Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome. Science 355, 89–92 (2017). Initial cryo-EM structures of HIV-1 strand transfer complex intasomes reveal a higher-order IN dodecamer architecture. sourceNot checked — no full text of the source
• cites [37] Bhatt, V. et al. Structural basis of host protein hijacking in human T-cell leukemia virus integration. Nat. Commun. 11, 3121 (2020). Together with Barski et al. (2020), cryo-EM studies reveal that deltaretrovirus intasomes are composed of IN tetramers and visualize details of the IN–PP2A interface. sourceSupported
• cites [38] Barski, M. S. et al. Cryo-EM structure of the deltaretroviral intasome in complex with the PP2A regulatory subunit B56γ. Nat. Commun. 11, 5043 (2020). sourceSupported

Some residues lining the ALLINI binding pocket are highly polymorphic among circulating HIV-1 strains.

“Some of the residues that line the ALLINI binding pocket are highly polymorphic among circulating HIV-1 strains, inspiring efforts to design molecules that remain active in the face of such amino acid sequence variability186,187.”

• cites [186] Bonnard, D. et al. Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration. J. Biol. Chem. 293, 6172–6186 (2018). sourceSupported
• cites [187] Peese, K. M. et al. 5,6,7,8-Tetrahydro-1,6-naphthyridine derivatives as potent HIV-1-integrase-allosteric-site inhibitors. J. Med. Chem. 62, 1348–1361 (2019). sourceNot checked — no full text of the source

ALLINI resistance maps to the integrase CCD dimer interface near the LEDGF/p75 binding site.

“Resistance to ALLINIs maps to the IN CCD dimer interface in the vicinity of the LEDGF/p75 binding site171,173,179,182,188.”

• cites [171] Christ, F. et al. Rational design of small-molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication. Nat. Chem. Biol. 6, 442–448 (2010). sourceNot checked — no full text of the source
• cites [173] Tsiang, M. et al. New class of HIV-1 integrase (IN) inhibitors with a dual mode of action. J. Biol. Chem. 287, 21189–21203 (2012). sourceSupported
• cites [179] Gupta, K. et al. Structural basis for inhibitor-induced aggregation of HIV Integrase. PLoS Biol. 14, e1002584 (2016). sourcePartly supported

  The Introduction places the LEDGF/p75 binding site at the integrase catalytic core domain (CCD) dimer interface, and the Discussion identifies some ALLINI-resistance substitutions that likely interfere with inhibitor binding there. But the Discussion also identifies resistance substitutions affecting the CCD–CTD interaction or residing in the N-terminal domain, so the claim overstates where resistance maps. Our copy provides section labels but no page numbers. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [182] Amadori, C. et al. The HIV-1 integrase-LEDGF allosteric inhibitor MUT-A: resistance profile, impairment of virus maturation and infectivity but without influence on RNA packaging or virus immunoreactivity. Retrovirology 14, 50 (2017). sourcePartly supported

  The Resistance profile and spectrum of activity of MUT-A section reports that most tested resistance mutations lie in the integrase catalytic core domain (CCD), close to the inhibitor binding site; the Background identifies that site as the LEDGF binding pocket. The copy does not locate those mutations specifically at the CCD dimer interface, and it notes an exception, N222K, in the C-terminal domain. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [188] Fenwick, C. et al. Preclinical profile of BI 224436, a novel HIV-1 non-catalytic-site integrase inhibitor. Antimicrob. Agents Chemother. 58, 3233–3244 (2014). sourceSupported

Mass spectrometry footprinting, mutagenesis, and computational chemistry support this mode of integrase aggregation by ALLINIs.

“Solution measurements using mass spectrometry footprinting techniques, mutagenesis and computational chemistry strongly support this mode of IN aggregation by ALLINIs178,189.”

• cites [178] Deng, N. et al. Allosteric HIV-1 integrase inhibitors promote aberrant protein multimerization by directly mediating inter-subunit interactions: structural and thermodynamic modeling studies. Protein Sci. 25, 1911–1917 (2016). sourceSupported
• cites [189] Shkriabai, N. et al. A critical role of the C-terminal segment for allosteric inhibitor-induced aberrant multimerization of HIV-1 integrase. J. Biol. Chem. 289, 26430–26440 (2014). sourcePartly supported

  The Results and Discussion report MS-based footprinting, tests of integrase mutants, and ALLINI-2-induced higher-order multimerization. The Discussion interprets these findings using structural models, but the passages do not establish that this paper performed computational chemistry; that part of the attributed claim is not supported by our copy. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

The ALLINI carboxylate forms a bidentate hydrogen bond with main-chain amides of integrase Glu170 and His171, mimicking Asp366 of LEDGF/p75.

“The carboxylate establishes a bidentate hydrogen bond with main chain amides of IN Glu170 and His171, perfectly recuperating the interactions made by Asp366 of LEDGF/p75 (Fig. 6C)109.”

• cites [109] Cherepanov, P., Ambrosio, A. L., Rahman, S., Ellenberger, T. & Engelman, A. Structural basis for the recognition between HIV-1 integrase and transcriptional coactivator p75. Proc. Natl Acad. Sci. USA 102, 17308–17313 (2005). sourcePartly supported

  The Results and Discussion section (page unavailable) reports that LEDGF/p75 Asp-366 forms a bidentate hydrogen bond with the main-chain amides of integrase Glu-170 and His-171. It does not establish that an ALLINI carboxylate makes the same bonds; its discussion of a small-molecule inhibitor is speculative. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

Recent work uncovered surprising differences in mechanisms targeting retroviral integration, even within a single genus.

“Recent work uncovered surprising differences between mechanisms involved in targeting retroviral integration, even within a single genus87.”

• cites [87] Li, W. et al. CPSF6-dependent targeting of speckle-associated domains distinguishes primate from nonprimate lentiviral integration. mBio 11, e02254-20 (2020). sourceSupported

The absence of structural similarity among ALLINIs outside the common warhead likely contributes to differential integrase aggregation mechanisms.

“The absence of structural similarity between ALLINIs outside of the common warhead (Fig. 6A) likely contributes to differential IN aggregation mechanisms reported for some of these compounds180, calling for more research and development of this fascinating class of antiviral agents.”

• cites [180] Koneru, P. C. et al. HIV-1 integrase tetramers are the antiviral target of pyridine-based allosteric integrase inhibitors. eLife 8, e46344 (2019). sourceSupported

All retroviral integrases contain three structural domains: an N-terminal domain, a catalytic core domain, and a C-terminal domain.

“All INs harbour three structural domains connected by flexible linkers: the N-terminal domain (NTD), the catalytic core domain (CCD), and the C-terminal domain (CTD).”

• cites [7] Schiff, R. D. & Grandgenett, D. P. Virus-coded origin of a 32,000-dalton protein from avian retrovirus cores: structural relatedness of p32 and the beta polypeptide of the avian retrovirus DNA polymerase. J. Virol. 28, 279–291 (1978). sourceNot checked — no full text of the source
• cites [22] Cai, M. et al. Solution structure of the N-terminal zinc binding domain of HIV-1 integrase. Nat. Struct. Biol. 4, 567–577 (1997). sourceNot checked — no full text of the source
• cites [23] Dyda, F. et al. Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science 266, 1981–1986 (1994). The first high-resolution structure of a retroviral IN CCD reveals similarity to other polynucleotidyltransferases, including DNA transposases and RNase H. sourceNot checked — no full text of the source

Because transesterification does not change the balance of high-energy bonds, the strand transfer reaction should be reversible.

“Because transesterification does not change the balance of high-energy bonds, the strand transfer reaction should be reversible.”

Retroviral integrases can cleave branched DNA constructs that mimic strand transfer products, in a reaction called disintegration.

“Indeed, retroviral INs can cleave branched DNA constructs that mimic strand transfer products, in a reaction termed disintegration50.”

• cites [50] Chow, S. A. & Brown, P. O. Juxtaposition of two viral DNA ends in a bimolecular disintegration reaction mediated by multimers of human immunodeficiency virus type 1 or murine leukemia virus integrase. J. Virol. 68, 7869–7878 (1994). sourceNot checked — no full text of the source

The driving force for the conformational change is thought to come from tension within sharply bent target DNA.

“The driving force for this conformational change is thought to originate from the tension within sharply bent target DNA.”

The mechanism that channels transesterification toward the product is likely conserved across this diverse family of enzymes.

“Thus, the mechanism of channelling transesterification towards the product is likely conserved throughout this highly diverse family of enzymes.”

Both integrase-catalyzed reactions follow a general SN2 nucleophilic substitution pathway at a phosphorus atom.

“Both reactions catalysed by IN follow the general SN2 nucleophilic substitution pathway at a phosphorus atom in vDNA (during 3’-processing) or target DNA (during strand transfer)49.”

• cites [49] Engelman, A., Mizuuchi, K. & Craigie, R. HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer. Cell 67, 1211–1221 (1991). sourceNot checked — no full text of the source

Retroviral integration into host genomes is non-random and is influenced by several factors, including the mode of viral nuclear entry and pre-integration complex-host interactions.

“Retroviral integration into host genomes is non-random and is influenced by several factors including the mode of viral nuclear entry and PIC-host interactions (see REF.68 for a recent in-depth review).”

• cites [68] Engelman, A. N., Maertens, G. N. in Retrovirus-Cell Interactions (ed. Parent, L. J.) Ch. 4, 163–199 (Elsevier Inc., 2018). sourceNot checked — no full text of the source

Partial deletions in the integrase coding region or point mutations such as H12N impair HIV-1 core formation.

“while partial deletions within the IN coding region or point mutations, such as H12N, additionally impaired HIV-1 core formation131.”

• cites [131] Engelman, A., Englund, G., Orenstein, J. M., Martin, M. A. & Craigie, R. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J. Virol. 69, 2729–2736 (1995). sourceNot checked — no full text of the source

The LEDGF/p75 IBD is an alpha-helical domain structurally related to HEAT repeat proteins.

“The LEDGF/p75 IBD is an α-helical domain topologically related to HEAT repeat proteins108.”

• cites [108] Cherepanov, P. et al. Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75. Nat. Struct. Mol. Biol. 12, 526–532 (2005). sourceNot checked — no full text of the source

Ablation of HIV-1 integrase by a premature stop in pol disrupts viral assembly and egress.

“Thus, ablation of HIV-1 IN via a premature stop within the pol gene disrupted viral assembly and egress130”

• cites [130] Bukovsky, A. & Gottlinger, H. Lack of integrase can markedly affect human immunodeficiency virus type 1 particle production in the presence of an active viral protease. J. Virol. 70, 6820–6825 (1996). sourceNot checked — no full text of the source

Most amino acid substitutions in the HIV-1 integrase D,D-35-E catalytic triad belong to class I.

“In particular, the majority of amino acid substitutions within the HIV-1 IN D,D-35-E catalytic triad fell into this category.”

• cites [131] Engelman, A., Englund, G., Orenstein, J. M., Martin, M. A. & Craigie, R. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J. Virol. 69, 2729–2736 (1995). sourceNot checked — no full text of the source

Accumulation of 2-LTR circles in cells infected with class I variants is consistent with a specific integration defect.

“Accumulation of 2-LTR circles, the products of end-to-end ligation of unintegrated vDNA ends, in cells infected with class I variants was consistent with a specific defect at the stage of integration131,134.”

• cites [131] Engelman, A., Englund, G., Orenstein, J. M., Martin, M. A. & Craigie, R. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J. Virol. 69, 2729–2736 (1995). sourceNot checked — no full text of the source
• cites [134] Leavitt, A. D., Robles, G., Alesandro, N. & Varmus, H. E. Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection. J. Virol. 70, 721–728 (1996). sourceNot checked — no full text of the source

The C-terminal peptide of MLV integrase folds onto the BET extraterminal domain, contributing two beta-strands to complete a three-stranded beta-sheet in a compact quaternary structure.

“The C-terminal peptide of MLV IN folds onto the extraterminal domain, contributing two β-strands to complete a 3-stranded β-sheet within a compact quaternary structure122,123.”

• cites [122] Crowe, B. L. et al. Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction. Proc. Natl Acad. Sci. USA 113, 2086–2091 (2016). sourceNot checked — no full text of the source
• cites [123] Aiyer, S. et al. A common binding motif in the ET domain of BRD3 forms polymorphic structural interfaces with host and viral proteins. Structure https://doi.org/10.1016/j.str.2021.01.010 (2021). sourceNot checked — no full text of the source

The INSTI pharmacophore consists of a metal-chelating core, generally a co-planar triad of oxygen atoms, and a halobenzyl side chain.

“The INSTI pharmacophore consists of a metal chelating core - generally a co-planar triad of oxygen atoms - and a halobenzyl side chain (Fig. 5A, shown in red and blue, respectively)156.”

• cites [156] Grobler, J. A. et al. Diketo acid inhibitor mechanism and HIV-1 integrase: implications for metal binding in the active site of phosphotransferase enzymes. Proc. Natl Acad. Sci. USA 99, 6661–6666 (2002). sourceNot checked — no full text of the source

Twenty-four years passed from the discovery of HIV-1 until raltegravir became available in clinical practice.

“Therefore, it is not surprising that 24 years transpired from the discovery of HIV-1 before the first IN strand transfer inhibitor (INSTI), raltegravir, became available in clinical practice146.”

• cites [146] Murray, J. M. et al. Antiretroviral therapy with the integrase inhibitor raltegravir alters decay kinetics of HIV, significantly reducing the second phase. AIDS 21, 2315–2321 (2007). sourceNot checked — no full text of the source

A successful integrase inhibitor would need an extremely slow dissociation rate to remain bound for the lifetime of the PIC.

“Thus, a successful inhibitor would have an extremely slow dissociation rate, remaining bound for the lifetime of the PIC.”

• AssumptionRests on an assumption: The HIV-1 integrase active site performs only one strand transfer event during infection.

MLV integrase mutations can strongly reduce reverse transcription.

“Similar to HIV-1, mutations in MLV IN can strongly reduce reverse transcription143.”

• cites [143] Steinrigl, A. et al. Mutations in the catalytic core or the C-terminus of murine leukemia virus (MLV) integrase disrupt virion infectivity and exert diverse effects on reverse transcription. Virology 362, 50–59 (2007). sourceNot checked — no full text of the source

INSTIs exploit the enhanced mobility of the 3' vDNA nucleotide that is separated from its Watson-Crick pair in the intasome.

“Thus, INSTIs take advantage of the enhanced mobility of the 3’ vDNA nucleotide that is separated from its Watson-Crick pair in the intasome.”

INSTIs display slow binding kinetics because the 3' adenosine must vacate its normal position for inhibitor binding.

“Because the 3’ adenosine must vacate its normal position to allow inhibitor binding, the INSTIs display slow binding kinetics163.”

• cites [163] Langley, D. R. et al. The terminal (catalytic) adenosine of the HIV LTR controls the kinetics of binding and dissociation of HIV integrase strand transfer inhibitors. Biochemistry 47, 13481–13488 (2008). sourceNot checked — no full text of the source

Integrase may play a role in reverse transcription, explaining some class II phenotypes.

“These observations suggest that IN may play a role in reverse transcription, accounting for some of the class II phenotypes.”

In avian alpha-retroviruses, the large RT subunit retains the entire integrase polypeptide due to alternative proteolytic processing.

“Moreover, in avian α-retroviruses, the large RT subunit retains the entire IN polypeptide due to alternative proteolytic processing7.”

• cites [7] Schiff, R. D. & Grandgenett, D. P. Virus-coded origin of a 32,000-dalton protein from avian retrovirus cores: structural relatedness of p32 and the beta polypeptide of the avian retrovirus DNA polymerase. J. Virol. 28, 279–291 (1978). sourceNot checked — no full text of the source

Because human cells lack a close integrase homolog, the viral enzyme appears to be an attractive drug target.

“Because human cells lack a close IN homolog, the viral enzyme would appear an ideal target for drug development.”

• AssumptionRests on an assumption: Human cells do not have a close homolog of HIV-1 integrase.

Because integrase tetramerization is important for RNA binding, lack of the IN-RNA complex in virions appears to be the unifying property of the three class II subclasses.

“Because HIV-1 IN tetramerization was important for RNA binding, the lack of IN-RNA complex within virions seems to be the unifying property of the three subclasses.”

Rising prevalence of HIV resistance to RT inhibitors underscores the urgency of developing new integrase inhibitors.

“The urgency is underscored by the rising prevalence of HIV resistance to RT inhibitors, which comprise the backbone of combination antiretroviral therapy170.”

• cites [170] Engone-Ondo, J. D. et al. High rate of virological failure and HIV drug resistance in semi-rural Gabon and implications for dolutegravir-based regimen efficacy. J. Antimicrob. Chemother. 76, 1051–1056 (2020). sourceNot checked — no full text of the source

ALLINIs inhibit HIV-1 maturation/morphogenesis.

“Accordingly, ALLINIs are inhibitors of HIV-1 maturation/morphogenesis139.”

• cites [139] Fontana, J. et al. Distribution and redistribution of HIV-1 nucleocapsid protein in immature, mature, and integrase-inhibited virions: a role for integrase in maturation. J. Virol. 89, 9765–9780 (2015). sourceNot checked — no full text of the source

Primate lentiviral intasome structures show that Gln148 and Asn155 participate in the secondary coordination spheres of the metal cations.

“The primate lentiviral intasome structures revealed that these residues participate in the secondary coordination spheres of the metal cations (Fig. 5B).”

The sensitivity of metal ions to the geometry and electronic properties of the ligand chelating cluster underpins the major mechanism of INSTI resistance.

“Thus, the sensitivity of metal ions for the geometry and electronic properties of the ligand chelating cluster underpins the major mechanism of INSTI resistance.”

Three main genetic pathways cause high-level raltegravir resistance, involving changes at HIV integrase residues Tyr143, Gln148, or Asn155.

“Three main genetic pathways lead to high-level resistance to raltegravir, involving changes of HIV IN residues Tyr143, Gln148, or Asn155 (REF.166).”

• cites [166] Grobler, J. A. & Hazuda, D. J. Resistance to HIV integrase strand transfer inhibitors: in vitro findings and clinical consequences. Curr. Opin. Virol. 8, 98–103 (2014). sourceNot checked — no full text of the source

INSTIs have unusually long residence in the active site due to metal ion interactions, vDNA base stacking, and extensive van der Waals contacts with integrase.

“Due to their interaction with the metal ions, stacking with vDNA bases and extensive van der Waals interactions with IN, INSTIs display unusually long residence in the active site.”

ALLINIs share a warhead with a carboxylate, a compact aliphatic side chain, and a bulky hydrophobic group.

“ALLINIs display limited structural diversity, sharing a warhead comprising three chemical functions: a carboxylate, a compact aliphatic (typically tert-butoxy) sidechain and a relatively bulky hydrophobic (usually aromatic) group (Fig. 6A, shown in red, green and blue, respectively).”

A more recent study detected branching of GSK1264-induced integrase polymers via homomeric CTD-CTD interactions.

“A more recent study detected branching of the GSK1264-induced IN polymers via homomeric (CTD-CTD) interactions181.”

• cites [181] Gupta, K. et al. Allosteric HIV integrase inhibitors promote formation of inactive branched polymers via homomeric carboxy-terminal domain interactions. Structure 29, 213–225.e5 (2021). sourceNot checked — no full text of the source