Understanding Capsid Assembly and Genome Packaging for Adeno-Associated Viruses

All serotypes tested, with the exception of AAV4, AAV5 and AAV11, require AAP in order to assemble their capsid.

“In the most recent studies, it has been shown that all serotypes tested, with the exception of AAV4, AAV5 and AAV11, require AAP in order to assemble their capsid [35,39, 45–47].”

• cites [35] Sonntag F, Schmidt K, Kleinschmidt JA. A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc. Natl Acad. Sci. USA 107(22), 10220– 10225 (2010). 20479244 PMC2890453 • The discovery of assembly-activating protein settled a decade-long controversy about which VP is essential for capsid assembly and the identification and characterization of the AAV scaffold protein. sourcePartly supported

  The Results and Discussion show that AAP is required for AAV2 capsid assembly. The paper does not establish that all tested serotypes require AAP or identify AAV4, AAV5, and AAV11 as exceptions; its discussion of other serotypes concerns sequence conservation, not assembly requirements. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [39] Naumer M, Sonntag F, Schmidt K Properties of the adeno-associated virus assembly-activating protein. J. Virol 86(23), 13038– 13048 (2012). 23015698 PMC3497687 sourceNot checked — no full text of the source
• cites [45] Earley LF, Powers JM, Adachi K Adeno-associated virus assembly-activating protein is not an essential requirement for capsid assembly of AAV serotypes 4, 5 and 11. J. Virol 85(23), 12686– 12697 (2016). PMC5244341 27852862 sourceSupported
• cites [46] Sonntag F, Kother K, Schmidt K The assembly-activating protein promotes capsid assembly of different adeno-associated virus serotypes. J. Virol 85(23), 12686– 12697 (2011). 21917944 PMC3209379 sourceDisputed

  The paper’s Results report that AAV5-VP3 capsid formation was restored by AAP-5, and its summary explicitly says AAV5 assembly required AAP-5. Thus AAV5 is not an exception to AAP dependence in this source. The paper discusses AAV4 and AAV11 sequence differences but does not test their assembly requirement. (LLM, conf=0.92)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [47] Popa-Wagner R, Sonntag F, Schmidt K, King J, Kleinschmidt JA. Nuclear translocation of adeno-associated virus type 2 capsid proteins for virion assembly. J. Gen. Virol 93(Pt 9), 1887– 1898 (2012). 22694902 sourceNot checked — no full text of the source

The cytoplasmic fraction contains VP monomers and oligomers ranging from 10 to 15S, consistent with trimers and pentamers of VP1, VP2 and VP3.

“Sedimentation sucrose gradient analysis of nuclear and cytoplasmic fractions of an AAV infection demonstrated that the cytoplasmic fraction contains a population of VP monomers and oligomers ranging in size from 10 to 15S consistent with the sedimentation coefficients for trimers and pentamers, respectively, of VP1, VP2 and VP3.”

• cites [27] Wistuba A, Weger S, Kern A, Kleinschmidt JA. Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins. J. Virol 69(9), 5311– 5319 (1995). 7636974 PMC189369 sourcePartly supported

  The Results (Fig. 3a–b; page not provided) report cytoplasmic VP monomers around 6.5S and a continuous distribution of VP1, VP2 and VP3 oligomers extending to 19S. The distinct 10–15S peak and the tentative pentamer interpretation instead concern nuclear VP2/VP3 oligomers; the paper does not identify cytoplasmic oligomers as trimers and pentamers. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Nucleolin and nucleophosmin associate with both VPs and assembled capsids in the nucleoli.

“Additionally, two nuclear proteins, nucleolin and nucleophosmin [43,44], have been shown to associate with both VPs and assembled capsids in the nucleoli.”

• cites [43] Bevington JM, Needham PG, Verrill KC, Collaco RF, Basrur V, Trempe JP. Adeno-associated virus interactions with B23/Nucleophosmin: identification of sub-nucleolar virion regions. Virology 357(1), 102– 113 (2007). 16959286 PMC1829415 sourcePartly supported

  The ‘NPM co-localization with Cap and Rep proteins’ section reports that nucleophosmin (NPM) co-localizes with AAV Cap proteins in nucleoli. The Discussion proposes a model in which Cap proteins assemble there and associate with NPM, but the paper does not establish an association with assembled capsids; it also does not address nucleolin. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [44] Qiu J, Brown KE. A 110-kDa nuclear shuttle protein, nucleolin, specifically binds to adeno-associated virus type 2 (AAV-2) capsid. Virology 257(2), 373– 382 (1999). 10329548 sourcePartly supported

  The Discussion (page unavailable) describes nucleolin binding to VP1 and VP2 and proposes that it may direct them to the nucleolus. The results section reports nucleolin coimmunoprecipitating with assembled capsids and colocalizing with them in the nucleus, while the Discussion says capsid assembly occurs in the nucleolus. The paper does not demonstrate that both associations occur specifically in the nucleoli; it addresses nucleolin, not the separately cited nucleophosmin. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

By replacing the D-sequence in one of the flanking ITRs with an unrelated sequence, packaging of genomes with only positive or negative polarity can be achieved.

“By replacing the D-sequence in one of the flanking ITRs with an unrelated sequence, the packaging of genomes with only positive or negative polarity can be achieved [49].”

• cites [49] Ling C, Wang Y, Lu Y Enhanced transgene expression from recombinant single-stranded D-sequence-substituted adeno-associated virus vectors in human cell lines in vitro and in murine hepatocytes in vivo. J. Virol 89(2), 952– 961 (2015). 25355884 PMC4300666 sourceSupported

Different types of contaminating DNA sequences, including fragments of the host cell genome from vector production cells, are copackaged with transgenes in AAV vectors.

“different types of contaminating DNA sequences have been described as being copackaged with the transgenes, including fragments of the host cell genome from the cells that are used during vector production [81].”

• cites [81] Levy HC, Bowman VD, Govindasamy L Heparin binding induces conformational changes in Adeno-associated virus serotype 2. J. Struct. Biol 165(3), 146– 156 (2009). 19121398 PMC3694330 sourcePartly supported

  The Results and Discussion section ('AAV2-Hep encapsidates an ordered, dodecahedral cage of DNA'; page unavailable) reports non-viral DNA inside AAV2 virus-like particles and suggests that random DNA fragments may be packaged during assembly. It does not identify those fragments as host-cell genomic DNA or show them copackaged with transgenes in AAV vectors; the particles studied lacked a viral genome. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

AAP functions as a scaffold protein that facilitates the localization of VP3 to the nucleus.

“The AAP is reported to function as a scaffold protein that facilitates the localization of VP3 to the nucleus [35].”

• cites [35] Sonntag F, Schmidt K, Kleinschmidt JA. A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc. Natl Acad. Sci. USA 107(22), 10220– 10225 (2010). 20479244 PMC2890453 • The discovery of assembly-activating protein settled a decade-long controversy about which VP is essential for capsid assembly and the identification and characterization of the AAV scaffold protein. sourcePartly supported

  The Results report that coexpressing AAP moves some VP3 into the nucleolus, a region within the nucleus. The Discussion says AAP *could* act as a scaffold during capsid assembly, but presents that as one possible mechanism, not an established function. The claim overstates the scaffold role and describes nucleolar targeting less precisely as nuclear localization. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Residues located at the two- and fivefold interfaces of the VP, secondary structure elements, and close to the fivefold channel play a role in capsid assembly.

“Residues located at the two- and fivefold interfaces of the VP, secondary structure elements and close to the fivefold channel play a role in capsid assembly (Table 1 & Figure 4) [36–37].”

• cites [36] Bleker S, Sonntag F, Kleinschmidt JA. Mutational analysis of narrow pores at the fivefold symmetry axes of adeno-associated virus type 2 capsids reveals a dual role in genome packaging and activation of phospholipase A2 activity. J. Virol 79(4), 2528– 2540 (2005). 15681453 PMC546590 sourcePartly supported

  The Discussion (page unavailable) reports that mutations in the VP beta-barrel impaired assembly and that several mutations near the fivefold pore impaired or abolished capsid formation. It does not establish a role in assembly for residues at the twofold interface; its mention of twofold axes concerns the location of VP N termini, not assembly. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [37] Popa-Wagner R, Porwal M, Kann M Impact of VP1-specific protein sequence motifs on adeno-associated virus type 2 intracellular trafficking and nuclear entry. J. Virol 86(17), 9163– 9174 (2012). 22696661 PMC3416132 sourceNot checked — no full text of the source

The nuclear fraction contained VP oligomers with sedimentation coefficients between 60 and 110S, consistent with empty and full capsids, respectively.

“The nuclear fraction contained VP oligomers with sedimentation coefficients between 60 and 110S consistent with empty and full capsids, respectively [27].”

• cites [27] Wistuba A, Weger S, Kern A, Kleinschmidt JA. Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins. J. Virol 69(9), 5311– 5319 (1995). 7636974 PMC189369 sourceSupported

In addition to VP3, the assembly-activating protein (AAP) is also essential for capsid assembly.

“However, in addition to VP3, the AAP is also essential for capsid assembly [35].”

• cites [35] Sonntag F, Schmidt K, Kleinschmidt JA. A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc. Natl Acad. Sci. USA 107(22), 10220– 10225 (2010). 20479244 PMC2890453 • The discovery of assembly-activating protein settled a decade-long controversy about which VP is essential for capsid assembly and the identification and characterization of the AAV scaffold protein. sourceSupported

For the majority of rAAV vectors, the ITRs and rep gene of AAV2 are utilized, resulting in pseudotyped rAAVs.

“For the majority of rAAV vectors, the ITRs and rep gene of serotype 2 (AAV2) are utilized resulting in pseudo-typed rAAVs [11].”

• cites [11] Grimm D, Kay MA, Kleinschmidt JA. Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6. Mol. Ther 7(6), 839– 850 (2003). 12788658 sourcePartly supported

  The Results describe pseudotyped particles containing AAV-2 vector DNA made with a helper that retains AAV-2 rep while substituting capsid genes from other serotypes. The Discussion says pseudotyping of non-type-2 vectors had been described only once, supporting the prominence of AAV-2-based pseudotyping. Neither passage establishes that the majority of all rAAV vectors use AAV-2 ITRs and rep or that most rAAV vectors are pseudotyped. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

Recent next-generation sequencing based technologies allow unbiased identification and quantification of DNA impurities.

“Recent next-generation sequencing based technologies allow an unbiased identification and quantification of DNA impurities [87].”

• cites [87] Lecomte E, Tournaire B, Cogne B Advanced characterization of DNA molecules in rAAV vector preparations by single-stranded virus next-generation sequencing. Mol. Ther. Nucleic Acids 4, e260 (2015). 26506038 PMC4881760 • Characterized the contamination of purified AAV vector preparation with packaged nonvector DNAs using next-generation sequencing. sourcePartly supported

  The Results section (‘Distribution of DNA contaminants identified by NGS correlates with qPCR data’; page not specified) reports relative proportions of contaminants measured by SSV-Seq, while the Introduction explains that it avoids qPCR’s need for target-specific assays. But the Discussion (page not specified) warns that uneven amplification can introduce quantitative bias into NGS data, so ‘unbiased’ quantification overstates the source. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Mutational studies on the entire cap gene showed that the VP3 common sequence is enough to assemble AAV capsids by knocking out the start codons of VP1 and VP2.

“Mutational studies on the entire cap gene showed that the VP3 common sequence is enough to assemble AAV capsids by knocking out the start codons of VP1 and VP2 [31–34].”

• cites [31] Ruffing M, Zentgraf H, Kleinschmidt JA. Assembly of viruslike particles by recombinant structural proteins of adeno-associated virus type 2 in insect cells. J. Virol 66(12), 6922– 6930 (1992). 1331503 PMC240317 sourceNot checked — no full text of the source
• cites [32] Steinbach S, Wistuba A, Bock T, Kleinschmidt JA. Assembly of adeno-associated virus type 2 capsids in vitro. J. Gen. Virol 78 (Pt 6) 1453– 1462 (1997). 9191943 sourceNot checked — no full text of the source
• cites [33] Rabinowitz JE, Xiao W, Samulski RJ. Insertional mutagenesis of AAV2 capsid and the production of recombinant virus. Virology 265(2), 274– 285 (1999). 10600599 sourceNot checked — no full text of the source
• cites [34] Warrington KH Jr, Gorbatyuk OS, Harrison JK, Opie SR, Zolotukhin S, Muzyczka N. Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus. J. Virol 78(12), 6595– 6609 (2004). 15163751 PMC416546 sourceSupported

For productive replication, AAVs depend on coinfection with helper viruses such as adenoviruses or herpesviruses.

“For productive replication, the AAVs depend on the coinfection with helper viruses, such as, adenoviruses or herpesviruses [2].”

• cites [2] Weitzman MD, Linden RM. Adeno-associated virus biology. Methods Mol. Biol 807, 1– 23 (2011). 22034024 sourceNot checked — no full text of the source

The viral capsid is assembled from the overlapping VP3 region.

“The viral capsid is assembled from the overlapping VP3 region [8].”

• cites [8] Mclaughlin SK, Collis P, Hermonat PL, Muzyczka N. Adeno-associated virus general transduction vectors: analysis of proviral structures. J. Virol 62(6), 1963– 1973 (1988). 2835501 PMC253280 sourceNot checked — no full text of the source

Helper viruses provide transcription factors for AAV promoters, replication factors, and factors that modify the host cell environment for efficient AAV transcription, translation and genome replication.

“The functions that these helper viruses provide include transcription factors for the AAV promoters, replication factors and factors that modify the host cell environment for efficient AAV transcription, translation and genome replication [2].”

• cites [2] Weitzman MD, Linden RM. Adeno-associated virus biology. Methods Mol. Biol 807, 1– 23 (2011). 22034024 sourceNot checked — no full text of the source

VP1 contains a phospholipase A2 site in its unique N-terminus (VP1u) that is important for endo/lysosomal escape during cellular trafficking and viral infectivity.

“VP1 is encoded within the entire cap ORF, it is approximately 735 aa in length, 87 kDa in molecular weight (MW) and contains a phospholipase A2 (PLA2) site that is important for endo/lysosomal escape during cellular trafficking and viral infectivity in its unique N-terminus (VP1u) [7].”

• cites [7] Girod A, Wobus CE, Zadori Z The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity. J. Gen.Virol 83(Pt 5), 973– 978 (2002). 11961250 sourceNot checked — no full text of the source

Mutation of the D-sequence region in both ITRs leads to a defect in replication and packaging.

“Mutation of this region in both ITRs leads to a defect in replication and packaging [50].”

• cites [50] Wang XS, Qing K, Ponnazhagan S, Srivastava A. Adeno-associated virus type 2 DNA replication in vivo: mutation analyses of the D sequence in viral inverted terminal repeats. J. Virol 71(4), 3077– 3082 (1997). 9060669 PMC191438 sourceNot checked — no full text of the source

Assembled capsids are first detected in the nucleus, specifically in the nucleoli during a productive infection.

“Assembled capsids are first detected in the nucleus, specifically in the nucleoli during a productive infection [26].”

• cites [26] Wistuba A, Kern A, Weger S, Grimm D, Kleinschmidt JA. Subcellular compartmentalization of adeno-associated virus type 2 assembly. J. Virol 71(2), 1341– 1352 (1997). 8995658 PMC191189 sourceNot checked — no full text of the source

Increasing the backbone of the vector genome plasmid to sizes larger than the packaging capacity of AAV vectors minimizes reverse packaging.

“the increase of the backbone of the vector genome plasmid to sizes larger than the packaging capacity of AAV vectors to minimize reverse packaging [86]”

• cites [86] Hauck B, Murphy SL, Smith PH Undetectable transcription of cap in a clinical AAV vector: implications for preformed capsid in immune responses. Mol. Ther 17(1), 144– 152 (2009). 18941440 PMC2834997 sourceNot checked — no full text of the source

Alterations of residues in the symmetry-related interfaces are predicted to abrogate the formation of assembly intermediates or building blocks that are important components of the assembly pathway.

“Alterations of residues in the symmetry-related interfaces are predicted to abrogate the formation of assembly intermediates or building blocks that are important components of the assembly pathway.”

For AAVs, specific regions of the capsid, including the two- and fivefold interfaces, have been identified as important determinants of assembly.

“For the AAVs, specific regions of the capsid have been identified as important determinants of assembly, including the two- and fivefold interfaces.”

In recombinant AAV vectors, a desired transgene expression cassette is packaged into the capsids instead of the wild-type viral genome.

“In recombinant AAV vectors (rAAVs), a desired transgene expression cassette is packaged into the capsids instead of the wild-type (WT) viral genome [10].”

• cites [10] Gray JT, Zolotukhin S. Design and construction of functional AAV vectors. Methods Mol. Biol 807, 25– 46 (2011). 22034025 sourceNot checked — no full text of the source

For packaging of a DNA molecule into AAV capsids, the DNA sequence must be flanked by the cis-acting ITRs.

“For the packaging of a certain DNA molecule into AAV capsids, the WT AAV genome or any transgene cassette, the DNA sequence must be flanked by the cis-acting ITRs.”

The translocation of the ssDNA into the capsid is believed to occur through the channel at the fivefold symmetry axis.

“The translocation of the ssDNA into the capsid is believed to occur through the channel at the fivefold symmetry axis.”

Single-stranded binding proteins E2A (adenovirus) or ICP8 (herpes virus) provided by helper viruses are involved in displacement of the ssDNA genome.

“In that process, the single-stranded binding proteins, E2A (adenovirus) or ICP8 (herpes virus) provided by the helper viruses are involved [2, 51].”

• cites [2] Weitzman MD, Linden RM. Adeno-associated virus biology. Methods Mol. Biol 807, 1– 23 (2011). 22034024 sourceNot checked — no full text of the source
• cites [51] Alex M, Weger S, Mietzsch M, Slanina H, Cathomen T, Heilbronn R. DNA-binding activity of adeno-associated virus Rep is required for inverted terminal repeat-dependent complex formation with herpes simplex virus ICP8. J. Virol 86(5), 2859– 2863 (2012). 22205745 PMC3302247 sourceNot checked — no full text of the source

The current model for AAV packaging couples the replication and packaging process.

“The current model for AAV packaging couples the replication and packaging process (Figure 5A).”

The T=1 icosahedral capsid of AAVs is assembled via two-, three- and fivefold interactions which form a dimer, trimer and pentamer, respectively.

“The T = 1 icosahedral capsid of the AAVs (Figure 2B) is assembled via two-, three- and fivefold interactions which form a dimer, trimer and pentamer, respectively (Figure 3).”

Removal of potential RBEs from helper constructs reduces packaging of nonvector DNA sequences.

“the removal of potential RBEs from helper constructs [85]”

• cites [85] Mietzsch M, Casteleyn V, Weger S, Zolotukhin S, Heilbronn R. OneBac 2.0: Sf9 cell lines for production of AAV5 vectors with enhanced infectivity and minimal encapsidation of foreign DNA. Hum. Gene Ther 26(10), 688– 697 (2015). 26134901 PMC4842952 sourceNot checked — no full text of the source

The Rep proteins, capsids and the AAV DNA colocalize in the nucleoplasm in infected cells.

“This is supported by the observation that the Rep proteins, capsids and the AAV DNA colocalize in the nucleoplasm in infected cells [26].”

• cites [26] Wistuba A, Kern A, Weger S, Grimm D, Kleinschmidt JA. Subcellular compartmentalization of adeno-associated virus type 2 assembly. J. Virol 71(2), 1341– 1352 (1997). 8995658 PMC191189 sourceNot checked — no full text of the source

Helper gene constructs transfected or infected into vector producer cells are a source of DNA impurities in AAV vector preparations.

“Another source of DNA impurities are the helper gene constructs that are transfected or infected into the vector producer cells.”

Reverse packaging results in the packaging of DNA sequences outside of the ITR-flanked vector genome cassette.

“Third, a phenomenon termed ‘reverse packaging’ has been described that results in the packaging of DNA sequences outside of the ITR-flanked vector genome cassette [84].”

• cites [84] Wright JF. Manufacturing and characterizing AAV-based vectors for use in clinical studies. Gene Ther. 15(11), 840– 848 (2008). 18418418 sourceNot checked — no full text of the source

Dimer and pentamer formation are important steps in AAV capsid assembly.

“This suggests that dimer and pentamer formation are important steps in this process, although further studies are required to determine their temporal order.”

Quantitative PCR limits quantification of foreign DNA contamination to sequences specifically detected by primers of known sequences.

“However, this limits the quantification to only those sequences that are specifically detected by primers of known sequences.”

The VP3 of AAV4, AAV5 and AAV11 enter the nucleus via an AAP-independent mechanism that is yet unknown.

“Thus, the VP3 of these exceptions (AAV4, AAV5 and AAV11) enter the nucleus via an AAP-independent mechanism that is yet unknown and challenge what is known about the role of AAP in AAV capsid assembly.”

After one single-stranded genome is fully displaced, the DNA is translocated into the capsid in a 3′ to 5′ direction by the helicase/ATPase domain of the small Rep proteins.

“After one single-stranded genome is fully displaced, the DNA is reported to be translocated into the capsid in a 3′ to 5′ direction by the helicase/ATPase domain of the small Rep proteins [53].”

• cites [53] King JA, Dubielzig R, Grimm D, Kleinschmidt JA. DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsids. Embo. J 20(12), 3282– 3291 (2001). 11406604 PMC150213 sourceNot checked — no full text of the source

Fidelity of the interactions between viral components dictates the ITR-mediated packaging of WT genomes as well as transgenes into AAV capsids.

“Fidelity of the interactions between these viral components dictate the ITR-mediated packaging of WT genomes as well as transgenes into AAV capsids.”

Genome sequences closely homologous to RBE, such as the AAVS1 site, have been detected in purified AAV vector preparations.

“It has been reported that genome sequences which are close homologs to RBE, such as, the AAVS1 site, were detected in purified AAV vector preparations [82].”

• cites [82] Hüser D, Weger S, Heilbronn R. Packaging of human chromosome 19-specific adeno-associated virus (AAV) integration sites in AAV virions during AAV wild-type and recombinant AAV vector production. J. Virol 77(8), 4881– 4887 (2003). 12663794 PMC152110 sourceNot checked — no full text of the source

Herpes virus directly interacts with AAV replication with its viral helicase-primase complex.

“Furthermore, herpes virus directly interacts with AAV replication with its viral helicase– primase complex [2].”

• cites [2] Weitzman MD, Linden RM. Adeno-associated virus biology. Methods Mol. Biol 807, 1– 23 (2011). 22034024 sourceNot checked — no full text of the source

The fivefold axis contains a cylindrical channel formed by the interaction of five DE loops.

“The fivefold axis contains a cylindrical channel formed by the interaction of five DE loops (β-ribbons; Figure 3C).”