CoChat
Sign Up
Cited by 12HIV Research and Treatmentinterferon and immune responsesVirus-based gene therapy research

HIV-1 Vpr combats the PU.1-driven antiviral response in primary human macrophages

Open access
Virgilio, Maria C., Ramnani, Barkha, Chen, Thomas, Disbennett, W. Miguel, Lubow, Jay, Welch, Joshua D., Collins, Kathleen L.·2024·Nature Communications
DOI 10.1038/s41467-024-49635-wPMC PMC11217462

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

HIV-1 Vpr promotes efficient spread of HIV-1 from macrophages to T cells by transcriptionally downmodulating restriction factors that target HIV-1 Envelope protein (Env). Here we find that Vpr induces broad transcriptomic changes by targeting PU.1, a transcription factor necessary for expression of host innate immune response genes, including those that target Env. Consistent with this, we find silencing PU.1 in infected macrophages lacking Vpr rescues Env. Vpr downmodulates PU.1 through a proteasomal degradation pathway that depends on physical interactions with PU.1 and DCAF1, a component of the Cul4A E3 ubiquitin ligase. The capacity for Vpr to target PU.1 is highly conserved across primate lentiviruses. In addition to impacting infected cells, we find that Vpr suppresses expression of innate immune response genes in uninfected bystander cells, and that virion-associated Vpr can degrade PU.1. Together, we demonstrate Vpr counteracts PU.1 in macrophages to blunt antiviral immune responses and promote viral spread.

AI snapshot

Written from the paper’s full text.

TL;DR

HIV-1 Vpr targets the myeloid transcription factor PU.1 for DCAF1-dependent proteasomal degradation in primary human macrophages, reducing PU.1-regulated antiviral genes such as MRC1 and IFITM3 and enhancing Env production/viral spread. Vpr also suppresses innate immune genes in uninfected bystander cells, and this PU.1-targeting activity is conserved across HIV-1, HIV-2, and SIV Vpr proteins.

Key findings

  • In scRNA-seq of primary MDMs infected with 89.6wt versus 89.6Δvpr, 3,150 genes showed statistically significant ≥2-fold differential expression; HOMER identified 316 PU.1-motif genes and 670 PU.1-IRF co-regulated genes, for 840 distinct PU.1-regulated genes among Vpr-downregulated genes. MRC1 showed log2FC = 0.932, FDR-corrected two-sided Wilcoxon p = 0.002, below the study’s two-fold cutoff.
  • Vpr reduced PU.1 protein via a proteasomal degradation pathway requiring physical interactions with PU.1 and DCAF1; the VprQ65R mutant defective for DCAF1 interaction failed to bind/degrade PU.1, and MG132 restored PU.1. Vpr did not reduce PU.1 mRNA when PU.1 was expressed from a heterologous promoter in HEK 293T cells.
  • Vpr suppressed PU.1-regulated innate immune genes, including ISG15, LY96, IFI6, and IFITM3, in infected and bystander cells; virion-associated Vpr degraded PU.1 in MDMs within five hours.
  • Silencing PU.1/SPI1 in Vpr-null HIV-infected MDMs rescued Env production; all tested Vprs from HIV-1, HIV-2, and SIV reduced PU.1, whereas HIV-2 Vpx did not.

Methods

  • Primary human CD14+ MDMs from three healthy donors were infected with replication-competent 89.6wt or 89.6Δvpr for 10 days and analyzed by 10X scRNA-seq; data were integrated with LIGER, visualized by UMAP, and infected cells were defined as gag+/tat+. Differential expression used two-sided Wilcoxon rank-sum with FDR correction; motif analysis used HOMER, and gene ontology used GOrilla/REVIGO.
  • Mechanistic assays used HEK 293T, K562, and primary MDMs, including co-immunoprecipitation, immunoblot, flow cytometry, immunofluorescence, and RT-qPCR; shRNA knockdown of DCAF1 or SPI1; proteasome inhibition with MG132; and five-hour treatment with virion-associated Vpr.
  • Non-single-cell statistics were performed in GraphPad Prism v10; scRNA-seq analysis and plotting used R/LIGER/Seurat, with details in figure legends.

Limitations

  • Stated: Vpr effects in uninfected bystander cells were not consistently detected with VSV-G-pseudotyped replication-defective virus at five days, and alternative explanations for the bystander phenotype remain possible. TET2 interaction with PU.1/Vpr/DCAF1 was not confirmed in MDMs, ubiquitylated PU.1 intermediates were not directly detected, and prior PU.1 suppression of HIV-LTR activity was not confirmed.
  • Stated: western blot detection of PU.1 degradation was challenging because PU.1 was overexpressed and/or Vpr was not expressed in enough PU.1-positive cells; more extensive cross-species comparisons of Vpr-PU.1 interactions are needed.
  • Evident from the text: scRNA-seq used three donors, and several key interaction/degradation experiments rely on HEK 293T or K562 overexpression systems, with primary MDM confirmation limited to selected assays.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41467-024-49635-w

Cites

92 of 92 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

45 of 69 cited sources checked · 24 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    Expression of genes involved in TLR-mediated IFN-I response, including ISG15, LY96, IFI6, and IFITM3, was very low in uninfected, unexposed cells.

    “As expected, expression of genes involved in the TLR-mediated IFN-I response to infection, including interferon stimulated gene 15 (ISG15), which is upregulated in response to IFN-I and TLR-signaling (reviewed in refs. 48,49), LY96, and interferon-inducible protein-6 (IFI6) (PU.1-IRF regulated genes) and IFITM3 were very low in uninfected, unexposed cells (Fig. 3C).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [48] Perng, Y. C. & Lenschow, D. J. ISG15 in antiviral immunity and beyond. Nat. Rev. Microbiol. 16, 423–439 (2018). source
    • cites [49] Schneider, W. M., Chevillotte, M. D. & Rice, C. M. Interferon-stimulated genes: a complex web of host defenses. Annu. Rev. Immunol. 32, 513–545 (2014). source
  • Premisecitation

    Vpr does not directly interact with MR.

    “Although the canonical targeting of host factors by Vpr involves the binding of Vpr to DCAF1, leading to ubiquitylation and degradation via the proteosome, Vpr does not directly interact with MR35.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [35] Lubow, J. et al. Mannose receptor is an HIV restriction factor counteracted by Vpr in macrophages. eLife 9, e51035 (2020). source

      The Results section reports that Vpr did not reduce MR expression in a 293T-cell experiment and concludes that Vpr does not degrade MR by the direct proteasomal mechanism used for UNG2. That experiment does not test whether Vpr physically binds MR, and the cited passages do not establish the claimed absence of a direct interaction. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
  • Findingcitation

    Vpr expression boosted HIV gene expression.

    “Vpr expression boosted HIV gene expression as previously reported41”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [41] Zhang, F. & Bieniasz, P. D. HIV-1 Vpr induces cell cycle arrest and enhances viral gene expression by depleting CCDC137. eLife 9, e55806 (2020). source
  • Interpretationcitation

    By targeting PU.1, Vpr systemically disrupted several antiviral factors, including MRC1 and IFITM3.

    “By targeting PU.1, Vpr systemically disrupted several antiviral factors, including MRC1, and IFITM3, a previously reported target of Vpr that is also capable of disrupting Env function38.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [38] Wang, Q. & Su, L. Vpr enhances HIV-1 Env processing and virion infectivity in macrophages by modulating TET2-dependent IFITM3 expression. mBio 10, e01344–19 (2019). source
  • Premisecitation

    Vpr suppresses expression of the MR gene, MRC1.

    “Instead, Vpr suppresses expression of the MR gene, MRC135.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [35] Lubow, J. et al. Mannose receptor is an HIV restriction factor counteracted by Vpr in macrophages. eLife 9, e51035 (2020). source
  • May combine two claims. 30
  • Classified as probably not a substantial claim. 24

  • May combine two claims.

    Premisecitation

    Vpr counteracts accelerated degradation of the HIV Env protein in HIV-infected macrophages but not T cells.

    “We previously reported that Vpr counteracts accelerated degradation of the HIV Env protein, which occurs in HIV-infected macrophages but not T cells33,34.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [33] Collins, D. R., Lubow, J., Lukic, Z., Mashiba, M. & Collins, K. L. Vpr promotes macrophage-dependent HIV-1 infection of CD4+ T lymphocytes. PLoS Pathog. 11, e1005054 (2015). source
    • cites [34] Mashiba, M., Collins, D. R., Terry, V. H. & Collins, K. L. Vpr overcomes macrophage-specific restriction of HIV-1 Env expression and virion production. Cell Host Microbe 16, 722–735 (2014). source

      The Results section ('Vpr prevents lysosomal degradation of HIV-1 Env in MDM'; page unavailable) shows that Vpr increases Env half-life in infected macrophages, and the Discussion describes the degradation pathway as macrophage-specific. The passages establish a contrast with epithelial cells, but do not establish the claim’s specific contrast with T cells. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The macrophage mannose receptor (MR) is highly expressed in macrophages but not T cells.

    “We recently identified this factor as the macrophage mannose receptor (MR)35, which is highly expressed in macrophages but not T cells36,37.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [35] Lubow, J. et al. Mannose receptor is an HIV restriction factor counteracted by Vpr in macrophages. eLife 9, e51035 (2020). source
    • cites [36] Liang, G. et al. Membrane metalloprotease TRABD2A restricts HIV-1 progeny production in resting CD4 + T cells by degrading viral Gag polyprotein. Nat. Immunol. https://doi.org/10.1038/s41590-019-0385-2 (2019) source
    • cites [37] Linehan, S. A., Martínez-Pomares, L., Stahl, P. D. & Gordon, S. Mannose receptor and its putative ligands in normal murine lymphoid and nonlymphoid organs: in situ expression of mannose receptor by selected macrophages, endothelial cells, perivascular microglia, and mesangial cells, but not dendritic cells. J. Exp. Med. 189, 1961–1972 (1999). source

      The Abstract and Discussion report MR expression in subsets of mature macrophages, rather than macrophages generally. The Results (Peripheral Lymph Node; page unavailable) find MR on medullary macrophages but not in T-cell areas, which supports the T-cell distinction in the tissues studied. The paper does not establish that MR is highly expressed across macrophages as a whole. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Through DCAF1, Vpr interacts with DDB1 as part of the Cul4A E3 ubiquitin ligase complex where host proteins recruited by Vpr are ubiquitylated and degraded.

    “Through DCAF1, Vpr interacts with damaged DNA binding protein 1 (DDB1) as part of the Cul4A E3 ubiquitin ligase complex where host proteins recruited by Vpr are ubiquitylated and degraded17–22.”
    Quote in the paperSubstantial claim5 of 6 sources checked
    • cites [17] McCall, C. M. et al. Human immunodeficiency virus type 1 Vpr-binding protein VprBP, a WD40 protein associated with the DDB1-CUL4 E3 ubiquitin ligase, is essential for DNA replication and embryonic development. Mol. Cell. Biol. 28, 5621–5633 (2008). source

      The Abstract (page unavailable) identifies DCAF1/VprBP as a Vpr-binding protein and reports that it binds DDB1 and, through DDB1, CUL4A. The Discussion (page unavailable) suggests that VprBP recruits substrates to the E3 ligase, but does not establish that Vpr recruits host proteins that are then ubiquitylated and degraded. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [18] Romani, B. & Cohen, É. A. Lentivirus Vpr and Vpx accessory proteins usurp the cullin4–DDB1 (DCAF1) E3 ubiquitin ligase. Curr. Opin. Virol. 2, 755–763 (2012). source

      The “Viral protein R” section (page unavailable) says Vpr engages the CUL4A–DDB1 E3 ligase through DCAF1 and can induce ubiquitination and proteasomal degradation of unidentified cellular proteins. It describes Vpr recruiting those proteins to the ligase as a model, however, and says whether Vpr instead enhances ubiquitination of natural DCAF1 substrates remains unresolved. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [19] Hakata, Y., Miyazawa, M. & Landau, N. R. Interactions with DCAF1 and DDB1 in the CRL4 E3 ubiquitin ligase are required for Vpr-mediated G2 arrest. Virol. J. 11, 108 (2014). source
    • cites [20] Schröfelbauer, B., Hakata, Y. & Landau, N. R. HIV-1 Vpr function is mediated by interaction with the damage-specific DNA-binding protein DDB1. Proc. Natl. Acad. Sci. USA. 104, 4130–4135 (2007). source
    • cites [21] Belzile, J.-P. et al. HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase. PLoS Pathog. 3, e85 (2007). source

      The Results and Discussion support Vpr’s association with the DDB1–CUL4A ligase complex, likely through VPRBP (DCAF1). But the Discussion describes ubiquitination and degradation of a protein recruited by Vpr as a proposed model involving a yet-unknown target, not an established outcome. Page numbers are unavailable in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [22] Hrecka, K. et al. HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins. Proc. Natl. Acad. Sci. USA. 113, E3921–E3930 (2016). source

  • May combine two claims.

    Interpretationcitation

    Vpr enhances infection of macrophages and increases viral burden in tissues containing macrophages.

    “These studies provide evidence that Vpr enhances infection of macrophages and increases viral burden in tissues containing macrophages.”
    Quote in the paperSubstantial claim2 of 5 sources checked
    • cites [1] Balliet, J. W. et al. Distinct effects in primary macrophages and lymphocytes of the human immunodeficiency virus type 1 accessory genes vpr, vpu, and nef: mutational analysis of a primary HIV-1 isolate. Virology 200, 623–631 (1994). source
    • cites [3] Eckstein, D. A. et al. HIV-1 Vpr enhances viral burden by facilitating infection of tissue macrophages but not nondividing CD4+ T cells. J. Exp. Med. 194, 1407–1419 (2001). source
    • cites [4] Connor, R. I., Chen, B. K., Choe, S. & Landau, N. R. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 206, 935–944 (1995). source
    • cites [5] Dedera, D., Hu, W., Vander Heyden, N. & Ratner, L. Viral protein R of human immunodeficiency virus types 1 and 2 is dispensable for replication and cytopathogenicity in lymphoid cells. J. Virol. 63, 3205–3208 (1989). source
    • cites [6] Rücker, E., Grivel, J.-C., Münch, J., Kirchhoff, F. & Margolis, L. Vpr and Vpu are important for efficient human immunodeficiency virus type 1 replication and CD4+ T-cell depletion in human lymphoid tissue ex vivo. J. Virol. 78, 12689–12693 (2004). source

      The paper states that Vpr enhances macrophage infection (introductory passage labeled Abstract, page unavailable) and reports lower viral replication in ex vivo human lymphoid tissue when vpr is deleted (results passage labeled Abstract, page unavailable). It does not establish that the increased viral burden in those tissues is due to macrophage infection or specifically characterize the result as occurring in tissues containing macrophages. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    PU.1 is a hematopoietic-specific ETS family transcription factor essential for lymphoid and myeloid development.

    “PU.1 is a hematopoietic-specific and ETS family transcription factor that is essential for lymphoid and myeloid development25–28.”
    Quote in the paperNot a substantial claim2 of 4 sources checked
    • cites [25] Turkistany, S. A. & Dekoter, R. P. The transcription factor PU.1 is a critical regulator of cellular communication in the immune system. Arch. Immunol. Ther. Exp. (Warsz) 59, 431–440 (2011). source
    • cites [26] Fisher, R. C. & Scott, E. W. Role of PU.1 in hematopoiesis. Stem Cells 16, 25–37 (1998). source
    • cites [27] Dakic, A. et al. PU.1 regulates the commitment of adult hematopoietic progenitors and restricts granulopoiesis. J. Exp. Med. 201, 1487–1502 (2005). source
    • cites [28] Gupta, P., Gurudutta, G. U., Saluja, D. & Tripathi, R. P. PU.1 and partners: regulation of haematopoietic stem cell fate in normal and malignant haematopoiesis. J. Cell. Mol. Med. 13, 4349–4363 (2009). source

  • May combine two claims.

    Interpretationcitation

    The effect of Vpr on MRC1 transcriptional expression is modest and combines synergistically with HIV Nef-dependent disruption of MR trafficking, reversing mannose receptor-dependent lysosomal degradation of Env in HIV-infected primary macrophages.

    “While the effect of Vpr on MRC1 transcriptional expression is modest, it combines synergistically with HIV Nef-dependent disruption of MR trafficking, reversing mannose receptor-dependent lysosomal degradation of Env in HIV-infected primary macrophages35.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [35] Lubow, J. et al. Mannose receptor is an HIV restriction factor counteracted by Vpr in macrophages. eLife 9, e51035 (2020). source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The relationship between DCAF1 and Vpr is well documented.

    “The relationship between DCAF1 and Vpr is well documented16–18.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [16] Zhang, S., Feng, Y., Narayan, O. & Zhao, L.-J. Cytoplasmic retention of HIV-1 regulatory protein Vpr by protein-protein interaction with a novel human cytoplasmic protein VprBP. Gene 263, 131–140 (2001). source
    • cites [17] McCall, C. M. et al. Human immunodeficiency virus type 1 Vpr-binding protein VprBP, a WD40 protein associated with the DDB1-CUL4 E3 ubiquitin ligase, is essential for DNA replication and embryonic development. Mol. Cell. Biol. 28, 5621–5633 (2008). source
    • cites [18] Romani, B. & Cohen, É. A. Lentivirus Vpr and Vpx accessory proteins usurp the cullin4–DDB1 (DCAF1) E3 ubiquitin ligase. Curr. Opin. Virol. 2, 755–763 (2012). source

  • Classified as probably not a substantial claim.

    Premisecitation

    PU.1 and TET2 form a complex to co-regulate myeloid-specific genes.

    “Interestingly, PU.1 and TET2 have been reported to form a complex to co-regulate myeloid-specific genes25,26,32.”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [25] Turkistany, S. A. & Dekoter, R. P. The transcription factor PU.1 is a critical regulator of cellular communication in the immune system. Arch. Immunol. Ther. Exp. (Warsz) 59, 431–440 (2011). source
    • cites [26] Fisher, R. C. & Scott, E. W. Role of PU.1 in hematopoiesis. Stem Cells 16, 25–37 (1998). source
    • cites [32] de la Rica, L. et al. PU.1 target genes undergo Tet2-coupled demethylation and DNMT3b-mediated methylation in monocyte-to-osteoclast differentiation. Genome Biol. 14, R99 (2013). source

      The Discussion (page not specified) reports a PU.1–TET2 interaction and their association with genes that become demethylated. The Results describe PU.1-dependent recruitment of TET2 and changes in gene expression during monocyte-to-osteoclast differentiation. The evidence supports joint regulation in that setting, but not the broader description of the targets as myeloid-specific genes generally. (LLM, conf=0.81)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    During HIV-1 infection of macrophages, in the absence of Vpr, TET2 demethylates the IFITM3 promoter, relieving suppression, contributing to the antiviral response.

    “During HIV-1 infection of macrophages, in the absence of Vpr, TET2 demethylates the IFITM3 promoter, relieving suppression, contributing to the antiviral response.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [38] Wang, Q. & Su, L. Vpr enhances HIV-1 Env processing and virion infectivity in macrophages by modulating TET2-dependent IFITM3 expression. mBio 10, e01344–19 (2019). source

  • May combine two claims.

    Premisecitation

    In monocytes, TET2 interacts with PU.1, leading to interaction with genetic targets.

    “In monocytes, TET2 interacts with PU.1, leading to interaction with genetic targets32.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [32] de la Rica, L. et al. PU.1 target genes undergo Tet2-coupled demethylation and DNMT3b-mediated methylation in monocyte-to-osteoclast differentiation. Genome Biol. 14, R99 (2013). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    HIV-2 contains Vpx, an accessory protein that shares a common genetic ancestor with Vpr but is lacking from all HIV-1 genomes.

    “HIV-2 differs from HIV-1 in that it contains Vpx, an accessory protein that shares a common genetic ancestor with Vpr55 but is lacking from all HIV-1 genomes.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [55] Sharp, P. M. & Hahn, B. H. Origins of HIV and the AIDS pandemic. Cold Spring Harb. Perspect. Med. 1, a006841 (2011). source

      The Origins of HIV-2 section (page unavailable) discusses HIV-2 lineages and their ancestry but not Vpx. Searches of our copy for Vpx, Vpr, and their proposed common ancestry found no passage addressing whether HIV-2 contains Vpx or whether it is absent from all HIV-1 genomes. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    TET2 is a DNA dioxygenase that demethylates the IFITM3 promoter during viral infection, inhibiting HIV Env trafficking and reducing viral spread.

    “TET2 is a DNA dioxygenase that demethylates the IFITM3 promoter during viral infection, inhibiting HIV Env trafficking and reducing viral spread61,62.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [61] Lu, J., Pan, Q., Rong, L., Liu, S.-L. & Liang, C. The IFITM proteins inhibit HIV-1 infection. J. Virol. 85, 2126–2137 (2011). source

      The Results report that IFITM3 suppresses HIV-1 infection and reduces the amount of infectious virus. They also show reduced HIV-1 entry, not inhibited HIV Env trafficking. The paper does not support the attributed mechanism involving TET2 or demethylation of the IFITM3 promoter. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [62] Compton, A. A. et al. IFITM proteins incorporated into HIV-1 virions impair viral fusion and spread. Cell Host Microbe 16, 736–747 (2014). source

      The Results section on endogenous IFITM proteins reports that they limit HIV-1 cell-to-cell spread. The Results section on IFITM3 reports reduced surface Env levels but does not establish inhibited Env trafficking; it says the decrease could reflect reduced spread or an effect on protein production or stability. The passages do not address TET2 or demethylation of the IFITM3 promoter. Page numbers were not available in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IFITM proteins are broad antiviral factors that inhibit viral entry and exit for HIV-1, SIV, MLV, VSV, EBOV, WNV, among other viruses.

    “IFITM proteins are broad antiviral factors that inhibit viral entry and exit for HIV-1, SIV, MLV, VSV, EBOV, WNV, among other viruses69.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [69] Tartour, K. et al. Interference with the production of infectious viral particles and bimodal inhibition of replication are broadly conserved antiviral properties of IFITMs. PLoS Pathog. 13, e1006610 (2017). source

      The Abstract identifies IFITMs as broad antiviral factors and lists HIV-1, SIV, MLV, VSV, EBOV, and WNV among viruses susceptible to reduced infectivity of newly produced particles. It also describes blocking incoming virions, but the Discussion says entry was not directly measured for most viruses studied. The paper supports effects at both ends of infection, not a claim that IFITMs inhibit the physical exit or release of those viruses. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Vpr is highly conserved amongst primate lentiviruses and promotes infection of nondividing cells, especially macrophages.

    “Vpr is highly conserved amongst primate lentiviruses and promotes infection of nondividing cells, especially macrophages34.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [34] Mashiba, M., Collins, D. R., Terry, V. H. & Collins, K. L. Vpr overcomes macrophage-specific restriction of HIV-1 Env expression and virion production. Cell Host Microbe 16, 722–735 (2014). source

      The Discussion and Results report that Vpr enhances HIV-1 infection and spread in primary macrophages, chiefly by increasing Env expression and virion production. The cited passages do not establish that Vpr is highly conserved among primate lentiviruses or that it promotes infection of nondividing cells generally. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Mannose receptor is a host restriction factor that reduces HIV spread in macrophages by binding to mannose residues on Env and directing Env to the lysosome for degradation.

    “We have previously shown that mannose receptor is a host restriction factor that reduces HIV spread in macrophages by binding to mannose residues on Env and directing Env to the lysosome for degradation35.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [35] Lubow, J. et al. Mannose receptor is an HIV restriction factor counteracted by Vpr in macrophages. eLife 9, e51035 (2020). source

  • May combine two claims.

    Premisecitation

    Vpr mediates the ubiquitylation and degradation of TET2, which in turn inhibits IFITM3 expression.

    “To prevent this, Vpr mediates the ubiquitylation and degradation of TET2, which in turn inhibits IFITM3 expression.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [38] Wang, Q. & Su, L. Vpr enhances HIV-1 Env processing and virion infectivity in macrophages by modulating TET2-dependent IFITM3 expression. mBio 10, e01344–19 (2019). source
    • cites [39] Lv, L. et al. Vpr Targets TET2 for degradation by CRL4VprBP E3 ligase to sustain IL-6 expression and enhance HIV-1 replication. Mol. Cell 70, 961–970.e5 (2018). source

      The Results section, “Vpr reprograms CRL4VprBP E3 ligase to catalyze polyubiquitylation of TET2” (page unavailable), supports Vpr-mediated TET2 polyubiquitylation and degradation. The copy does not support the further claim that this inhibits IFITM3 expression; its reported downstream expression effect concerns sustained IL-6 expression (Results, “Vpr degrades TET2 to sustain IL-6 expression and enhance HIV-1 replication”; page unavailable). Searches for IFITM3 expression and interferon-induced transmembrane protein 3 did not yield a passage addressing that link. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    PU.1 is the master transcriptional regulator necessary for macrophage differentiation and silencing PU.1 prior to infection results in a change in the cellular phenotype that causes resistance to HIV infection.

    “This approach was necessary because PU.1 is the master transcriptional regulator necessary for macrophage differentiation and silencing PU.1 prior to infection results in a change in the cellular phenotype that causes resistance to HIV infection72,73.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [72] Laguette, N. et al. SAMHD1 is the dendritic– and myeloid–cell–specific HIV–1 restriction factor counteracted by Vpx. Nature 474, 654–657 (2011). source

      The Abstract discusses SAMHD1, not PU.1: it reports that silencing SAMHD1 increases susceptibility to HIV-1 infection. Searches for PU.1 and for PU.1 silencing before infection found no passage addressing PU.1’s role in macrophage differentiation or the claimed phenotype change and resistance. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [73] Hrecka, K. et al. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 474, 658–661 (2011). source

      The paper’s Abstract attributes reduced HIV-1 infection of macrophages to SAMHD1-mediated restriction, not to PU.1 silencing. A search for PU.1, macrophage differentiation, and silencing before infection found no passage in our copy addressing either part of the attributed claim. (LLM, conf=0.77)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    In the scRNA-seq data set, MRC1 transcripts showed a Vpr-dependent change (log2FC = 0.932) that is statistically significant (FDR p = 0.002) but fell below a two-fold change cutoff.

    “In the scRNA-seq data set shown here, we identified a Vpr-dependent change in MRC1 transcripts (log2FC = 0.932) that is statistically significant (false-discovery rate corrected two-sided Wilcoxon rank-sum p-value = 0.002) but fell just below our arbitrary cutoff of a two-fold change (Figs. 1H and 2C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Vpr-mediated downmodulation of PU.1-regulated gene expression is mediated by protein-protein interaction between Vpr and PU.1, resulting in accelerated proteasomal degradation of PU.1.

    “Finally, we found that Vpr-mediated downmodulation of PU.1-regulated gene expression is mediated by protein-protein interaction between Vpr and PU.1, resulting in accelerated proteasomal degradation of PU.1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both the interaction between PU.1 and Vpr and PU.1's subsequent degradation require DCAF1.

    “Remarkably, both the interaction between PU.1 and Vpr as well PU.1’s subsequent degradation requires the Vpr interacting protein, DCAF1.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    The capacity for Vpr to target PU.1 is highly conserved across primate lentiviruses.

    “The capacity for Vpr to target PU.1 is highly conserved across primate lentiviruses.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Transient transfection confirmed a significant decrease in PU.1 for cells expressing VprROD or Vpr89.6 but not VpxROD.

    “Transient transfection of HEK 293T cells with a PU.1 expression vector and either HIV-2RODVpx, HIV-2RODVpr, or HIV-189.6Vpr confirmed a significant decrease in PU.1 for cells expressing VprROD or Vpr89.6 but not VpxROD (Fig. 5B, C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Vpr induces broad transcriptomic changes by targeting PU.1, a transcription factor necessary for expression of host innate immune response genes, including those that target Env.

    “Here we find that Vpr induces broad transcriptomic changes by targeting PU.1, a transcription factor necessary for expression of host innate immune response genes, including those that target Env.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mutation VprQ65R resulted in loss of DCAF1 association and disrupted formation of the Vpr-PU.1 complex.

    “To our surprise, mutation of the glutamine residue at position 65 of Vpr (VprQ65R) not only resulted in the loss of DCAF1 association but also disrupted formation of the Vpr-PU.1 complex.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In MDMs, virion-associated Vpr delivered to primary MDMs resulted in Vpr-dependent degradation of PU.1 with wild type HIV-1 but not with Vpr mutant defective at DCAF1 interaction or Vpr-null.

    “Using this approach, we observed Vpr-dependent degradation of PU.1 treated with wild type HIV-1 but not an HIV harboring a Vpr mutant defective at interacting with DCAF1 (89.6-VprQ65R) or 89.6-Vpr-null (Fig. 7A, B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HIV-2 Vpr has a unique function to degrade PU.1 that is not shared by Vpx.

    “We identified a unique function of HIV-2 Vpr to degrade PU.1 that was not shared by Vpx.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In MDMs, endogenous PU.1 and DCAF1 interact with Vpr.

    “As shown in Fig. 6C, we determined that both endogenous PU.1 and DCAF1 interact with Vpr in this physiologically relevant target of HIV-1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    PU.1 was not degraded in the presence of proteasome inhibitors or by a Vpr mutant defective at interacting with DCAF1 in primary MDMs.

    “Consistent with this model, PU.1 was not degraded in the presence of proteasome inhibitors or by a Vpr mutant defective at interacting with DCAF1 in primary MDMs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    In macrophages, Vpr likely exerts negative effects directly on PU.1 protein and indirectly on PU.1 RNA through PU.1 binding sites in the SPI1 promoter.

    “Moreover, in macrophages Vpr likely exerts negative effects directly on PU.1 protein and indirectly on PU.1 RNA through PU.1 binding sites in the SPI1 promoter52.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [52] Chen, H. et al. PU.1 (Spi-1) autoregulates its expression in myeloid cells. Oncogene 11, 1549–1560 (1995).

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Vpr requires DCAF1 to target host factors involved in DNA repair.

    “To target these factors, Vpr requires the host protein DCAF1 (also known as Vpr binding protein (VprBP))16.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Zhang, S., Feng, Y., Narayan, O. & Zhao, L.-J. Cytoplasmic retention of HIV-1 regulatory protein Vpr by protein-protein interaction with a novel human cytoplasmic protein VprBP. Gene 263, 131–140 (2001). source

  • May combine two claims.

    Interpretationown data

    Vpr counteracts PU.1 in macrophages to blunt antiviral immune responses and promote viral spread.

    “Together, we demonstrate Vpr counteracts PU.1 in macrophages to blunt antiviral immune responses and promote viral spread.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Macrophages require early and continuously high levels of PU.1 expression to maintain normal functionality.

    “Macrophages require early and continuously high levels of PU.1 expression (reviewed in refs. 25,26) to maintain normal functionality.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [25] Turkistany, S. A. & Dekoter, R. P. The transcription factor PU.1 is a critical regulator of cellular communication in the immune system. Arch. Immunol. Ther. Exp. (Warsz) 59, 431–440 (2011). source
    • cites [26] Fisher, R. C. & Scott, E. W. Role of PU.1 in hematopoiesis. Stem Cells 16, 25–37 (1998). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A requirement for Vpr to achieve maximal replication and persistence in vivo was first discovered using an SIV molecular clone in rhesus monkeys.

    “In addition, a requirement for Vpr to achieve maximal replication and persistence in vivo was first discovered using an SIV molecular clone in rhesus monkeys74.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [74] Lang, S. M. et al. Importance of vpr for infection of rhesus monkeys with simian immunodeficiency virus. J. Virol. 67, 902–912 (1993). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TET2 is ubiquitously expressed in the nuclei of all cells.

    “TET2 is ubiquitously expressed in the nuclei of all cells71”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [71] Lorsbach, R. B. et al. TET1, a member of a novel protein family, is fused to MLL in acute myeloid leukemia containing the t(10;11)(q22;q23). Leukemia 17, 637–641 (2003). source

  • May combine two claims.

    Interpretationreasoning

    In primary HIV-infected macrophages, Vpr mainly targets PU.1 to counteract its anti-viral defense response rather than to counteract an inhibitory effect on HIV gene expression.

    “Thus, in primary HIV-infected macrophages, Vpr mainly targets PU.1 to counteract its anti-viral defense response rather than to counteract an inhibitory effect on HIV gene expression.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    The study utilized wild type HIV-infected primary macrophages and characterized Vpr-dependent transcriptomic changes comparing fully infected and uninfected (HIV-RNA-negative) bystander cells.

    “Our study differed from this prior report in that we utilized wild type HIV-infected primary macrophages and characterized Vpr-dependent transcriptomic changes comparing fully infected and uninfected (HIV-RNA-negative), bystander cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The primary selective pressure for Vpr in lentiviruses is to disrupt the macrophage innate antiviral response to infection, which is achieved by reducing PU.1 levels in infected cells.

    “Although the role of Vpr in HIV infection has remained largely undefined, we provide evidence that the primary selective pressure for Vpr in lentiviruses is to disrupt the macrophage innate antiviral response to infection, which is achieved by reducing PU.1 levels in infected cells.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    For single-cell RNA sequencing, 89.6WT and 89.6Δvpr conditions with similar percentages of infected cells were selected using flow cytometry data.

    “The resulting flow cytometry data was used to select 89.6WT and 89.6Δvpr conditions with similar percentages of infected cells.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    K562 cells were inoculated with varying amounts of 3xFLAG-tagged Vpr/Vpx expression viruses so as to achieve equal FLAG expression.

    “K562 cells were inoculated with varying amounts of 3xFLAG-tagged Vpr/Vpx expression viruses to achieve equal FLAG expression.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Total corrected fluorescence per cell was divided by the number of nuclei to normalize for cell volume and account for multinucleated syncytia.

    “Total corrected fluorescence per cell was divided by the number of nuclei to normalize cell volume to account for multinucleated syncytia.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    In all flow cytometry experiments, cells were gated sequentially by forward scatter vs. side scatter and then by forward scatter area vs. height to exclude doublets.

    “In all experiments, cells were gated sequentially by forward scatter vs. side scatter for cells and then by forward scatter area vs. height to exclude doublets.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Transductions were performed with equal virus amounts, determined by Gag p24 mass, across conditions.

    “All transductions were performed via spin inoculation at 1050 x g for 2 hr at 25°C with equal virus amounts determined by Gag p24 mass in medium containing 4 µg/mL polybrene (Sigma).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    DMSO-only control wells were treated with the same volume of DMSO as contained in the MG132 treatment conditions.

    “DMSO-only control treatment wells (Vehicle) were treated with the same volume of DMSO as contained in the MG132 treatment conditions.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Identical laser and gain settings were used across all images for each individual replicate of the immunofluorescence experiment.

    “Identical laser and gain settings were used across all images for each individual replicate of the experiment.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For RT-qPCR, cell samples were diluted so that all conditions contained the same cell numbers as input.

    “HEK 293 T cells sorted as described in ‘Flow Cytometry’ below were counted using the Countess II Cell Counter (Thermo Fisher Scientific), and cell samples were diluted such that all conditions contained the same cell numbers as input.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Studies using anonymized leukocytes were determined to be exempt from human studies requirements by the University of Michigan Institutional Review Board.

    “Studies using these cells were determined to be exempt from human studies requirements by the University of Michigan Institutional Review Board because the project involves only biological specimens that cannot be linked to a specific individual by the investigator(s) directly or indirectly through a coding system.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    HEK 293T and K562 cells were obtained from ATCC and independently authenticated by STR profiling.

    “HEK 293Ts (CRL-3216) and K562 (CCL-243) cells were obtained from ATCC and independently authenticated by STR profiling.”
    Quote in the paperNot a substantial claim
47 of 47

  • The paper relies on this without support.

    Vpr is packaged in the virus particle at high levels through specific interactions with Gag p62.

    “In addition, it is unique amongst HIV accessory proteins in that it is packaged in the virus particle at high levels through specific interactions with Gag p62.”

  • The paper relies on this without support.

    Vpr is efficiently packaged into the virus particle through specific interactions with Gag p62.

    “Because Vpr is efficiently packaged into the virus particle through specific interactions with Gag p62, the Vpr phenotype we observed in bystander cells could be due to a low and/or transient presence of Vpr in MDMs that have taken up viral particles but remained uninfected.”

  • The paper relies on this without support.

    MG132 is a specific inhibitor of the proteasome.

    “PU.1 levels were restored with MG132, a specific inhibitor of the proteasome.”

  • The paper relies on this without support.

    NL4-3-VprQ65R is defective at interactions with DCAF-1.

    “In addition, the Vpr mutant that is defective at interactions with DCAF-1 (NL4-3-VprQ65R) was also defective at co-precipitating PU.1 (Fig. 6F), further confirming a role for DCAF1 in the interaction between Vpr and PU.1.”
4 of 4

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Conflicting adjusted-p-value cutoff for motif analysis

    Minor

    The methods say genes with adjusted p-values greater than 0.05 were selected for HOMER analysis, while the figure legend defines significant differential expression using p-values below 0.05. These opposite cutoffs leave unclear which genes were used for the promoter-motif analysis underpinning the PU.1 finding.

1 of 1

Cites

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Virgilio, M. C., Ramnani, B., Chen, T., Disbennett, W. M., Lubow, J., Welch, J. D., & Collins, K. L.
Affiliations
University of Michigan (US)
Corresponding author
Collins, Kathleen L.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2024-06-29
Volume
15
Issue
1
Pages
5514-5514
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-024-49635-w
PMC
PMC11217462
OpenAlex
W4400199045
Cited by
10
References
92
Citations by year
2026: 4 · 2025: 6 · 2024: 2
FWCI
2.18
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
  • PubMed Central (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Division of Intramural Research, National Institute of Allergy and Infectious Diseases (R01AI149669, R21AI32379, F31AI15504, F31AI125090-01)
  • U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (T32GM007315, 5T32GM008353-27)
  • U.S. Department of Health & Human Services | NIH | National Institute on Drug Abuse (1R61DA059916-01)
Topics
  • Life Sciences › Immunology and Microbiology › Virology › HIV Research and Treatment
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
Keywords
HIV-1 Vpr, PU.1, macrophages, host restriction factors, HIV-1 envelope glycoprotein, proteasomal degradation, DCAF1, transcriptional downregulation
MeSH terms
Proto-Oncogene Protein Spi-1, Humans, Immunity, Innate, Macrophages

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review