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In humans, sterile alpha motif (SAM) domain- and histidine-aspartic acid (HD) domain-containing protein 1 (SAMHD1) is a dNTPase enzyme that prevents HIV-1 infection in non-cycling cells, such as differentiated THP-1 cells and human primary macrophages. Although phosphorylation of threonine 592 (T592) in SAMHD1 is recognized as the primary regulator of the ability to prevent HIV-1 infection, the contributions of SAMHD1 acetylation to this ability remain unknown. Mass spectrometry analysis of SAMHD1 proteins derived from cycling and non-cycling THP-1 cells, primary cycling B cells, and primary macrophages revealed that SAMHD1 is preferentially acetylated at lysine residues 354, 494, and 580 (K354, K494, and K580). In non-cycling cells, SAMHD1 is preferentially acetylated at K580, suggesting that this post-translational modification may contribute to the ability of SAMHD1 to block HIV-1 infection. Consistent with this finding, we found that mutations in K580 disrupted the ability of SAMHD1 to block HIV-1 infection without affecting the ability of SAMHD1 to deplete cellular dNTP levels. Gene editing of SAMHD1 in macrophage-like cells revealed that an intact K580 is required for HIV-1 restriction. This finding suggests that K580 acetylation in SAMHD1 is essential for blocking HIV-1 infection. More importantly, we found that a larger proportion of SAMHD1 featuring K580 acetylation could be detected in human primary macrophages when compared to human primary monocytes. In agreement, we found that SAMHD1 is acetylated during the monocyte-to-macrophage differentiation process. This finding agrees with the idea that the blockade of HIV-1 infection in macrophages requires SAMHD1 acetylation.IMPORTANCEThe natural inhibitor of HIV-1, sterile alpha motif (SAM) domain- and histidine-aspartic acid (HD) domain-containing protein 1 (SAMHD1), plays a pivotal role in preventing HIV-1 infection of macrophages and dendritic cells, which are vital components of the immune system. This study unveils that SAMHD1 undergoes post-translational modifications, specifically acetylation at lysines 354, 494, and 580. Our research underscores the significance of these modifications, demonstrating that acetylation at residue K580 is indispensable for SAMHD1's efficacy in blocking HIV-1 infection. Notably, K580 is found in a critical regulatory domain of SAMHD1, highlighting acetylation as a novel layer of SAMHD1 regulation for HIV-1 restriction in humans. A comprehensive understanding of the regulation mechanisms governing this anti-HIV-1 protein is crucial for leveraging nature's defense mechanisms against HIV-1 and could pave the way for innovative therapeutic strategies., Competing Interests: The authors declare no conflict of interest.

Cleavage and polyadenylation specificity factor subunit 6 (CPSF6, also known as CFIm68) is a 68 kDa component of the mammalian cleavage factor I (CFIm) complex that modulates mRNA alternative polyadenylation (APA) and determines 3' untranslated region (UTR) length, an important gene expression control mechanism. CPSF6 directly interacts with the HIV-1 core during infection, suggesting involvement in HIV-1 replication. Here, we review the contributions of CPSF6 to every stage of the HIV-1 replication cycle. Recently, several groups described the ability of HIV-1 infection to induce CPSF6 translocation to nuclear speckles, which are biomolecular condensates. We discuss the implications for CPSF6 localization in condensates and the potential role of condensate-localized CPSF6 in the ability of HIV-1 to control the protein expression pattern of the cell., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The initial stages of HIV-1 infection involve the transport of the viral core into the nuclear compartment. The presence of the HIV-1 core in the nucleus triggers the translocation of CPSF6/CPSF5 from paraspeckles into nuclear speckles, forming puncta-like structures. While this phenomenon is well-documented, the efficiency of CPSF6 translocation to nuclear speckles upon HIV-1 infection varies depending on the type of cell used. In some human cell lines, only 1-2% of the cells translocate CPSF6 to nuclear speckles when exposed to a 95% infection rate. To address the issue that only 1-2% of cells translocate CPSF6 to nuclear speckles when a 95% infection rate is achieved, we screened several human cell lines and identified a human a cell line in which approximately 85% of the cells translocate CPSF6 to nuclear speckles when 95% infection rate is achieved. This cellular system has enabled the development of a robust fluorescence microscopy method to quantify the translocation of CPSF6 into nuclear speckles following HIV-1 infection. This assay holds the potential to support studies aimed at understanding the role of CPSF6 translocation to nuclear speckles in HIV-1 infection. Additionally, since the translocation of CPSF6 into nuclear speckles depends on the physical presence of the viral core in the nucleus, our method also serves as a reporter of HIV-1 nuclear import., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Recent evidence has shown that uncoating and reverse transcription precede nuclear import. These recent breakthroughs have been made possible through the development of innovative biochemical and imaging techniques. This method outlines the biochemical assay used for detecting the presence of the HIV-1 core in the nuclear compartment. In this procedure, human cells are infected with HIV-1 NL4-3 , with or without the inclusion of PF74, a small molecule that inhibits core entry into the nuclear compartment. Subsequently, cells are separated into cytosolic and nuclear fractions. To assess whether the capsid protein has reached the nuclear compartment, cytosolic and nuclear fractions are subjected to Western blot analysis, utilizing antibodies specific to the HIV-1 capsid protein p24. To validate the true origin of these fractions, Western blot analysis employing antibodies against cytosolic and nuclear markers are also performed. In summary, this assay provides a reliable and efficient means to detect the presence of the HIV-1 capsid protein in the nucleus during infection under various conditions., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The early events of HIV-1 infection involve the transport of the viral core into the nucleus. This event triggers the translocation of CPSF6 from paraspeckles into nuclear speckles forming puncta-like structures. Our investigations revealed that neither HIV-1 integration nor reverse transcription is required for the formation of puncta-like structures. Moreover, HIV-1 viruses without viral genome are competent for the induction of CPSF6 puncta-like structures. In agreement with the notion that HIV-1 induced CPSF6 puncta-like structures are biomolecular condensates, we showed that osmotic stress and 1,6-hexanediol induced the disassembly of CPSF6 condensates. Interestingly, replacing the osmotic stress by isotonic media re-assemble CPSF6 condensates in the cytoplasm of the cell. To test whether CPSF6 condensates were important for infection we utilized hypertonic stress, which prevents formation of CPSF6 condensates, during infection. Remarkably, preventing the formation of CPSF6 condensates inhibits the infection of wild type HIV-1 but not of HIV-1 viruses bearing the capsid changes N74D and A77V, which do not form CPSF6 condensates during infection 1,2 . We also investigated whether the functional partners of CPSF6 are recruited to the condensates upon infection. Our experiments revealed that CPSF5, but not CPSF7, co-localized with CPSF6 upon HIV-1 infection. We found condensates containing CPSF6/CPSF5 in human T cells and human primary macrophages upon HIV-1 infection. Additionally, we observed that the integration cofactor LEDGF/p75 changes distribution upon HIV-1 infection and surrounds the CPSF6/CPSF5 condensates. Overall, our work demonstrated that CPSF6 and CPSF5 are forming biomolecular condensates that are important for infection of wild type HIV-1 viruses., (© 2023. The Author(s).)

The movement of viruses and other large macromolecular cargo through nuclear pore complexes (NPCs) is poorly understood. The human immunodeficiency virus type 1 (HIV-1) provides an attractive model to interrogate this process. HIV-1 capsid (CA), the chief structural component of the viral core, is a critical determinant in nuclear transport of the virus. HIV-1 interactions with NPCs are dependent on CA, which makes direct contact with nucleoporins (Nups). Here we identify Nup35, Nup153, and POM121 to coordinately support HIV-1 nuclear entry. For Nup35 and POM121, this dependence was dependent cyclophilin A (CypA) interaction with CA. Mutation of CA or removal of soluble host factors changed the interaction with the NPC. Nup35 and POM121 make direct interactions with HIV-1 CA via regions containing phenylalanine glycine motifs (FG-motifs). Collectively, these findings provide additional evidence that the HIV-1 CA core functions as a macromolecular nuclear transport receptor (NTR) that exploits soluble host factors to modulate NPC requirements during nuclear invasion., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The rapid spread and dominance of the Omicron SARS-CoV-2 lineages have posed severe health challenges worldwide. While extensive research on the role of the Receptor Binding Domain (RBD) in promoting viral infectivity and vaccine sensitivity has been well documented, the functional significance of the 681 PRRAR/SV 687 polybasic motif of the viral spike is less clear. In this work, we monitored the infectivity levels and neutralization potential of the wild-type human coronavirus 2019 (hCoV-19), Delta, and Omicron SARS-CoV-2 pseudoviruses against sera samples drawn four months post administration of a third dose of the BNT162b2 mRNA vaccine. Our findings show that in comparison to hCoV-19 and Delta SARS-CoV-2, Omicron lineages BA.1 and BA.2 exhibit enhanced infectivity and a sharp decline in their sensitivity to vaccine-induced neutralizing antibodies. Interestingly, P681 mutations within the viral spike do not play a role in the neutralization potential or infectivity of SARS Cov-2 pseudoviruses carrying mutations in this position. The P681 residue however, dictates the ability of the spike protein to promote fusion and syncytia formation between infected cells. While spike from hCoV-19 (P681) and Omicron (H681) promote only modest cell fusion and formation of syncytia between cells that express the spike-protein, Delta spike (R681) displays enhanced fusogenic activity and promotes syncytia formation. Additional analysis shows that a single P681R mutation within the hCoV-19 spike, or H681R within the Omicron spike, restores fusion potential to similar levels observed for the Delta R681 spike. Conversely, R681P point mutation within the spike of Delta pseudovirus abolishes efficient fusion and syncytia formation. Our investigation also demonstrates that spike proteins from hCoV-19 and Delta SARS-CoV-2 are efficiently incorporated into viral particles relative to the spike of Omicron lineages. We conclude that the third dose of the Pfizer-BNT162b2 provides appreciable protection against the newly emerged Omicron sub-lineages. However, the neutralization sensitivity of these new variants is diminished relative to that of the hCoV-19 or Delta SARS-CoV-2. We further show that the P681 residue within spike dictates cell fusion and syncytia formation with no effects on the infectivity of the specific viral variant and on its sensitivity to vaccine-mediated neutralization., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier Ltd.)

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 6 million deaths worldwide. The high variability in COVID-19 symptoms remains one of the most interesting mysteries of the pandemic. Genetic and environmental factors are likely to be key determinants of COVID-19 symptomatology. Here, we explored ACE2 as a genetic determinant for SARS-CoV-2 infection and COVID-19 symptomatology. Each human genome encodes two alleles of ACE2, which encodes the cell entry receptor for SARS-CoV-2. Here, we determined whether naturally occurring human ACE2 (hACE2) polymorphisms in the human population affect SARS-CoV-2 infection and the severity of COVID-19 symptoms. ACE2 variants S19P, I21V, E23K, K26R, K31R, N33I, H34R, E35K, and T92I showed increased virus infection compared to wild-type ACE2; thus, these variants could increase the risk for COVID-19. In contrast, variants D38V, Y83H, I468V, and N638S showed reduced infection, indicating a potential protective effect. hACE2 variants K26R and T92I increased infection by three-fold without changing the levels of ACE2 on the surface of the cells, suggesting that these variants may increase the risk of severe COVID-19. On the contrary, hACE2 variants D38V and Y83H decreased SARS-CoV-2 infection by four- and ten-fold, respectively, without changing surface expression, suggesting that these variants may protect against severe COVID-19. Remarkably, all protective hACE2 Polymorphisms were found almost exclusively in Asian populations, which may provide a partial explanation for the low COVID-19 mortality rates in Asian countries. Thus, hACE2 polymorphisms may modulate susceptibility to SARS-CoV-2 in the host and partially account for the differences in severity of COVID-19 among different ethnic groups.

The core of HIV-1 viruses bearing the capsid change N74D (HIV-1-N74D) do not bind the human protein CPSF6. In primary human CD4 + T cells, HIV-1-N74D viruses exhibit an infectivity defect when compared to wild-type. We first investigated whether loss of CPSF6 binding accounts for the loss of infectivity. Depletion of CPSF6 in human CD4 + T cells did not affect the early stages of wild-type HIV-1 replication, suggesting that defective infectivity in the case of HIV-1-N74D viruses is not due to the loss of CPSF6 binding. Based on our previous result that cyclophilin A (Cyp A) protected HIV-1 from human tripartite motif-containing protein 5α (TRIM5α hu ) restriction in CD4 + T cells, we found that depletion of TRIM5α hu in CD4 + T cells rescued the infectivity of HIV-1-N74D, suggesting that HIV-1-N74D cores interacted with TRIM5α hu . Accordingly, TRIM5α hu binding to HIV-1-N74D cores was increased compared with that of wild-type cores, and consistently, HIV-1-N74D cores lost their ability to bind Cyp A. In agreement with the notion that N74D capsids are defective in their ability to bind Cyp A, we found that HIV-1-N74D viruses were 20-fold less sensitive to TRIMCyp restriction when compared to wild-type viruses in OMK cells. Structural analysis revealed that N74D hexameric capsid protein in complex with PF74 is different from wild-type hexameric capsid protein in complex with PF74, which explains the defect of N74D capsids to interact with Cyp A. In conclusion, we showed that the decreased infectivity of HIV-1-N74D in CD4 + T cells is due to a loss of Cyp A protection from TRIM5α hu restriction activity.

The HIV-1 capsid is the target for the antiviral drugs GS-CA1 and Lenacapavir (GS-6207). We investigated the mechanism by which GS-CA1 and GS-6207 inhibit HIV-1 infection. HIV-1 inhibition by GS-CA1 did not require CPSF6 in CD4 + T cells. Contrary to PF74 that accelerates uncoating of HIV-1, GS-CA1 and GS-6207 stabilized the core. GS-CA1, unlike PF74, allowed the core to enter the nucleus, which agrees with the fact that GS-CA1 inhibits infection after reverse transcription. Unlike PF74, GS-CA1 did not disaggregate preformed CPSF6 complexes in nuclear speckles, suggesting that PF74 and GS-CA1 have different mechanisms of action. GS-CA1 stabilized the HIV-1 core, possibly by inducing a conformational shift in the core; in agreement, HIV-1 cores bearing N74D regained their ability to bind CPSF6 in the presence of GS-CA1. We showed that GS-CA1 binds to the HIV-1 core, changes its conformation, stabilizes the core, and thereby prevents viral uncoating and infection., Competing Interests: The authors declare no competing interests., (© 2021.)

Elevated intracellular levels of dNTPs have been shown to be a biochemical marker of cancer cells. Recently, a series of mutations in the multifunctional dNTP triphosphohydrolase (dNTPase), sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), have been reported in various cancers. Here, we investigated the structure and functions of SAMHD1 R366C/H mutants, found in colon cancer and leukemia. Unlike many other cancer-specific mutations, the SAMHD1 R366 mutations do not alter cellular protein levels of the enzyme. However, R366C/H mutant proteins exhibit a loss of dNTPase activity, and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely because of a loss of interaction with the γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1 replication, functions of SAMHD1 that are dependent on the ability of the enzyme to hydrolyze dNTPs. However, these mutants retain dNTPase-independent functions, including mediating dsDNA break repair, interacting with CtIP and cyclin A2, and suppressing innate immune responses. Finally, SAMHD1 degradation in human primary-activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study suggests that the loss of SAMHD1 dNTPase activity induced by R366 mutations can mechanistically contribute to the elevated dNTP levels commonly found in cancer cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Overexpression of the human Sad-1-Unc-84 homology protein 2 (SUN2) blocks HIV-1 infection in a capsid-dependent manner. In agreement, we showed that overexpression of SUN1 (Sad1 and UNC-84a) also blocks HIV-1 infection in a capsid-dependent manner. SUN2 and the related protein SUN1 are transmembrane proteins located in the inner membrane of the nuclear envelope. The N-terminal domains of SUN1/2 localizes to the nucleoplasm while the C-terminal domains are localized in the nuclear lamina. Because the N-terminal domains of SUN1/2 are located in the nucleoplasm, we hypothesized that SUN1/2 might be interacting with the HIV-1 replication complex in the nucleus leading to HIV-1 inhibition. Our results demonstrated that SUN1/2 interacts with the HIV-1 capsid, and in agreement with our hypothesis, the use of N-terminal deletion mutants showed that SUN1/2 proteins bind to the viral capsid by using its N-terminal domain. SUN1/2 deletion mutants correlated restriction of HIV-1 with capsid binding. Interestingly, the ability of SUN1/2 to restrict HIV-1 also correlated with perinuclear localization of these proteins. In agreement with the notion that SUN proteins interact with the HIV-1 capsid in the nucleus, we found that restriction of HIV-1 by overexpression of SUN proteins do not block the entry of the HIV-1 core into the nucleus. Our results showed that HIV-1 restriction is mediated by the interaction of SUN1/2N-terminal domains with the HIV-1 core in the nuclear compartment., (© 2021. The Author(s).)

SAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T 592 E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells., (© 2021. The Author(s).)

To understand the role of the HIV-1 capsid in viral replication, we developed a protocol to biochemically track capsid in the nucleus during infection. To this end, we separated HIV-1-infected cells into nuclear and cytosolic fractions. Fractions were analyzed by western blotting for HIV-1 capsid content as well as for nuclear and cytosolic markers to assess the bona fide origin of the fractions. This protocol can be applied in both cycling and non-cycling human cells. For complete details on the use and execution of this protocol, please refer to Selyutina et al. (2020a)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

SAMHD1 impedes infection of myeloid cells and resting T lymphocytes by retroviruses, and the enzymatic activity of the protein-dephosphorylation of deoxynucleotide triphosphates (dNTPs)-implicates enzymatic dNTP depletion in innate antiviral immunity. Here we show that the allosteric binding sites of the enzyme are plastic and can accommodate oligonucleotides in place of the allosteric activators, GTP and dNTP. SAMHD1 displays a preference for oligonucleotides containing phosphorothioate bonds in the Rp configuration located 3' to G nucleotides (GpsN), the modification pattern that occurs in a mechanism of antiviral defense in prokaryotes. In the presence of GTP and dNTPs, binding of GpsN-containing oligonucleotides promotes formation of a distinct tetramer with mixed occupancy of the allosteric sites. Mutations that impair formation of the mixed-occupancy complex abolish the antiretroviral activity of SAMHD1, but not its ability to deplete dNTPs. The findings link nucleic acid binding to the antiretroviral activity of SAMHD1, shed light on the immunomodulatory effects of synthetic phosphorothioated oligonucleotides and raise questions about the role of nucleic acid phosphorothioation in human innate immunity.

HIV-1 reverse transcription (RT) occurs before or during uncoating, but the cellular compartment where RT and uncoating occurs is unknown. Using imaging and biochemical assays to track HIV-1 capsids in the nucleus during infection, we demonstrated that higher-order capsid complexes and/or complete cores containing the viral genome are imported into the nucleus. Inhibition of RT does not prevent capsid nuclear import; thus, RT may occur in nuclear compartments. Cytosolic and nuclear fractions of infected cells reveal that most RT intermediates are enriched in nuclear fractions, suggesting that HIV-1 RT occurs in the nucleus alongside uncoating. In agreement, we find that capsid in the nucleus induces recruitment of cleavage and polyadenylation specific factor 6 (CPSF6) to SC35 nuclear speckles, which are highly active transcription sites, suggesting that CPSF6 through capsid is recruiting viral complexes to SC35 speckles for the occurrence of RT. Thus, nuclear import precedes RT and uncoating, which fundamentally changes our understanding of HIV-1 infection., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Disruption of cyclophilin A (CypA)-capsid interactions affects HIV-1 replication in human lymphocytes. To understand this mechanism, we utilize human Jurkat cells, peripheral blood mononuclear cells (PBMCs), and CD4 + T cells. Our results show that inhibition of HIV-1 infection caused by disrupting CypA-capsid interactions is dependent on human tripartite motif 5α (TRIM5α hu ), showing that TRIM5α hu restricts HIV-1 in CD4 + T cells. Accordingly, depletion of TRIM5α hu in CD4 + T cells rescues HIV-1 that fail to interact with CypA, such as HIV-1-P90A. We found that TRIM5α hu binds to the HIV-1 core. Disruption of CypA-capsid interactions fail to affect HIV-1-A92E/G94D infection, correlating with the loss of TRIM5α hu binding to HIV-1-A92E/G94D cores. Disruption of CypA-capsid interactions in primary cells has a greater inhibitory effect on HIV-1 when compared to Jurkat cells. Consistent with TRIM5α restriction, disruption of CypA-capsid interactions in CD4 + T cells inhibits reverse transcription. Overall, our results reveal that CypA binding to the core protects HIV-1 from TRIM5α hu restriction., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Flaviviruses such as Zika cause sporadic pandemic outbreaks worldwide. There is an urgent need for anti-Zika virus (ZIKV) drugs to prevent mother-to-child transmission of ZIKV, new infections in high-risk populations, and the infection of medical personnel in ZIKV-affected areas., Methods: Here, we showed that the small molecule 6-deoxyglucose-diphyllin (DGP) exhibited anti-ZIKV activity both in vitro and in vivo. DGP potently blocked ZIKV infection across all human and monkey cell lines tested. DGP also displayed broad-spectrum antiviral activity against other flaviviruses. Remarkably, DGP prevented ZIKV-induced mortality in mice lacking the type I interferon receptor (Ifnar1 -/- ). Cellular and virological experiments showed that DGP blocked ZIKV at a pre-fusion step or during fusion, which prevented the delivery of viral contents into the cytosol of the target cell. Mechanistic studies revealed that DGP prevented the acidification of endosomal/lysosomal compartments in target cells, thus inhibiting ZIKV fusion with cellular membranes and infection., Findings: These investigations revealed that DGP inhibits ZIKV infection in vitro and in vivo., Interpretation: The small molecule DGP has great potential for preclinical studies and the ability to inhibit ZIKV infection in humans., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Cellular SAMHD1 inhibits replication of many viruses by limiting intracellular deoxynucleoside triphosphate (dNTP) pools. We investigate the influence of SAMHD1 on human cytomegalovirus (HCMV). During HCMV infection, we observe SAMHD1 induction, accompanied by phosphorylation via viral kinase UL97. SAMHD1 depletion increases HCMV replication in permissive fibroblasts and conditionally permissive myeloid cells. We show this is due to enhanced gene expression from the major immediate-early (MIE) promoter and is independent of dNTP levels. SAMHD1 suppresses innate immune responses by inhibiting nuclear factor κB (NF-κB) activation. We show that SAMHD1 regulates the HCMV MIE promoter through NF-κB activation. Chromatin immunoprecipitation reveals increased RELA and RNA polymerase II on the HCMV MIE promoter in the absence of SAMHD1. Our studies reveal a mechanism of HCMV virus restriction by SAMHD1 and show how SAMHD1 deficiency activates an innate immune pathway that paradoxically results in increased viral replication through transcriptional activation of the HCMV MIE gene promoter., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

TRIM5α is an antiviral restriction factor that inhibits retroviral infection in a species-specific fashion. TRIM5α binds to and forms assemblies around the retroviral capsid. Following binding, poorly understood, ubiquitin-dependent events lead to the disassembly of the viral core, prior to the accumulation of viral reverse transcription products in the target cell. It is also known that assemblies of TRIM5α and other TRIM family proteins can be targets of autophagic degradation. The goal of this study was to define the role of specific ubiquitin linkages in the retroviral restriction and autophagic degradation of TRIM5α and delineate any connection between these two processes. To this end, we generated fusion proteins in which the catalytic domains of different deubiquitinase (DUB) enzymes, with different specificities for polyubiquitinated linkages, were fused to the N-terminal RING domain of Rhesus macaque TRIM5α. We assessed the role of ubiquitination in restriction and the degree to which specific types of ubiquitination are required for the association of TRIM5α with autophagic proteins. We determined that K63-linked ubiquitination by TRIM5α is required to induce capsid disassembly and to inhibit reverse transcription of HIV, while the ability to inhibit HIV-1 infection was not dependent on K63-linked ubiquitination. We also observed that K63-linked ubiquitination is required for the association of TRIM5α with autophagosomal membranes and the autophagic adapter protein p62. IMPORTANCE Although the mechanisms by which TRIM5α can induce the abortive disassembly of retroviral capsids have remained obscure, numerous studies have suggested a role for ubiquitination and cellular degradative pathways. These studies have typically relied on global perturbation of cellular degradative pathways. Here, through the use of linkage-specific deubiquitinating enzymes tethered to TRIM5α, we delineate the ubiquitin linkages which drive specific steps in restriction and degradation by TRIM5α, providing evidence for a noncanonical role for K63-linked ubiquitin in the process of retroviral restriction by TRIM5α and potentially providing insight into the mechanism of action of other TRIM family proteins., (Copyright © 2019 American Society for Microbiology.)

SAMHD1 is a human restriction factor known to prevent infection of macrophages, resting CD4 + T cells, and dendritic cells by HIV-1. To test the contribution of MxB to the ability of SAMHD1 to block HIV-1 infection, we created human THP-1 cell lines that were knocked out for expression of MxB, SAMHD1, or both. Interestingly, MxB depletion renders SAMHD1 ineffective against HIV-1 but not SIVmac. We observed similar results in human primary macrophages that were knockdown for the expression of MxB. To understand how MxB assists SAMHD1 restriction of HIV-1, we examined direct interaction between SAMHD1 and MxB in pull-down experiments. In addition, we investigated several properties of SAMHD1 in the absence of MxB expression, including subcellular localization, phosphorylation of the SAMHD1 residue T592, and dNTPs levels. These experiments showed that SAMHD1 restriction of HIV-1 requires expression of MxB., (Copyright © 2019. Published by Elsevier Inc.)

HIV integrates into the host genome to create a persistent viral reservoir. Stimulation of CD4 + memory T lymphocytes with common γc-chain cytokines renders these cells more susceptible to HIV infection, making them a key component of the reservoir itself. IL-15 is up-regulated during primary HIV infection, a time when the HIV reservoir established. Therefore, we investigated the molecular and cellular impact of IL-15 on CD4 + T-cell infection. We found that IL-15 stimulation induces SAM domain and HD domain-containing protein 1 (SAMHD1) phosphorylation due to cell cycle entry, relieving an early block to infection. Perturbation of the pathways downstream of IL-15 receptor (IL-15R) indicated that SAMHD1 phosphorylation after IL-15 stimulation is JAK dependent. Treating CD4 + T cells with Ruxolitinib, an inhibitor of JAK1 and JAK2, effectively blocked IL-15-induced SAMHD1 phosphorylation and protected CD4 + T cells from HIV infection. Using high-resolution single-cell immune profiling using mass cytometry by TOF (CyTOF), we found that IL-15 stimulation altered the composition of CD4 + T-cell memory populations by increasing proliferation of memory CD4 + T cells, including CD4 + T memory stem cells (T SCM ). IL-15-stimulated CD4 + T SCM , harboring phosphorylated SAMHD1, were preferentially infected. We propose that IL-15 plays a pivotal role in creating a self-renewing, persistent HIV reservoir by facilitating infection of CD4 + T cells with stem cell-like properties. Time-limited interventions with JAK1 inhibitors, such as Ruxolitinib, should prevent the inactivation of the endogenous restriction factor SAMHD1 and protect this long-lived CD4 + T-memory cell population from HIV infection., Competing Interests: The authors declare no conflict of interest.

The capsid-binding assay is an in vitro experiment used to determine whether cellular proteins interact with the HIV-1 core. In vitro assembled HIV-1 capsids recapitulate the surface of the HIV-1 core. The assay involves the incubation of in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes with the protein in question. Subsequently, the mixture is spun through a sucrose cushion using an ultracentrifuge, and the pellet is analyzed for the presence of the protein in question. Although this binding assay is reliable, it is labor intensive and does not contain washing steps. Here we have developed a simpler and faster assay to measure whether a cellular protein is binding to capsid. More importantly, this novel capsid-binding assay contains washing steps. In this assay, we took advantage of the HIV-1 capsid mutant A14C/E45C protein, which is stabilized by disulfide bonds, and is resistant to washing steps. We validated the reliability and specificity of this novel assay by testing the capsid binding ability of TRIMCyp, CPSF6 and MxB with their corresponding controls. Overall, this novel assay provides a reliable and fast methodology to search for novel capsid binders., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Human immunodeficiency virus type 1 (HIV-1) displays the unique ability to infect nondividing cells. The capsid of HIV-1 is the viral determinant for viral nuclear import. To understand the cellular factors involved in the ability of HIV-1 to infect nondividing cells, we sought to find capsid mutations that allow the virus to infect dividing but not nondividing cells. Because the interaction of capsid with the nucleoporin protein 153 (Nup153) is important for nuclear import of HIV-1, we solved new crystal structures of hexameric HIV-1 capsid in complex with a Nup153-derived peptide containing a phenylalanine-glycine repeat (FG repeat), which we used to guide structure-based mutagenesis of the capsid-binding interface. HIV-1 viruses with mutations in these capsid residues were tested for their ability to infect dividing and nondividing cells. HIV-1 viruses with capsid N57 substitutions infected dividing but not nondividing cells. Interestingly, HIV-1 viruses with N57 mutations underwent reverse transcription but not nuclear translocation. The mutant capsids also lost the ability to interact with Nup153 and CPSF6. The use of small molecules PF74 and BI-2 prevented the interaction of FG-containing nucleoporins (Nups), such as Nup153, with the HIV-1 core. Analysis of integration sites in HIV-1 viruses with N57 mutations revealed diminished integration into transcriptionally active genes in a manner resembling that of HIV-1 in CPSF6 knockout cells or that of HIV-1-N74D. The integration pattern of the N57 mutant HIV-1 can be explained by loss of capsid interaction with CPSF6, whereas capsid interaction with Nup153 is required for HIV-1 to infect nondividing cells. Additionally, the observed viral integration profiles suggested that integration site selection is a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin. IMPORTANCE One of the key advantages that distinguish lentiviruses, such as HIV-1, from all other retroviruses is its ability to infect nondividing cells. Interaction of the HIV-1 capsid with Nup153 and CPSF6 is important for nuclear entry and integration; however, the contribution of each of these proteins to nuclear import and integration is not clear. Using genetics, we demonstrated that these proteins contribute to different processes: Nup153 is essential for the HIV-1 nuclear import in nondividing cells, and CPSF6 is important for HIV-1 integration. In addition, nuclear factors such as CPSF6 and the state of the chromatin are known to be important for integration site selection; nevertheless, the preferential determinant influencing integration site selection is not known. This work demonstrates that integration site selection is a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin., (Copyright © 2018 American Society for Microbiology.)

Germline mutations in the human SAMHD1 gene cause the development of Aicardi-Goutières Syndrome (AGS), with a dominant feature being increased systemic type I interferon(IFN) production. Here we tested the state of type I IFN induction and response to, in SAMHD1 knockout (KO) human monocytic cells. SAMHD1 KO cells exhibited spontaneous transcription and translation of IFN-β and subsequent interferon-stimulated genes (ISGs) as compared to parental wild-type cells. This elevation of IFN-β and ISGs was abrogated via inhibition of the TBK1-IRF3 pathway in the SAMHD1 KO cells. In agreement, we found that SAMHD1 KO cells present high levels of phosphorylated TBK1 when compared to control cells. Moreover, addition of blocking antibody against type I IFN also reversed elevation of ISGs. These experiments suggested that SAMHD1 KO cells are persistently auto-stimulating the TBK1-IRF3 pathway, leading to an enhanced production of type I IFN and subsequent self-induction of ISGs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

SAMHD1 is a dNTP triphosphohydrolase (dNTPase) that impairs retroviral replication in a subset of non-cycling immune cells. Here we show that SAMHD1 is a redox-sensitive enzyme and identify three redox-active cysteines within the protein: C341, C350, and C522. The three cysteines reside near one another and the allosteric nucleotide binding site. Mutations C341S and C522S abolish the ability of SAMHD1 to restrict HIV replication, whereas the C350S mutant remains restriction competent. The C522S mutation makes the protein resistant to inhibition by hydrogen peroxide but has no effect on the tetramerization-dependent dNTPase activity of SAMHD1 in vitro or on the ability of SAMHD1 to deplete cellular dNTPs. Our results reveal that enzymatic activation of SAMHD1 via nucleotide-dependent tetramerization is not sufficient for the establishment of the antiviral state and that retroviral restriction depends on the ability of the protein to undergo redox transformations., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

SAMHD1 is a critical restriction factor for HIV-1 in non-cycling cells and its antiviral activity is regulated by T592 phosphorylation. Here, we show that SAMHD1 dephosphorylation at T592 is controlled during the cell cycle, occurring during M/G 1 transition in proliferating cells. Using several complementary proteomics and biochemical approaches, we identify the phosphatase PP2A-B55α responsible for rendering SAMHD1 antivirally active. SAMHD1 is specifically targeted by PP2A-B55α holoenzymes during mitotic exit, in line with observations that PP2A-B55α is a key mitotic exit phosphatase in mammalian cells. Strikingly, as HeLa or activated primary CD4 + T cells enter the G 1 phase, pronounced reduction of RT products is observed upon HIV-1 infection dependent on the presence of dephosphorylated SAMHD1. Moreover, PP2A controls SAMHD1 pT592 level in non-cycling monocyte-derived macrophages (MDMs). Thus, the PP2A-B55α holoenzyme is a key regulator to switch on the antiviral activity of SAMHD1.

The recent Zika virus (ZIKV) outbreak in Brazil has suggested associations of this virus infection with neurological disorders, including microcephaly in newborn infants and Guillian-Barré syndrome in adults. Previous reports have shown that AXL, a transmembrane receptor tyrosine kinase protein, is essential for ZIKV infection of mammalian cells, but this remains controversial. Here, we have assessed the involvement of AXL in the ability of ZIKV to infect mammalian cells, and also the requirement for endocytosis and acidic pH. We demonstrated that AXL is essential for ZIKV infection of human fibroblast cell line HT1080 as the targeted deletion of the gene for AXL in HT1080 cells made them no longer susceptible to ZIKV infection. Our results also showed that infection was prevented by lysosomotropic agents such as ammonium chloride, chloroquine and bafilomycin A1, which neutralize the normally acidic pH of endosomal compartments. Infection by ZIKV was also blocked by chlorpromazine, indicating a requirement for clathrin-mediated endocytosis. Taken together, our findings suggest that AXL most likely serves as an attachment factor for ZIKV on the cell surface, and that productive infection requires endocytosis and delivery of the virus to acidified intracellular compartments., (Copyright © 2018 Elsevier Inc. All rights reserved.)

[This corrects the article DOI: 10.1371/journal.ppat.1006906.].

SERINC5(S5) is a multi-span transmembrane protein that potently blocks the infectivity of HIV-1 produced by human T-cells. The ability of S5 to restrict infectivity correlates with its presence in the virion, but the exact mechanism by which S5 restricts HIV-1 is unknown. Here we tested whether the core from HIV-1 virions containing S5 is delivered to the cytoplasm. Using the "fate of the capsid" assay, we demonstrated that the viral core of S5-restricted HIV-1 does not reach the cytoplasm of target cells, suggesting a block in the delivery of the core to the cytoplasm. In agreement with evidence suggesting that the viral determinants for S5 restriction map to the envelope of HIV-1, we observed that S5 induces conformational changes to the HIV-1 envelope. Further, we demonstrated that S5 localizes to detergent-resistant membranes (DRMs), as has been shown previously for the HIV-1 envelope in producer cells. In order to identify the determinants of S5 restriction, we explored the ability of all human SERINC proteins to restrict HIV-1. In contrast to human S5, we observed that human SERINC2(S2) did not restrict HIV-1, and was inefficiently incorporated into HIV-1 virions when compared to S5. Experiments using S5-S2 chimeric proteins revealed two functional domains for restriction: one necessary for S5 incorporation into virions, which does not seem to be necessary for restriction, and a second one necessary to change the HIV-1 envelope conformation, localize to DRMs, and block infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Numerous viruses, including HIV-1, exploit the microtubule network to traffic toward the nucleus during infection. Although numerous studies have observed a role for the minus-end microtubule motor dynein in HIV-1 infection, the mechanism by which the viral core containing the viral genome associates with dynein and induces its perinuclear trafficking has remained unclear. Here, we report that the dynein adapter protein bicaudal D2 (BICD2) is able to interact with HIV-1 viral cores in target cells. We also observe that BICD2 can bind in vitro-assembled capsid tubes through its CC3 domain. We observe that BICD2 facilitates infection by promoting the trafficking of viral cores to the nucleus, thereby promoting nuclear entry of the viral genome and infection. Finally, we observe that depletion of BICD2 in the monocytic cell line THP-1 results in an induction of IFN-stimulated genes in these cells. Collectively, these results identify a microtubule adapter protein critical for trafficking of HIV-1 in the cytoplasm of target cells and evasion of innate sensing mechanisms in macrophages., Competing Interests: The authors declare no conflict of interest.

Mutations in the human SAMHD1 gene are known to correlate with the development of the Aicardi-Goutières syndrome (AGS), which is an inflammatory encephalopathy that exhibits neurological dysfunction characterized by increased production of type I interferon (IFN); this evidence has led to the concept that the SAMHD1 protein negatively regulates the type I IFN response. Additionally, the SAMHD1 protein has been shown to prevent efficient HIV-1 infection of macrophages, dendritic cells, and resting CD4+ T cells. To gain insights on the SAMHD1 molecular determinants that are responsible for the deregulated production of type I IFN, we explored the biochemical, cellular, and antiviral properties of human SAMHD1 mutants known to correlate with the development of AGS. Most of the studied SAMHD1 AGS mutants exhibit defects in the ability to oligomerize, decrease the levels of cellular deoxynucleotide triphosphates in human cells, localize exclusively to the nucleus, and restrict HIV-1 infection. At least half of the tested variants preserved the ability to be degraded by the lentiviral protein Vpx, and all of them interacted with RNA. Our investigations revealed that the SAMHD1 AGS variant p.G209S preserve all tested biochemical, cellular, and antiviral properties, suggesting that this residue is a determinant for the ability of SAMHD1 to negatively regulate the type I IFN response in human patients with AGS. Overall, our work genetically separated the ability of SAMHD1 to negatively regulate the type I IFN response from its ability to restrict HIV-1., (© 2017 Wiley Periodicals, Inc.)

The small-molecule 6-(tert-butyl)-4-phenyl-4-(trifluoromethyl)-1H,3H-1,3,5-triazin-2-one (3G11) inhibits HIV-1 replication in the human T cell line MT-2. Here, we showed that 3G11 specifically and potently blocks HIV-1 infection. By contrast, 3G11 did not block other retroviruses such as HIV-2, simian immunodeficiency virus (SIV mac ), bovine immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, N-tropic murine leukemia virus, B-tropic murine leukemia virus, and Moloney murine leukemia virus. Analysis of DNA metabolism by real-time PCR revealed that 3G11 blocks the formation of HIV-1 late reverse transcripts during infection prior to the first-strand transfer step. In agreement, an in vitro assay revealed that 3G11 blocks the enzymatic activity of HIV-1 reverse transcriptase as strong as nevirapine. Docking of 3G11 to the HIV-1 reverse transcriptase enzyme suggested a direct interaction between residue L100 and 3G11. In agreement, an HIV-1 virus bearing the reverse transcriptase change L100I renders HIV-1 resistant to 3G11, which suggested that the reverse transcriptase enzyme is the viral determinant for HIV-1 sensitivity to 3G11. Although NMR experiments revealed that 3G11 binds to the HIV-1 capsid, functional experiments suggested that capsid is not the viral determinant for sensitivity to 3G11. Overall, we described a novel non-nucleoside reverse transcription inhibitor that blocks HIV-1 infection., (© 2016 John Wiley & Sons A/S.)

The HIV-1 restriction factor SAMHD1 has the ability to negatively modulate retrotransposition of the long interspersed element 1(LINE-1). By exploring the ability of human SAMHD1 polymorphisms to inhibit LINE-1, we found that the single nucleotide polymorphism S33A present in the Korean population lose the ability to inhibit LINE-1 retrotransposition. Because SAMHD1 residue S33 is phosphorylated in human cycling and non-cycling cells, we demonstrated that SAMHD1 requires to be either phosphorylated on position 33 or to contain a bulky residue in order to inhibit LINE-1 retrotransposition. Therefore this unique mutation uncouples functions in this important restriction factor.

SAMHD1 is a dNTP hydrolase, whose activity is required for maintaining low dNTP concentrations in non-cycling T cells, dendritic cells, and macrophages. SAMHD1-dependent dNTP depletion is thought to impair retroviral replication in these cells, but the relationship between the dNTPase activity and retroviral restriction is not fully understood. In this study, we investigate allosteric activation of SAMHD1 by deoxynucleotide-dependent tetramerization and measure how the lifetime of the enzymatically active tetramer is affected by different dNTP ligands bound in the allosteric site. The EC 50 dNTP values for SAMHD1 activation by dNTPs are in the 2-20 μm range, and the half-life of the assembled tetramer after deoxynucleotide depletion varies from minutes to hours depending on what dNTP is bound in the A2 allosteric site. Comparison of the wild-type SAMHD1 and the T592D mutant reveals that the phosphomimetic mutation affects the rates of tetramer dissociation, but has no effect on the equilibrium of allosteric activation by deoxynucleotides. Collectively, our data suggest that deoxynucleotide-dependent tetramerization contributes to regulation of deoxynucleotide levels in cycling cells, whereas in non-cycling cells restrictive to retroviral replication, SAMHD1 activation is likely to be achieved through a distinct mechanism., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

SAMHD1, a dNTP triphosphohydrolase, contributes to interferon signaling and restriction of retroviral replication. SAMHD1-mediated retroviral restriction is thought to result from the depletion of cellular dNTP pools, but it remains controversial whether the dNTPase activity of SAMHD1 is sufficient for restriction. The restriction ability of SAMHD1 is regulated in cells by phosphorylation on T592. Phosphomimetic mutations of T592 are not restriction competent, but appear intact in their ability to deplete cellular dNTPs. Here we use analytical ultracentrifugation, fluorescence polarization and NMR-based enzymatic assays to investigate the impact of phosphomimetic mutations on SAMHD1 tetramerization and dNTPase activity in vitro. We find that phosphomimetic mutations affect kinetics of tetramer assembly and disassembly, but their effects on tetramerization equilibrium and dNTPase activity are insignificant. In contrast, the Y146S/Y154S dimerization-defective mutant displays a severe dNTPase defect in vitro, but is indistinguishable from WT in its ability to deplete cellular dNTP pools and to restrict HIV replication. Our data suggest that the effect of T592 phosphorylation on SAMHD1 tetramerization is not likely to explain the retroviral restriction defect, and we hypothesize that enzymatic activity of SAMHD1 is subject to additional cellular regulatory mechanisms that have not yet been recapitulated in vitro.

Background: HIV-1 capsid influences viral uncoating and nuclear import. Some capsid is detected in the nucleus but it is unclear if it has any function. We reported that the antibiotic Coumermycin-A1 (C-A1) inhibits HIV-1 integration and that a capsid mutation confers resistance to C-A1, suggesting that capsid might affect post-nuclear entry steps., Results: Here we report that C-A1 inhibits HIV-1 integration in a capsid-dependent way. Using molecular docking, we identify an extended binding pocket delimited by two adjacent capsid monomers where C-A1 is predicted to bind. Isothermal titration calorimetry confirmed that C-A1 binds to hexameric capsid. Cyclosporine washout assays in Jurkat CD4+ T cells expressing engineered human TRIMCyp showed that C-A1 causes faster and greater escape from TRIMCyp restriction. Sub-cellular fractionation showed that small amounts of capsid accumulated in the nuclei of infected cells and C-A1 reduced the nuclear capsid. A105S and N74D capsid mutant viruses did not accumulate capsid in the nucleus, irrespective of C-A1 treatment. Depletion of Nup153, a nucleoporin located at the nuclear side of the nuclear pore that binds to HIV-1 capsid, made the virus less susceptible to TRIMCyp restriction, suggesting that Nup153 may help maintain some integrity of the viral core in the nucleus. Furthermore C-A1 increased binding of CPSF6, a nuclear protein, to capsid., Conclusions: Our results indicate that capsid is involved in post-nuclear entry steps preceding integration.

Unlabelled: In a previous screen of putative interferon-stimulated genes, SUN2 was shown to inhibit HIV-1 infection in an uncharacterized manner. SUN2 is an inner nuclear membrane protein belonging to the linker of nucleoskeleton and cytoskeleton complex. We have analyzed here the role of SUN2 in HIV infection. We report that in contrast to what was initially thought, SUN2 is not induced by type I interferon, and that SUN2 silencing does not modulate HIV infection. However, SUN2 overexpression in cell lines and in primary monocyte-derived dendritic cells inhibits the replication of HIV but not murine leukemia virus or chikungunya virus. We identified HIV-1 and HIV-2 strains that are unaffected by SUN2, suggesting that the effect is specific to particular viral components or cofactors. Intriguingly, SUN2 overexpression induces a multilobular flower-like nuclear shape that does not impact cell viability and is similar to that of cells isolated from patients with HTLV-I-associated adult T-cell leukemia or with progeria. Nuclear shape changes and HIV inhibition both mapped to the nucleoplasmic domain of SUN2 that interacts with the nuclear lamina. This block to HIV replication occurs between reverse transcription and nuclear entry, and passaging experiments selected for a single-amino-acid change in capsid (CA) that leads to resistance to overexpressed SUN2. Furthermore, using chemical inhibition or silencing of cyclophilin A (CypA), as well as CA mutant viruses, we implicated CypA in the SUN2-imposed block to HIV infection. Our results demonstrate that SUN2 overexpression perturbs both nuclear shape and early events of HIV infection., Importance: Cells encode proteins that interfere with viral replication, a number of which have been identified in overexpression screens. SUN2 is a nuclear membrane protein that was shown to inhibit HIV infection in such a screen, but how it blocked HIV infection was not known. We show that SUN2 overexpression blocks the infection of certain strains of HIV before nuclear entry. Mutation of the viral capsid protein yielded SUN2-resistant HIV. Additionally, the inhibition of HIV infection by SUN2 involves cyclophilin A, a protein that binds the HIV capsid and directs subsequent steps of infection. We also found that SUN2 overexpression substantially changes the shape of the cell's nucleus, resulting in many flower-like nuclei. Both HIV inhibition and deformation of nuclear shape required the domain of SUN2 that interacts with the nuclear lamina. Our results demonstrate that SUN2 interferes with HIV infection and highlight novel links between nuclear shape and viral infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)