Human Immunodeficiency Virus Type 1 Restriction Research Articles

The envelope proteins from SARS-CoV-2 and SARS-CoV potently reduce the infectivity of human immunodeficiency virus type 1 (HIV-1)

BackgroundViroporins are virally encoded ion channels involved in virus assembly and release. Human immunodeficiency virus type 1 (HIV-1) and influenza A virus encode for viroporins. The human coronavirus SARS-CoV-2 encodes for at least two viroporins, a small 75 amino acid transmembrane protein known as the envelope (E) protein and a larger 275 amino acid protein known as Orf3a. Here, we compared the replication of HIV-1 in the presence of four different β-coronavirus E proteins.ResultsWe observed that the SARS-CoV-2 and SARS-CoV E proteins reduced the release of infectious HIV-1 yields by approximately 100-fold while MERS-CoV or HCoV-OC43 E proteins restricted HIV-1 infectivity to a lesser extent. Mechanistically, neither reverse transcription nor mRNA synthesis was involved in the restriction. We also show that all four E proteins caused phosphorylation of eIF2-α at similar levels and that lipidation of LC3-I could not account for the differences in restriction. However, the level of caspase 3 activity in transfected cells correlated with HIV-1 restriction in cells. Finally, we show that unlike the Vpu protein of HIV-1, the four E proteins did not significantly down-regulate bone marrow stromal cell antigen 2 (BST-2).ConclusionsThe results of this study indicate that while viroporins from homologous viruses can enhance virus release, we show that a viroporin from a heterologous virus can suppress HIV-1 protein synthesis and release of infectious virus.

Hemin and Iron Modulated Intrinsic Cellular Factors Regulate HIV-1 Replication in Sickle Cell Disease

Human immunodeficiency virus type 1 (HIV-1) is challenged by intrinsic antiviral restriction factors which are counteracted with its own viral proteins. We recently showed that in Sickle Cell Disease (SCD), anti-viral restriction factor SAMHD1 is activated and inhibits ex vivo HIV-1 infection. Activation of SAMHD1 is caused by a reduced phosphorylation by CDK2, that are inhibited by reduced levels of intracellular iron as found in SCD peripheral blood mononuclear cells (PBMCs). The present study was aimed at identifying additional HIV-1 restriction factors in SCD PBMCs. We used a customized array that included 43 known anti-HIV-1 restriction factors to measure mRNA expression of anti-viral factors. The identified factors were further validated with shRNA-mediated knockdowns of the identified genes and their effect on HIV-1 replication in cultured and primary cells.We also mimicked SCD conditions by treating PBMCs from healthy donors with hemin and iron chelators. We observed an upregulation of 19 genes in SCD PBMCs including APOBEC3A, APOBEC3C, TRIM5α, TRIM22, MX2 and RSAD2 which were significantly upregulated and were further validated using real-time q-PCR. In PBMCs treated with hemin, APOBEC3A, RASD2 and MX2 mRNAs were found to be overexpressed, whereas TRIM5α expression was increased in PBMC treated with either hemin or iron chelators. Knockdowns by shRNA of APOBEC3C and TRIM5α, but not APOBEC3A or TRIM22 induced HIV-1 replication. In conclusion, APOBEC3A, APOBEC3C, TRIM5α, TRIM22, MX2 and RSAD2 may also contribute to HIV-1 restriction in SCD. Similar to SAMHD1, these factors are likely to be modulated by hemolysis and intracellular iron levels. Our findings further point to the contribution of hemolysis and deregulated iron metabolism to potential HIV-1 restriction in SCD.This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005, and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. DisclosuresNo relevant conflicts of interest to declare.

Potential Utilization of APOBEC3-Mediated Mutagenesis for an HIV-1 Functional Cure.

The introduction of combination antiretroviral therapy (cART) has managed to control the replication of human immunodeficiency virus type 1 (HIV-1) in infected patients. However, a complete HIV-1 cure, including a functional cure for or eradication of HIV-1, has yet to be achieved because of the persistence of latent HIV-1 reservoirs in adherent patients. The primary source of these viral reservoirs is integrated proviral DNA in CD4+ T cells and other non-T cells. Although a small fraction of this proviral DNA is replication-competent and contributes to viral rebound after the cessation of cART, >90% of latent viral reservoirs are replication-defective and some contain high rates of G-to-A mutations in proviral DNA. At least in part, these high rates of G-to-A mutations arise from the APOBEC3 (A3) family proteins of cytosine deaminases. A general model has shown that the HIV-1 virus infectivity factor (Vif) degrades A3 family proteins by proteasome-mediated pathways and inactivates their antiviral activities. However, Vif does not fully counteract the HIV-1 restriction activity of A3 family proteins in vivo, as indicated by observations of A3-mediated G-to-A hypermutation in the proviral DNA of HIV-1-infected patients. The frequency of A3-mediated hypermutation potentially contributes to slower HIV-1/AIDS disease progression and virus evolution including the emergence of cytotoxic T lymphocyte escape mutants. Therefore, combined with other strategies, the manipulation of A3-mediated mutagenesis may contribute to an HIV-1 functional cure aimed at cART-free remission. In this mini-review, we discuss the possibility of an HIV-1 functional cure arising from manipulation of A3 mutagenic activity.

Interferon Induction By HbS, SERINC5 Upregulation and Reduction of HIV-1 Nef and AP2 Contribute to HIV-1 Restriction in Sickle Cell Disease

Concerted action of numerous positively acting cellular factors is essential for Human immunodeficiency virus type 1 (HIV-1) replication but in turn is challenged by anti-viral restriction factors. Previously we showed that ex vivo one round HIV-1 replication and replication of fully competent T-tropic HIV-1(IIIB) is significantly reduced in peripheral blood mononuclear cells (PBMCs) obtained from patients with Sickle Cell Disease (SCD). Further, we identified and confirmed CDKN1A (p21) and CH25H as host restriction factors expressed in SCD PBMCs that may contribute to the HIV-1 inhibition, in addition to the previously reported SAMHD1 and IKBα. Since CH25H is an interferon stimulated gene (ISG), we analyzed IRFs and interferon expression in SCD PBMCs. Higher levels of IRF7 and IFNβ mRNA were observed in SCD PBMCs compared to controls. We probed further to ascertain if hemin or sickle Hb was responsible for interferon response. We found upregulation of IFNβ in THP-1 - derived macrophages treated with lysates of HbSS RBCs or purified HbS as compared to untreated or HbA treated controls. HbSS RBCs lysates and purified HbS inhibited HIV-1 gag mRNA expression in monocyte-derived macrophages infected with HIV-1(Ba-L). Recent clinical study showed increased levels of CD4 in HIV-1 infected SCD patients in Africa. Thus we analyzed CD4 levels in HIV-1 IIIB infected SCD PBMCs, and found them to be higher compared to controls. Levels of HIV-1 nef mRNA, that controls CD4 expression was lower in HIV-1 IIIB infected SCD PBMCs. As Nef counteracts SERINC3/5 restriction factor, we analyzed its expression as well as the expression of AP2 clathrin adaptor that is required for Nef mediated internalization of CD4. AP2 expression was lower and SERINC5 expression was higher in SCD PBMCs. CONCLUSIONS: SCD PBMCs could resist HIV-1 infection because of the increased IFNβ production by macrophages exposed to HbSS or sickle cell RBCs. SCD PBMC have increased levels of SERNIC5 and lower levels of HIV-1 Nef and host AP2 expression that, culumlatively, can increased CD4 levels and lead to the overall improved immunological health of SCD patients. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 1SC1HL150685, 5U54MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures No relevant conflicts of interest to declare.

Transcriptome Profiling of Human Monocyte-Derived Macrophages Upon CCL2 Neutralization Reveals an Association Between Activation of Innate Immune Pathways and Restriction of HIV-1 Gene Expression.

Macrophages are key targets of human immunodeficiency virus type 1 (HIV-1) infection and main producers of the proinflammatory chemokine CC chemokine ligand 2 (CCL2), whose expression is induced by HIV-1 both in vitro and in vivo. We previously found that CCL2 neutralization in monocyte-derived macrophages (MDMs) strongly inhibited HIV-1 replication affecting post-entry steps of the viral life cycle. Here, we used RNA-sequencing to deeply characterize the cellular factors and pathways modulated by CCL2 blocking in MDMs and involved in HIV-1 replication restriction. We report that exposure to CCL2 neutralizing antibody profoundly affected the MDM transcriptome. Functional annotation clustering of up-regulated genes identified two clusters enriched for antiviral defense and immune response pathways, comprising several interferon-stimulated, and restriction factor coding genes. Transcripts in the clusters were enriched for RELA and NFKB1 targets, suggesting the activation of the canonical nuclear factor κB pathway as part of a regulatory network involving miR-155 up-regulation. Furthermore, while HIV-1 infection caused small changes to the MDM transcriptome, with no evidence of host defense gene expression and type I interferon signature, CCL2 blocking enabled the activation of a strong host innate response in infected macrophage cultures, and potently inhibited viral genes expression. Notably, an inverse correlation was found between levels of viral transcripts and of the restriction factors APOBEC3A (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 A), ISG15, and MX1. These findings highlight an association between activation of innate immune pathways and HIV-1 restriction upon CCL2 blocking and identify this chemokine as an endogenous factor contributing to the defective macrophage response to HIV-1. Therapeutic targeting of CCL2 may thus strengthen host innate immunity and restrict HIV-1 replication.

Kinetics of Early Innate Immune Activation during HIV-1 Infection of Humanized Mice.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with aberrant immune activation; however, most model systems for HIV-1 have been used during established infection. Here, we utilize ultrasensitive HIV-1 quantification to delineate early events during the eclipse, burst, and chronic phases of HIV-1 infection in humanized mice. We show that very early in infection, HIV-1 suppresses peripheral type I interferon (IFN) and interferon-stimulated gene (ISG) responses, including the HIV-1 restriction factor IFI44. At the peak of innate immune activation, prior to CD4 T cell loss, HIV-1 infection differentially affects peripheral and lymphoid Toll-like receptor (TLR) expression profiles in T cells and macrophages. This results in a trend toward an altered activation of nuclear factor κB (NF-κB), TANK-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3). The subsequent type I and III IFN responses result in preferential induction of peripheral ISG responses. Following this initial innate immune activation, peripheral expression of the HIV-1 restriction factor SAM domain- and HD domain-containing protein 1 (SAMHD1) returns to levels below those observed in uninfected mice, suggesting that HIV-1 interferes with their basal expression. However, peripheral cells still retain their responsiveness to exogenous type I IFN, whereas splenic cells show a reduction in select ISGs in response to IFN. This demonstrates the highly dynamic nature of very early HIV-1 infection and suggests that blocks to the induction of HIV-1 restriction factors contribute to the establishment of viral persistence.IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) infection is restricted to humans and some nonhuman primates (e.g., chimpanzee and gorilla). Alternative model systems based on simian immunodeficiency virus (SIV) infection of macaques are available but do not recapitulate all aspects of HIV-1 infection and disease. Humanized mice, which contain a human immune system, can be used to study HIV-1, but only limited information on early events and immune responses is available to date. Here, we describe very early immune responses to HIV-1 and demonstrate a suppression of cell-intrinsic innate immunity. Furthermore, we show that HIV-1 infection interacts differently with innate immune responses in blood and lymphoid organs.

Restriction of HIV-1 Infection in Sickle Cell Disease and Trait

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) replication is controlled by host intrinsic antiviral restriction factors some of which are counteracted by HIV-1 accessory proteins. We recently showed that ex vivo HIV-1 infection is suppressed in PBMCs obtained from Sickle Cell Disease (SCD) patients. The inhibition was mediated in part by SAMHD1 and NF-κB inhibitor, IkBα and triggered by ferroportin, an iron export protein. Ferroportin expression reduced intracellular iron levels and inhibited cellular CDK2 activity leading to reduction of SAMHD1 phosphorylation and increased expression of IkBα.OBJECTIVES: The study was designed to further clarify the mechanism of HIV-1 inhibition in SCD and identify additional HIV-1 restriction factors that may contribute to the restriction mechanism.METHODS: A customized array was utilized to determine the expression of restriction factors in SCD PBMCs. The shRNA-mediated knockdowns of the identified genes were used to further validate the role of these factors in HIV-1 replication in cultured and primary cells. HIV-1(IIIB strain) and VSVG-pseudotyped pNL4-3.Luc.R-E-virus was used to analyze HIV-1 replication.RESULTS: Overexpression of ferroportin in THP-1 cells led to increased expression of HO-1 and p21 and reduced phosphorylation of SAMHD1. Inhibition of HO-1 and p21 by small molecules increased HIV-1 replication in SCD PBMCs suggesting that these factors contributed to the restriction mechanism. Knocking down SAMHD1 in SCD PBMC did not fully restore HIV-1 replication pointing to additional restriction factors. Analysis of HIV-1 restriction factors in SCD PBMCs using customized array showed increased expression of APOBECs, TRIMs, CH25H, CPSF6, CTR9, EIF2ak2, IFI16, MX2, PML and RTF1 mRNAs. We next tested the effect of SCD trait on HIV-1 infection ex vivo and in vivo. PBMCs obtained from SCD trait individuals showed restricted HIV-1 infection with HIV-1(IIIB strain). To further validate these findings, we compared 9 trait (HbAS) HIV-1 infected individuals with 107 non-SCD (HbAA or HbAC) HIV-1 infected individuals from Howard University HIV clinic. HIV-1 viral load and levels of HIV-1 env and gag were significantly lower in HIV-1 infected SCD trait subjects. Expression of HO-1, p21 were increased and RNR2 expression was reduced in SCD trait PBMCs. Small molecule inhibitors of HO-1 and p21 induced HIV-1 replication in SCD trait PBMCs.CONCLUSIONS: Our findings point to HO-1 and p21 as factors that, in addition to SAMHD1 and IKBα, mediate HIV-1 restriction in SCD. In SCD trait, HO-1, p21 and RNR2 play a key role in ex vivo HIV-1 restriction. Thus HIV-1 infection is deregulated not only in SCD patients but also in SCD trait individuals. The HIV-1 restriction is mediated by iron-activated antiviral restriction factors.ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 5G12MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. DisclosuresNekhai:NIMHD, NIH: Research Funding; NHLBI, NIH: Research Funding; NIAID, NIH: Research Funding.

Multiple components of the nuclear pore complex interact with the amino-terminus of MX2 to facilitate HIV-1 restriction.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced post-entry inhibitor of human immunodeficiency virus type-1 (HIV-1) infection. While the precise mechanism of viral inhibition remains unclear, MX2 is localized to the nuclear envelope, and blocks the nuclear import of viral cDNAs. The amino-terminus of MX2 (N-MX2) is essential for anti-viral function, and mutation of a triple arginine motif at residues 11 to 13 abrogates anti-HIV-1 activity. In this study, we sought to investigate the role of N-MX2 in anti-viral activity by identifying functionally relevant host-encoded interaction partners through yeast-two-hybrid screening. Remarkably, five out of seven primary candidate interactors were nucleoporins or nucleoporin-like proteins, though none of these candidates were identified when screening with a mutant RRR11-13A N-MX2 fragment. Interactions were confirmed by co-immunoprecipitation, and RNA silencing experiments in cell lines and primary CD4+ T cells demonstrated that multiple components of the nuclear pore complex and nuclear import machinery can impact MX2 anti-viral activity. In particular, the phenylalanine-glycine (FG) repeat containing cytoplasmic filament nucleoporin NUP214, and transport receptor transportin-1 (TNPO1) were consistently required for full MX2, and interferon-mediated, anti-viral function. Both proteins were shown to interact with the triple arginine motif, and confocal fluorescence microscopy revealed that their simultaneous depletion resulted in diminished MX2 accumulation at the nuclear envelope. We therefore propose a model whereby multiple components of the nuclear import machinery and nuclear pore complex help position MX2 at the nuclear envelope to promote MX2-mediated restriction of HIV-1.

CRISPR/Cas9-mediated deletion of miR-146a enhances antiviral response in HIV-1 infected cells.

The human immunodeficiency virus type 1 (HIV-1) causes persistent infection in human and induces miR-146a expression in infected cells. miR-146a represses the innate immune response by inhibiting the expression of TRAF6 and IRAK1 genes, thus negatively controls the NF-κB-related cytokines and interferon stimulated genes. Here we reported that lentiviral CRISPR/Cas9 system was highly efficient in introducing mutations in the precursor miR-146a genomic sequences, resulting in a loss of miR-146a expression and function. miR-146a ablation led to increasing cytokines production in LPS-stimulated A549 cells. Moreover, miR-146a knockout in HIV-1 infected MT2 cells markedly increased the expression of cytokines and HIV-1 restriction factors and reversed T cell exhaustion markers expression, thus influencing HIV-1 replication. Our study indicates that lentiviral CRISPR/Cas9-mediated gene editing is an effective approach to abrogate miR-146a expression, which consequently inhibits HIV-1 replication as well as proviral reactivation by enhancing the expression of cytokines and HIV-1 restriction factors.

SAMHD1 Phosphorylation Coordinates the Anti-HIV-1 Response by Diverse Interferons and Tyrosine Kinase Inhibition.

ABSTRACTMacrophages are susceptible to human immunodeficiency virus type 1 (HIV-1) infection despite abundant expression of antiviral proteins. Perhaps the most important antiviral protein is the restriction factor sterile alpha motif domain and histidine/aspartic acid domain-containing protein 1 (SAMHD1). We investigated the role of SAMHD1 and its phospho-dependent regulation in the context of HIV-1 infection in primary human monocyte-derived macrophages and the ability of various interferons (IFNs) and pharmacologic agents to modulate SAMHD1. Here we show that stimulation by type I, type II, and to a lesser degree, type III interferons share activation of SAMHD1 via dephosphorylation at threonine-592 as a consequence of signaling. Cyclin-dependent kinase 1 (CDK1), a known effector kinase for SAMHD1, was downregulated at the protein level by all IFN types tested. Pharmacologic inhibition or small interfering RNA (siRNA)-mediated knockdown of CDK1 phenocopied the effects of IFN on SAMHD1. A panel of FDA-approved tyrosine kinase inhibitors potently induced activation of SAMHD1 and subsequent HIV-1 inhibition. The viral restriction imposed via IFNs or dasatinib could be overcome through depletion of SAMHD1, indicating that their effects are exerted primarily through this pathway. Our results demonstrate that SAMHD1 activation, but not transcriptional upregulation or protein induction, is the predominant mechanism of HIV-1 restriction induced by type I, type II, and type III IFN signaling in macrophages. Furthermore, SAMHD1 activation presents a pharmacologically actionable target through which HIV-1 infection can be subverted.

Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G

APOBEC3G (A3G) is a human enzyme that inhibits human immunodeficiency virus type 1 (HIV-1) infectivity, in the absence of the viral infectivity factor Vif, through deoxycytidine deamination and a deamination-independent mechanism. A3G converts from a fast to a slow binding state through oligomerization, which suggests that large A3G oligomers could block HIV-1 reverse transcriptase-mediated DNA synthesis, thereby inhibiting HIV-1 replication. However, it is unclear how the small number of A3G molecules found in the virus could form large oligomers. Here we measure the single-stranded DNA binding and oligomerization kinetics of wild-type and oligomerization-deficient A3G, and find that A3G first transiently binds DNA as a monomer. Subsequently, A3G forms N-terminal domain-mediated dimers, whose dissociation from DNA is reduced and their deaminase activity inhibited. Overall, our results suggest that the A3G molecules packaged in the virion first deaminate viral DNA as monomers before dimerizing to form multiple enzymatically deficient roadblocks that may inhibit reverse transcription.

TRIM5 Retroviral Restriction Activity Correlates with the Ability To Induce Innate Immune Signaling.

ABSTRACTHost restriction factor TRIM5 inhibits retroviral transduction in a species-specific manner by binding to and destabilizing the retroviral capsid lattice before reverse transcription is completed. However, the restriction mechanism may not be that simple since TRIM5 E3 ubiquitin ligase activity, the proteasome, autophagy, and TAK1-dependent AP-1 signaling have been suggested to contribute to restriction. Here, we show that, among a panel of seven primate and Carnivora TRIM5 orthologues, each of which has potential for potent retroviral restriction activity, all activated AP-1 signaling. In contrast, TRIM family paralogues most closely related to TRIM5 did not. While each primate species has a single TRIM5 gene, mice have at least seven TRIM5 homologues that cluster into two groups, Trim12a, -b, and -c and Trim30a, -b, -c, and -d. The three Trim12 proteins activated innate immune signaling, while the Trim30 proteins did not, though none of the murine Trim5 homologues restricted any of a panel of cloned retroviruses. To determine if any mouse TRIM5 homologues had potential for restriction activity, each was fused to the human immunodeficiency virus type 1 (HIV-1) CA binding protein cyclophilin A (CypA). The three Trim12-CypA fusions all activated AP-1 and restricted HIV-1 transduction, whereas the Trim30-CypA fusions did neither. AP-1 activation and HIV-1 restriction by the Trim12-CypA fusions were inhibited by disruption of TAK1. Overall then, these experiments demonstrate that there is a strong correlation between TRIM5 retroviral restriction activity and the ability to activate TAK1-dependent innate immune signaling.IMPORTANCE The importance of retroviruses for the evolution of susceptible host organisms cannot be overestimated. Eight percent of the human genome is retrovirus sequence, fixed in the germ line during past infection. Understanding how metazoa protect their genomes from mutagenic retrovirus infection is therefore of fundamental importance to biology. TRIM5 is a cellular protein that protects host genome integrity by disrupting the retroviral capsid as it transports viral nucleic acid to the host cell nucleus. Previous data suggest that innate immune signaling contributes to TRIM5-mediated restriction. Here, we show that activation of innate immune signaling is conserved among primate and carnivore TRIM5 orthologues and among 3 of the 7 mouse Trim5 homologues and that such activity is required for TRIM5-mediated restriction activity.

Phospholipase D1 Couples CD4+ T Cell Activation to c-Myc-Dependent Deoxyribonucleotide Pool Expansion and HIV-1 Replication.

Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their de novo synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for de novo dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibitors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis.

Identification of Cellular Proteins Interacting with the Retroviral Restriction Factor SAMHD1

Human and mouse SAMHD1 proteins block human immunodeficiency virus type 1 (HIV-1) infection in noncycling human monocytic cells by reducing the intracellular deoxynucleoside triphosphate (dNTP) concentrations. Phosphorylation of human SAMHD1 at threonine 592 (T592) by cyclin-dependent kinase 1 (CDK1) and cyclin A2 impairs its HIV-1 restriction activity, but not the dNTP hydrolase activity, suggesting that dNTP depletion is not the sole mechanism of SAMHD1-mediated HIV-1 restriction. Using coimmunoprecipitation and mass spectrometry, we identified and validated two additional host proteins interacting with human SAMHD1, namely, cyclin-dependent kinase 2 (CDK2) and S-phase kinase-associated protein 2 (SKP2). We observed that mouse SAMHD1 specifically interacted with cyclin A2, cyclin B1, CDK1, and CDK2. Given the role of these SAMHD1-interacting proteins in cell cycle progression, we investigated the regulation of these host proteins by monocyte differentiation and activation of CD4+ T cells and examined their effect on the phosphorylation of human SAMHD1 at T592. Our results indicate that primary monocyte differentiation and CD4+ T-cell activation regulate the expression of these SAMHD1-interacting proteins. Furthermore, our results suggest that, in addition to CDK1 and cyclin A2, CDK2 phosphorylates T592 of human SAMHD1 and thereby regulates its HIV-1 restriction function. SAMHD1 is the first dNTP triphosphohydrolase found in mammalian cells. Human and mouse SAMHD1 proteins block HIV-1 infection in noncycling cells. Previous studies suggested that phosphorylation of human SAMHD1 at threonine 592 by CDK1 and cyclin A2 negatively regulates its HIV-1 restriction activity. However, it is unclear whether human SAMHD1 interacts with other host proteins in the cyclin A2 and CDK1 complex and whether mouse SAMHD1 shares similar cellular interacting partners. Here, we identify five cell cycle-related host proteins that interact with human and mouse SAMHD1, including three previously unknown cellular proteins (CDK2, cyclin B1, and SKP2). Our results demonstrate that several SAMHD1-interacting cellular proteins regulate phosphorylation of SAMHD1 and play an important role in HIV-1 restriction function. Our findings help define the role of these cellular interacting partners of SAMHD1 that regulate its HIV-1 restriction function.

APOBEC3 Multimerization Correlates with HIV-1 Packaging and Restriction Activity in Living Cells

APOBEC3G belongs to a family of DNA cytosine deaminases that are involved in the restriction of a broad number of retroviruses including human immunodeficiency virus type 1 (HIV-1). Prior studies have identified two distinct mechanistic steps in Vif-deficient HIV-1 restriction: packaging into virions and deaminating viral cDNA. APOBEC3A, for example, although highly active, is not packaged and is therefore not restrictive. APOBEC3G, on the other hand, although having weaker enzymatic activity, is packaged into virions and is strongly restrictive. Although a number of studies have described the propensity for APOBEC3 oligomerization, its relevance to HIV-1 restriction remains unclear. Here, we address this problem by examining APOBEC3 oligomerization in living cells using molecular brightness analysis. We find that APOBEC3G forms high-order multimers as a function of protein concentration. In contrast, APOBEC3A, APOBEC3C and APOBEC2 are monomers at all tested concentrations. Among other members of the APOBEC3 family, we show that the multimerization propensities of APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3H (haplotype II) bear more resemblance to APOBEC3G than to APOBEC3A/3C/2. Prior studies have shown that all of these multimerizing APOBEC3 proteins, but not the monomeric family members, have the capacity to package into HIV-1 particles and restrict viral infectivity. This correlation between oligomerization and restriction is further evidenced by two different APOBEC3G mutants, which are each compromised for multimerization, packaging and HIV-1 restriction. Overall, our results imply that multimerization of APOBEC3 proteins may be related to the packaging mechanism and ultimately to virus restriction.

Slower Uncoating Is Associated with Impaired Replicative Capability of Simian-Tropic HIV-1

Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees, but not Old World monkeys, such as rhesus and cynomolgus (CM) monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously generated a simian-tropic HIV-1 that replicates efficiently in CM cells. This virus encodes a capsid protein (CA) with SIVmac239-derived loops between α-helices 4 and 5 (L4/5) and between α-helices 6 and 7 (L6/7), along with the entire vif from SIVmac239 (NL-4/5S6/7SvifS). These SIVmac239-derived sequences were expected to protect the virus from HIV-1 restriction factors in monkey cells. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired. By long-term cultivation of human CEM-SS cells infected with NL-4/5S6/7SvifS, we succeeded in partially rescuing the impaired replicative capability of the virus in human cells. This adapted virus encoded a G-to-E substitution at the 116th position of the CA (NL-4/5SG116E6/7SvifS). In the work described here, we explored the mechanism by which the replicative capability of NL-4/5S6/7SvifS was impaired in human cells. Quantitative analysis (by real-time PCR) of viral DNA synthesis from infected cells revealed that NL-4/5S6/7SvifS had a major defect in nuclear entry. Mutations in CA are known to affect viral core stability and result in deleterious effects in HIV-1 infection; therefore, we measured the kinetics of uncoating of these viruses. The uncoating of NL-4/5S6/7SvifS was significantly slower than that of wild type HIV-1 (WT), whereas the uncoating of NL-4/5SG116E6/7SvifS was similar to that of WT. Our results suggested that the lower replicative capability of NL-4/5S6/7SvifS in human cells was, at least in part, due to the slower uncoating of this virus.

Breaking Barriers to an AIDS Model with Macaque-Tropic HIV-1 Derivatives.

The development of an animal model of human immunodeficiency virus type 1 (HIV-1)/AIDS that is suitable for preclinical testing of antiretroviral therapy, vaccines, curative strategies, and studies of pathogenesis has been hampered by the human-specific tropism of HIV-1. Although simian immunodeficiency virus (SIV) or HIV-1/SIV chimeric viruses (SHIVs)-rhesus macaque models are excellent surrogates for AIDS research, the genetic differences between SIV or SHIV and HIV-1 limit their utility as model systems. The identification of innate retroviral restriction factors has increased our understanding about blockades to HIV-1 replication in macaques and provided a guide for the construction of macaque-tropic HIV-1 clones. However, while these viruses replicate in macaque cells in vitro, they are easily controlled and have not caused AIDS in host animals, indicating that we may not fully understand the restrictive barriers of innate immunity. In this review, we discuss recent findings regarding HIV-1 restriction factors, particularly as they apply to cross-species transmission of primate lentiviruses and the development of a macaque model of HIV-1/AIDS.

Signals in APOBEC3F N-terminal and C-terminal Deaminase Domains Each Contribute to Encapsidation in HIV-1 Virions and Are Both Required for HIV-1 Restriction

Human cytidine deaminases APOBEC3F (A3F) and APOBEC3G (A3G) inhibit human immunodeficiency virus type-1 (HIV-1) replication. In the absence of HIV-1 Vif, A3F and/or A3G are incorporated into assembling virions and exert antiviral functions in subsequently infected target cells. Encapsidation of A3F or A3G within the protease-matured virion core following their incorporation into virions is hypothesized to be important for the antiviral function of these proteins. In this report, we demonstrated that A3F was quantitatively encapsidated in the mature virion core. In distinct contrast, A3G was distributed both within and outside of the virion core. Analysis of a series of A3F-A3G chimeras comprised of exchanged N- and C-terminal deaminase domains identified a 14 amino acid segment in the A3F C-terminal deaminase domain that contributed to preferential encapsidation and anti-HIV activity. Amino acid residue L306 in this C-terminal segment was determined to be necessary, but not sufficient, for these effects. Amino acid residue W126 in the N-terminal deaminase domain was determined also to contribute to preferential encapsidation and antiviral activity of A3F. Analysis of the A3F (W126A L306A) double mutant revealed that both residues are required for full anti-HIV function. The results reported here advance our understanding of the mechanisms of A3F virion encapsidation and antiviral function and may lead to innovative strategies to inhibit HIV-1 replication.

Oligomerization of HIV-1 Restriction Factor APOBEC3G Transforms it from a Fast Enzyme to a Slow Nucleic Acid Binding Protein

Human APOBEC3G (A3G) is a cellular protein that inhibits reverse transcription and replication of human immunodeficiency virus type-1 (HIV-1) in the absence of the viral protein Vif. A3G impairs viral replication by two different mechanisms, which both rely on its ability to bind single-stranded nucleic acids. First, A3G deaminates cytidine bases of viral single-stranded DNA (ssDNA). Secondly, A3G blocks DNA synthesis by reverse transcriptase (RT), the viral DNA polymerase, by a mechanism independent of catalytic activity. Seven A3G proteins are packaged per HIV-1 virion, requiring that each molecule rapidly locate deamination sites on viral ssDNA, which is a transient intermediate during reverse transcription. In contrast, the roadblock mechanism, a model in which A3G oligomerizes on the viral template strand and blocks RT-catalyzed DNA elongation, requires an extremely slow off-rate from single-stranded nucleic acids. We hypothesize that A3G exhibits fast binding kinetics as a dimer, enabling rapid deamination activity, and slow kinetics as an oligomer, preventing RT from elongating viral DNA. We use optical tweezers, in combination with fluorescence anisotropy and surface plasmon resonance, to quantify both types of binding kinetics. DNA stretching experiments reveal that the time constant for oligomerization, ranging from 200 to 1000 s, is inversely dependent on protein concentration. The apparent dissociation constant of A3G oligomerization decreases exponentially with ssDNA incubation time, dropping by an order of magnitude in 1000 s, which suggests that fast binding of catalytically active dimers converts to oligomerization on this timescale. This slow association and dissociation of A3G oligomers, which is consistent with ensemble methods, supports the roadblock hypothesis. Collectively, our measurements quantitatively characterize the complex, highly unusual nucleic acid binding kinetics of A3G responsible for its dual mechanism for inhibiting viral replication.

Vif hijacks CBF-β to degrade APOBEC3G and promote HIV-1 infection

Restriction factors, such as the retroviral complementary DNA deaminase APOBEC3G, are cellular proteins that dominantly block virus replication. The AIDS virus, human immunodeficiency virus type 1 (HIV-1), produces the accessory factor Vif, which counteracts the host's antiviral defence by hijacking a ubiquitin ligase complex, containing CUL5, ELOC, ELOB and a RING-box protein, and targeting APOBEC3G for degradation. Here we reveal, using an affinity tag/purification mass spectrometry approach, that Vif additionally recruits the transcription cofactor CBF-β to this ubiquitin ligase complex. CBF-β, which normally functions in concert with RUNX DNA binding proteins, allows the reconstitution of a recombinant six-protein assembly that elicits specific polyubiquitination activity with APOBEC3G, but not the related deaminase APOBEC3A. Using RNA knockdown and genetic complementation studies, we also demonstrate that CBF-β is required for Vif-mediated degradation of APOBEC3G and therefore for preserving HIV-1 infectivity. Finally, simian immunodeficiency virus (SIV) Vif also binds to and requires CBF-β to degrade rhesus macaque APOBEC3G, indicating functional conservation. Methods of disrupting the CBF-β-Vif interaction might enable HIV-1 restriction and provide a supplement to current antiviral therapies that primarily target viral proteins.