Human Immunodeficiency Virus Type 1 Particles Research Articles

HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C560-649) and examined the consequences on Env trafficking and incorporation into particles. FIP1C560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane.IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes trapped inside the cell within the endosomal recycling compartment. Intracellular trapping resulted in a loss of envelope protein on released particles and a corresponding loss of infectivity. Mutations of specific trafficking motifs in the envelope protein tail prevented its trapping in the recycling compartment. These results establish that trafficking to the endosomal recycling compartment is an essential step in HIV envelope protein particle incorporation.

Interferon-Inducible CD169/Siglec1 Attenuates Anti-HIV-1 Effects of Alpha Interferon.

A hallmark of human immunodeficiency virus type 1 (HIV-1) infection in vivo is chronic immune activation concomitant with type I interferon (IFN) production. Although type I IFN induces an antiviral state in many cell types, HIV-1 can replicate in vivo via mechanisms that have remained unclear. We have recently identified a type I IFN-inducible protein, CD169, as the HIV-1 attachment factor on dendritic cells (DCs) that can mediate robust infection of CD4+ T cells in trans Since CD169 expression on macrophages is also induced by type I IFN, we hypothesized that type I IFN-inducible CD169 could facilitate productive HIV-1 infection in myeloid cells in cis and CD4+ T cells in trans and thus offset antiviral effects of type I IFN. In support of this hypothesis, infection of HIV-1 or murine leukemia virus Env (MLV-Env)-pseudotyped HIV-1 particles was enhanced in IFN-α-treated THP-1 monocytoid cells, and this enhancement was primarily dependent on CD169-mediated enhancement at the virus entry step, a phenomenon phenocopied in HIV-1 infections of IFN-α-treated primary monocyte-derived macrophages (MDMs). Furthermore, expression of CD169, a marker of type I IFN-induced immune activation in vivo, was enhanced in lymph nodes from pigtailed macaques infected with simian immunodeficiency virus (SIV) carrying HIV-1 reverse transcriptase (RT-SHIV), compared to uninfected macaques, and interestingly, there was extensive colocalization of p27gag and CD169, suggesting productive infection of CD169+ myeloid cells in vivo While cell-free HIV-1 infection of IFN-α-treated CD4+ T cells was robustly decreased, initiation of infection in trans via coculture with CD169+ IFN-α-treated DCs restored infection, suggesting that HIV-1 exploits CD169 in cis and in trans to attenuate a type I IFN-induced antiviral state.IMPORTANCE HIV-1 infection in humans causes immune activation characterized by elevated levels of proinflammatory cytokines, including type I interferons (IFN). Although type I IFN induces an antiviral state in many cell types in vitro, HIV-1 can replicate in vivo via mechanisms that have remained unclear. In this study, we tested the hypothesis that CD169, a type I IFN-inducible HIV-1 attachment factor, offsets antiviral effects of type I IFN. Infection of HIV-1 was rescued in IFN-α-treated myeloid cells via upregulation of CD169 and a subsequent increase in CD169-dependent virus entry. Furthermore, extensive colocalization of viral Gag and CD169 was observed in lymph nodes of infected pigtailed macaques, suggesting productive infection of CD169+ cells in vivo Treatment of dendritic cell (DC)-T cell cocultures with IFN-α upregulated CD169 expression on DCs and rescued HIV-1 infection of CD4+ T cells in trans, suggesting that HIV-1 exploits CD169 to attenuate type I IFN-induced restrictions.

Rab3a-Bound CD63 Is Degraded and Rab3a-Free CD63 Is Incorporated into HIV-1 Particles.

CD63, a member of the tetraspanin family, is involved in virion production by human immunodeficiency virus type 1 (HIV-1), but its mechanism is unknown. In this study, we showed that a small GTP-binding protein, Rab3a, interacts with CD63. When Rab3a was exogenously expressed, the amounts of CD63 decreased in cells. The Rab3a-mediated reduction of CD63 was suppressed by lysosomal and proteasomal inhibitors. The amount of CD63 was increased by reducing the endogenous Rab3a level using a specific shRNA. These results indicate that Rab3a binds to CD63 to induce the degradation of CD63. Rab3a is thought to be involved in exocytosis, but we found that another function of Rab3a affects the fate of CD63 in lysosomes. CD63 interacted with Rab3a and was incorporated into HIV-1 particles. However, Rab3a was not detected in HIV-1 virions, thereby indicating that Rab3a-free CD63, but not Rab3a-bound CD63, is incorporated into HIV-1 particles. Overexpression or silencing of Rab3a moderately reduced HIV-1 virion formation. Overexpression of Rab3a decreased CD63 levels, but did not affect the incorporation of CD63 into HIV-1 particles. This study showed that Rab3a binds to CD63 to induce the degradation of CD63, and only Rab3a-free CD63 is incorporated into HIV-1 particles.

Human immunodeficiency virus type-1 induces a regulatory B cell-like phenotype in vitro.

Individuals infected with human immunodeficiency virus type-1 (HIV-1) usually show a general dysregulation and hyper-activation of the immune system. A direct influence of HIV-1 particles on B-cell phenotypes and functions has been previously described. However, the consequences of B-cell dysregulation are still poorly understood. We evaluated the phenotypic changes in primary B cells after direct contact with HIV-1 particles in comparison with different types of stimuli. The functionality of treated B cells was challenged in co-culture experiments with autologous CD4+ and CD8+ T cells. We demonstrated that HIV-1 induces a phenotypic change in B cells towards a regulatory B-cell phenotype, showing a higher level of IL-10, TGF-β1, EBI3 or IL-12(p35) mRNA expression and acquiring an immunosuppressive profile. The acquisition of a Breg phenotype was confirmed by co-culture experiments where HIV-treated B cells reduced the proliferation and the TNFα production of CD4+ or CD8+ T cells. This suppressive ability of HIV-treated B cells was dependent on cell-to-cell contact between these B cells and effector cells. To our knowledge, these data provide the first evidence that HIV-1 can directly induce a regulatory B cell-like immunosuppressive phenotype, which could have the ability to impair specific immune responses. This dysregulation could constitute one of the mechanisms underlying unsuccessful efforts to develop an efficient vaccine against HIV-1.

Inhibition of HIV-1 Maturation via Small-Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein.

The human immunodeficiency virus type 1 (HIV-1) capsid protein is an attractive therapeutic target, owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsids to HIV-1 infectivity. To date, small-molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here, we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, Boehringer-Ingelheim compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant cross-links in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle.IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here, we show that one such compound, compound 1, interferes with assembly of the conical viral capsid during virion maturation and results in perturbations at a specific protein-protein interface in the capsid lattice. We also identify and characterize a mutation in the capsid protein that confers resistance to the inhibitor. This study reveals a novel mechanism by which a capsid-targeting small molecule can inhibit HIV-1 replication.

Different Expression of Interferon-Stimulated Genes in Response to HIV-1 Infection in Dendritic Cells Based on Their Maturation State.

Dendritic cells (DCs) are professional antigen-presenting cells whose functions are dependent on their degree of differentiation. In their immature state, DCs capture pathogens and migrate to the lymph nodes. During this process, DCs become resident mature cells specialized in antigen presentation. DCs are characterized by a highly limiting environment for human immunodeficiency virus type 1 (HIV-1) replication due to the expression of restriction factors such as SAMHD1 and APOBEC3G. However, uninfected DCs capture and transfer viral particles to CD4 lymphocytes through a trans-enhancement mechanism in which chemokines are involved. We analyzed changes in gene expression with whole-genome microarrays when immature DCs (IDCs) or mature DCs (MDCs) were productively infected using Vpx-loaded HIV-1 particles. Whereas productive HIV infection of IDCs induced expression of interferon-stimulated genes (ISGs), such induction was not produced in MDCs, in which a sharp decrease in ISG- and CXCR3-binding chemokines was observed, lessening trans-infection of CD4 lymphocytes. Similar patterns of gene expression were found when DCs were infected with HIV-2 that naturally expresses Vpx. Differences were also observed under conditions of restrictive HIV-1 infection, in the absence of Vpx. ISG expression was not modified in IDCs, whereas an increase of ISG- and CXCR3-binding chemokines was observed in MDCs. Overall these results suggest that sensing and restriction of HIV-1 infection are different in IDCs and MDCs. We propose that restrictive infection results in increased virulence through different mechanisms. In IDCs avoidance of sensing and induction of ISGs, whereas in MDCs increased production of CXCR3-binding chemokines, would result in lymphocyte attraction and enhanced infection at the immune synapse.IMPORTANCE In this work we describe for the first time the activation of a different genetic program during HIV-1 infection depending on the state of maturation of DCs. This represents a breakthrough in the understanding of the restriction to HIV-1 infection of DCs. The results show that infection of DCs by HIV-1 reprograms their gene expression pattern. In immature cells, productive HIV-1 infection activates interferon-related genes involved in the control of viral replication, thus inducing an antiviral state in surrounding cells. Paradoxically, restriction of HIV-1 by SAMHD1 would result in lack of sensing and IFN activation, thus favoring initial HIV-1 escape from the innate immune response. In mature DCs, restrictive infection results in HIV-1 sensing and induction of ISGs, in particular CXCR3-binding chemokines, which could favor the transmission of HIV to lymphocytes. Our data support the hypothesis that genetic DC reprograming by HIV-1 infection favors viral escape and dissemination, thus increasing HIV-1 virulence.

HIV-1 gp41-targeting fusion inhibitory peptides enhance the gp120-targeting protein-mediated inactivation of HIV-1 virions

Protein- or peptide-based viral inactivators are being developed as novel antiviral drugs with improved efficacy, pharmacokinetics and toxicity profiles because they actively inactivate cell-free human immunodeficiency virus type 1 (HIV-1) virions before attachment to host cells. By contrast, most clinically used antiviral drugs must penetrate host cells to inhibit viral replication. In this study, we pre-treated HIV-1 particles with a gp120-targeting bispecific multivalent protein, 2Dm2m or 4Dm2m, in the presence or absence of the gp41-targeting HIV-1 fusion inhibitory peptides enfuvirtide (T20), T2635, or sifuvirtide (SFT). HIV-1 virions were separated from the inhibitors using PEG-6000, followed by testing of the residual infectivity of the HIV-1 virions. 2Dm2m and 4Dm2m exhibited significant inactivation activity against all HIV-1 strains tested with EC50 values at the low nanomolar level, whereas none of the gp41-targeting peptides showed inactivation activity at concentrations up to 250 nM. Notably, these three peptides significantly enhanced protein-mediated inactivation against cell-free HIV-1 virions, including HIV-1 laboratory-adapted and primary HIV-1 strains, as well as those resistant to T20 or T2635 and virions released from reactivated latently HIV-1-infected cells. These results indicate that the gp120-targeting bispecific multivalent proteins 2Dm2m and 4Dm2m have potential for further development as HIV-1 inactivator-based antiviral drugs for use in the clinic, either alone or in combination with a gp41-targeting HIV-1 fusion inhibitor such as T20, to treat patients with HIV-1 infection and AIDS.Emerging Microbes & Infections (2017) 6, e59; doi:10.1038/emi.2017.46; published online 21 June 2017

Human Immunodeficiency Virus-1 Impairs IFN-Alpha Production Induced by TLR-7 Agonist in Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) play an important role in innate immune response against viruses, mainly through interferon-α (IFN-α) secretion. Impaired IFN-α secretion has been observed in patients with acute human immunodeficiency virus type 1 (HIV-1) infection and the reasons for this impairment are still obscure. To know the grounds behind this situation, HIV-1 viral copy numbers similar to those found in primary HIV-1 infection were used to stimulate peripheral blood mononuclear cells (PBMCs) and pDCs in this study. Intracellular IFN-α production was seen as early as 2 h in pDCs with TLR-7 agonist (imiquimod) stimulation, but HIV-1 required 48 h to induce secretion of IFN-α in supernatants and it was 10 times less compared to imiquimod. Thus, it shows that HIV-1 delays and impairs IFN-α production from pDCs. Furthermore, the IFN-α inhibitory activity of HIV-1 was checked by stimulating PBMCs and pDCs with imiquimod either simultaneously with HIV-1 or after 2 h pre-exposure to HIV-1. Pre-exposure to HIV-1 resulted in significant reduction in IFN-α secretion by pDCs and PBMCs when compared to imiquimod alone. In addition, simultaneous stimulation of these populations with HIV-1 and imiquimod resulted in significant impairment in IFN-α production in pDCs but not in PBMCs. HIV-1 not only fails to induce IFN-α in adequate quantities but also inhibits IFN-α secretary capacity of pDCs. HIV-1 particles were found to bind CD303 receptor on pDC surface probably blocking initiation of cascade leading to IFN-α impairment. The understanding of the pathways that lead to this suppression may help in devising the HIV control strategies.

The C-terminal domain of glyceraldehyde 3-phosphate dehydrogenase plays an important role in suppression of tRNALys3 packaging into human immunodeficiency virus type-1 particles

Human immunodeficiency virus type-1 (HIV-1) requires the packaging of human tRNALys3 as a primer for effective viral reverse transcription. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) suppresses the packaging efficiency of tRNALys3. Although the binding of GAPDH to Pr55gag is important for the suppression mechanism, it remains unclear which domain of GAPDH is responsible for the interaction with Pr55gag. In this study, we show that Asp256, Lys260, Lys263 and Glu267 of GAPDH are important for the suppression of tRNALys3 packaging. Yeast two-hybrid analysis demonstrated that the C-terminal domain of GAPDH (151–335) interacts with both the matrix region (MA; 1–132) and capsid N-terminal domain (CA-NTD; 133–282). The D256R, K263E or E267R mutation of GAPDH led to the loss of the ability to bind to wild-type (WT) MA, and the D256R/K260E double mutation of GAPDH resulted in the loss of detectable binding activity to WT CA-NTD. In contrast, R58E, Q59A or Q63A of MA, and E76R or R82E of CA-NTD abrogated the interaction with the C-terminal domain of GAPDH. Multiple-substituted GAPDH mutant (D256R/K260E/K263E/E267R) retained the oligomeric formation with WT GAPDH in HIV-1 producing cells, but the incorporation level of the hetero-oligomer was decreased in viral particles. Furthermore, the viruses produced from cells expressing the D256R/K260E/K263E/E267R mutant restored tRNALys3 packaging efficiency because the mutant exerted a dominant negative effect by preventing WT GAPDH from binding to MA and CA-NTD and improved the reverse transcription. These findings indicate that the amino acids Asp256, Lys260, Lys263 and Glu267 of GAPDH is essential for the mechanism of tRNALys3-packaging suppression and the D256R/K260E/K263E/E267R mutant of GAPDH acts in a dominant negative manner to suppress tRNALys3 packaging.

Functional organization of the HIV lipid envelope.

The chemical composition of the human immunodeficiency virus type 1 (HIV-1) membrane is critical for fusion and entry into target cells, suggesting that preservation of a functional lipid bilayer organization may be required for efficient infection. HIV-1 acquires its envelope from the host cell plasma membrane at sites enriched in raft-type lipids. Furthermore, infectious particles display aminophospholipids on their surface, indicative of dissipation of the inter-leaflet lipid asymmetry metabolically generated at cellular membranes. By combining two-photon excited Laurdan fluorescence imaging and atomic force microscopy, we have obtained unprecedented insights into the phase state of membranes reconstituted from viral lipids (i.e., extracted from infectious HIV-1 particles), established the role played by the different specimens in the mixtures, and characterized the effects of membrane-active virucidal agents on membrane organization. In determining the molecular basis underlying lipid packing and lateral heterogeneity of the HIV-1 membrane, our results may help develop compounds with antiviral activity acting by perturbing the functional organization of the lipid envelope.

Glycosylphosphatidylinositol Anchor Deficiency Attenuates the Production of Infectious HIV-1 and Renders Virions Sensitive to Complement Attack.

Human immunodeficiency virus type 1 (HIV-1) escapes complement-mediated lysis (CML) by incorporating host regulators of complement activation (RCA) into its envelope. CD59, a key member of RCA, is incorporated into HIV-1 virions at levels that protect against CML. Since CD59 is a glycosylphosphatidylinositol-anchored protein (GPI-AP), we used GPI anchor-deficient Jurkat cells (Jurkat-7) that express intracellular CD59, but not surface CD59, to study the molecular mechanisms underlying CD59 incorporation into HIV-1 virions and the role of host proteins in virus replication. Compared to Jurkat cells, Jurkat-7 cells were less supportive to HIV-1 replication and more sensitive to CML. Jurkat-7 cells exhibited similar capacities of HIV-1 binding and entry to Jurkat cells, but were less supportive to viral RNA and DNA biosynthesis as infected Jurkat-7 cells produced reduced amounts of HIV-1 RNA and DNA. HIV-1 virions produced from Jurkat-7 cells were CD59 negative, suggesting that viral particles acquire CD59, and probably other host proteins, from the cell membrane rather than intracellular compartments. As a result, CD59-negative virions were sensitive to CML. Strikingly, these virions exhibited reduced activity of virus binding and were less infectious, implicating that GPI-APs may be also important in ensuring the integrity of HIV-1 particles. Transient expression of the PIG-A gene restored CD59 expression on the surface of Jurkat-7 cells. After HIV-1 infection, the restored CD59 was colocalized with viral envelope glycoprotein gp120/gp41 within lipid rafts, which is identical to that on infected Jurkat cells. Thus, HIV-1 virions acquire RCA from the cell surface, likely lipid rafts, to escape CML and ensure viral infectivity.

Promiscuous RNA binding ensures effective encapsidation of APOBEC3 proteins by HIV-1.

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G) and APOBEC3F (A3F), act as potent human immunodeficiency virus type-1 (HIV-1) restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC) region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA) and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC) fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.

Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture.

CD169-dependent cell-associated HIV-1 transmission: a driver of virus dissemination.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) occurs across mucosal surfaces of the genital and gastrointestinal tracts and accounts for the vast majority of newly acquired infections worldwide. In the absence of an effective vaccine, interventional strategies such as microbicides that target viral attachment and entry into mucosa-resident target cells are particularly attractive and might have the greatest impact on reducing the HIV-1 pandemic. Rational development of microbicides would be greatly aided with a better understanding of several key questions of mucosal HIV-1 transmission, including the molecular mechanism(s) of how HIV-1 traverses mucosal barriers, the type of cells that it initially infects to gain a foothold in the naive host, and how it is disseminated from local sites of infection to draining lymph nodes. In this review, we discuss the role of myeloid dendritic cells (DCs) in cell-associated HIV-1 transmission and in facilitating systemic HIV-1 dissemination. We will evaluate the role of CD169 as a DC-associated HIV-1 attachment factor, investigate the molecular mechanisms by which HIV-1 particles are transferred from DCs to CD4(+) T cells across virological synapses, and provide arguments for inclusion of molecules in microbicides that can effectively target HIV-1 attachment to DCs and DC-mediated virus transfer.

Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopy.

Human immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells. Importance: HIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.

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.

Induction of heat-shock protein 70 by prostaglandin A1 inhibits HIV-1 Vif-mediated degradation of APOBEC3G

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. This antiviral activity has been associated with the induction of heat-shock protein 70 (HSP70) in infected cells. We investigated a new role of prostaglandin A1 (PGA1) in the replication of HIV-1 in non-permissive cells. Because overexpression of HSP70 blocks the viral infectivity factor (Vif)-mediated degradation of APOBEC3G (A3G) via the ubiquitin–proteasome pathway, we examined the effects of PGA1 on A3G and HIV-1 replication. The induction of HSP70 synthesis by PGA1 blocked Vif-mediated A3G degradation and enhanced the incorporation of A3G into both wild-type and Vif-deficient viruses. Furthermore, we determined the viral titer of HIV-1 particles produced from PGA1-treated 293T cells. The induction of HSP70 synthesis by PGA1 significantly reduced the viral titer in the presence of A3G. Additionally, the p24 Gag antigen levels were dramatically reduced in non-permissive cells treated once or repeatedly with PGA1. Thus, we showed that PGA1 inhibits HIV-1 replication, at least in part, by blocking Vif-mediated A3G degradation.

Interferon-Inducible Mechanism of Dendritic Cell-Mediated HIV-1 Dissemination Is Dependent on Siglec-1/CD169

Human immunodeficiency virus type 1 (HIV-1) interactions with myeloid dendritic cells (DCs) can result in virus dissemination to CD4+ T cells via a trans infection pathway dependent on virion incorporation of the host cell derived glycosphingolipid (GSL), GM3. The mechanism of DC-mediated trans infection is extremely efficacious and can result in infection of multiple CD4+ T cells as these cells make exploratory contacts on the DC surface. While it has long been appreciated that activation of DCs with ligands that induce type I IFN signaling pathway dramatically enhances DC-mediated T cell trans infection, the mechanism by which this occurs has remained unclear until now. Here, we demonstrate that the type I IFN-inducible Siglec-1, CD169, is the DC receptor that captures HIV in a GM3-dependent manner. Selective downregulation of CD169 expression, neutralizing CD169 function, or depletion of GSLs from virions, abrogated DC-mediated HIV-1 capture and trans infection, while exogenous expression of CD169 in receptor-naïve cells rescued GSL-dependent capture and trans infection. HIV-1 particles co-localized with CD169 on DC surface immediately following capture and subsequently within non-lysosomal compartments that redistributed to the DC – T cell infectious synapses upon initiation of T cell contact. Together, these findings describe a novel mechanism of pathogen parasitization of host encoded cellular recognition machinery (GM3 – CD169 interaction) for DC-dependent HIV dissemination.

Reevaluation of the Requirement for TIP47 in Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Incorporation

Incorporation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins into assembling particles is crucial for virion infectivity. Genetic and biochemical data indicate that the matrix (MA) domain of Gag and the cytoplasmic tail of the transmembrane glycoprotein gp41 play an important role in coordinating Env incorporation; however, the molecular mechanism and possible role of host factors in this process remain to be defined. Recent studies suggested that Env incorporation is mediated by interactions between matrix and tail-interacting protein of 47 kDa (TIP47; also known as perilipin-3 and mannose-6-phosphate receptor-binding protein 1), a member of the perilipin, adipophilin, TIP47 (PAT) family of proteins implicated in protein sorting and lipid droplet biogenesis. We have confirmed by nuclear magnetic resonance spectroscopy titration experiments and surface plasmon resonance that MA binds TIP47. We also reevaluated the role of TIP47 in HIV-1 Env incorporation in HeLa cells and in the Jurkat T-cell line. In HeLa cells, TIP47 overexpression or RNA interference (RNAi)-mediated depletion had no significant effect on HIV-1 Env incorporation, virus release, or particle infectivity. Similarly, depletion of TIP47 in Jurkat cells did not impair HIV-1 Env incorporation, virus release, infectivity, or replication. Our results thus do not support a role for TIP47 in HIV-1 Env incorporation or virion infectivity.

Comparative Proteomic Analysis of HIV-1 Particles Reveals a Role for Ezrin and EHD4 in the Nef-Dependent Increase of Virus Infectivity

Nef is a human immunodeficiency virus type 1 (HIV-1) auxiliary protein that plays an important role in virus replication and the onset of acquired immunodeficiency. Although known functions of Nef might explain its contribution to HIV-1-associated pathogenesis, how Nef increases virus infectivity is still an open question. In vitro, Nef-deleted viruses have a defect that prevents efficient completion of early steps of replication. We have previously shown that this restriction is not due to the absence of Nef in viral particles. Rather, a loss of function in virus-producing cells accounts for the lower infectivity of nef-deleted viruses compared to wild-type (WT) viruses. Here we used DiGE and iTRAQ to identify differences between the proteomes of WT and nef-deleted viruses. We observe that glucosidase II is enriched in WT virions, whereas Ezrin, ALG-2, CD81, and EHD4 are enriched in nef-deleted virions. Functional analysis shows that glucosidase II, ALG-2, and CD81 have no or only Nef-independent effect on infectivity. In contrast, Ezrin and EHD4 are involved in the ability of Nef to increase virus infectivity (referred to thereafter as Nef potency). Indeed, simultaneous Ezrin and EHD4 depletion in SupT1 and 293T virus-producing cells result in an ∼30 and ∼70% decrease of Nef potency, respectively. Finally, while Ezrin behaves as an inhibitory factor counteracted by Nef, EHD4 should be considered as a cofactors required by Nef to increase virus infectivity.