Relative resistance of patient-derived envelope sequences to SERINC5-mediated restriction of HIV-1 infectivity.

IMPORTANCE: Pathogenesis of HIV-1 is enhanced through several viral-encoded proteins that counteract a range of host restriction molecules. HIV-1 Nef counteracts the cell membrane protein SERINC5 by downregulating it from the cell surface, thereby enhancing virion infectivity. Some subtype B reference Envelope sequences have shown the ability to bypass SERINC5 infectivity restriction independent of Nef. However, it is not clear if and to what extent circulating HIV-1 strains can exhibit resistance to SERINC5 restriction. Using a panel of Envelope sequences isolated from 50 Tanzanians infected with non-B HIV-1 subtypes, we show that the lentiviral reporters pseudotyped with patient-derived Envelopes have reduced sensitivity to SERINC5 and that this sensitivity differed among viral subtypes. Moreover, we found that SERINC5 sensitivity within patient-derived Envelopes can be modulated by separate regions, highlighting the complexity of viral/host interactions.

HIV-1 subtype C Nef-mediated SERINC5 down-regulation significantly contributes to overall Nef activity.

BACKGROUND: Nef performs multiple cellular activities that enhance HIV-1 pathogenesis. The role of Nef-mediated down-regulation of the host restriction factor SERINC5 in HIV-1 pathogenesis is not well-defined. We aimed to investigate if SERINC5 down-regulation activity contributes to HIV-1 subtype C disease progression, to assess the relative contribution of this activity to overall Nef function, and to identify amino acids required for optimal activity. We measured the SERINC5 down-regulation activity of 106 subtype C Nef clones, isolated from individuals in early infection, for which the Nef activities of CD4 and HLA-I down-regulation as well as alteration of TCR signalling were previously measured. The relationship between SERINC5 down-regulation and markers of disease progression, and the relative contribution of SERINC5 down-regulation to a Nef fitness model-derived E value (a proxy for overall Nef fitness in vivo), were assessed. RESULTS: No overall relationship was found between SERINC5 down-regulation and viral load set point (p = 0.28) or rate of CD4+ T cell decline (p = 0.45). CD4 down-regulation (p = 0.02) and SERINC5 down-regulation (p = 0.003) were significant determinants of E values in univariate analyses, with the greatest relative contribution for SERINC5 down-regulation, and only SERINC5 down-regulation remained significant in the multivariate analysis (p = 0.003). Using a codon-by-codon analysis, several amino acids were significantly associated with increased (10I, 11V, 38D, 51T, 65D, 101V, 188H and, 191H) or decreased (10K, 38E, 65E, 135F, 173T, 176T and, 191R) SERINC5 down-regulation activity. Site-directed mutagenesis experiments of selected mutants confirmed a substantial reduction in SERINC5 down-regulation activity associated with the mutation 173T, while mutations 10K, 135F, and 176T were associated with more modest reductions in activity that were not statistically significant. CONCLUSIONS: These results suggest that SERINC5 down-regulation is a significant contributor to overall Nef function and identify potential genetic determinants of this Nef function that may have relevance for vaccines or therapeutics.

Substitutions in Nef That Uncouple Tetherin and SERINC5 Antagonism Impair Simian Immunodeficiency Virus Replication in Primary Rhesus Macaque Lymphocytes.

Most simian immunodeficiency viruses (SIVs) use Nef to counteract restriction by the tetherin proteins of their nonhuman primate hosts. In addition to counteracting tetherin, SIV Nef has a number of other functions, including the downmodulation of CD3, CD4, and major histocompatibility complex class I (MHC I) molecules from the surface of SIV-infected cells and the enhancement of viral infectivity by preventing the incorporation of SERINC5 into virions. Although these activities require different surfaces of Nef, they can be difficult to separate because of their dependence on similar interactions with AP-1 or AP-2 for clathrin-mediated endocytosis. We previously observed extensive overlap of the SIV Nef residues required for counteracting tetherin and SERINC5. Here, we define substitutions in Nef that separate anti-tetherin activity from SERINC5 antagonism and other activities of Nef. This information was used to engineer an infectious molecular clone of SIV (SIVmac239nefSA) that is sensitive to tetherin but retains CD3, CD4, MHC I, and SERINC5 downmodulation. In primary rhesus macaque CD4+ T cells, SIVmac239nefSA exhibits impaired replication compared to wild-type SIVmac239 under conditions of interferon-induced upregulation of tetherin. These results demonstrate that tetherin antagonism can be separated from other Nef functions and that resistance to tetherin is essential for optimal replication in primary CD4+ T cells. IMPORTANCE Tetherin is an interferon-inducible transmembrane protein that prevents the detachment of enveloped viruses from infected cells by physically tethering nascent virions to cellular membranes. SIV Nef downmodulates simian tetherin to overcome this restriction in nonhuman primate hosts. Nef also enhances virus infectivity by preventing the incorporation of SERINC5 into virions and contributes to immune evasion by downmodulating other proteins from the cell surface. To assess the contribution of tetherin antagonism to virus replication, we engineered an infectious molecular clone of SIV with substitutions in Nef that uncouple tetherin antagonism from other Nef functions. These substitutions impaired virus replication in interferon-treated macaque CD4+ T cells, revealing the impact of tetherin on SIV replication under physiological conditions in primary CD4+ lymphocytes.

HIV-1 restriction by SERINC5.

Serine incorporator 5 (SERINC5 or SER5) is a multipass transmembrane protein with ill-defined cellular activities. SER5 was recently described as a human immunodeficiency virus 1 (HIV-1) restriction factor capable of inhibiting HIV-1 that does not express its accessory protein Nef (Δ Nef). SER5 incorporated into the viral membrane impairs the entry of HIV-1 by disrupting the fusion between the viral and the plasma membrane after envelope receptor interaction induced the first steps of the fusion process. The mechanisms of how SER5 prevents membrane fusion are not fully understood and viral envelope proteins were identified that escape the SER5-mediated restriction. Primate lentiviruses, such as HIV-1 and simian immunodeficiency viruses (SIVs), use their accessory protein Nef to downregulate SER5 from the plasma membrane by inducing an endocytic pathway. In addition to being directly antiviral, recent data suggest that SER5 is an important adapter protein in innate signaling pathways leading to the induction of inflammatory cytokines. This review discusses the current knowledge about HIV-1 restriction by SER5.

HIV-1 Nef hijacks both exocytic and endocytic pathways of host intracellular trafficking through differential regulation of Rab GTPases.

BACKGROUND: HIV-1 Nef regulates several cellular functions in an infected cell which results in viral persistence and AIDS pathogenesis. The currently understood molecular mechanism(s) underlying Nef-dependent cellular function(s) are unable to explain how events are coordinately regulated in the host cell. Intracellular membranous trafficking maintains cellular homeostasis and is regulated by Rab GTPases - a member of the Ras superfamily. RESULTS: In the current study, we tried to decipher the role of Nef on the Rab GTPases-dependent complex and vesicular trafficking. Expression profiling of Rabs in Nef-expressing cells showed that Nef differentially regulates the expression of individual Rabs in a cell-specific manner. Further analysis of Rabs in HIV-1NL4-3 or ΔNef infected cells demonstrated that the Nef protein is responsible for variation in Rabs expression. Using a panel of competitive peptide inhibitors against Nef, we identified the critical domain of HIV-1 Nef involved in modulation of Rabs expression. The molecular function of Nef-mediated upregulation of Rab5 and Rab7 and downregulation of Rab11 increased the transport of SERINC5 from the cell surface to the lysosomal compartment. Moreover, the Nef-dependent increase in Rab27 expression assists exosome release. Reversal of Rabs expression using competitive inhibitors against Nef and manipulation of Rabs expression reduced viral release and infectivity of progeny virions. CONCLUSION: This study demonstrates that Nef differentially regulates the expression of Rab proteins in HIV-1 infected cells to hijack the host intracellular trafficking, which augments viral replication and HIV-1 pathogenesis. SIGNIFICANCE: Our study emphasized the indispensable role of HIV-1 protein Nef on various aspects of the intracellular trafficking regulated by Rabs GTPases, which explained how HIV-1 Nef may hijack membrane trafficking pathways in infected cells.

Aromatic Side Chain at Position 412 of SERINC5 Exerts Restriction Activity toward HIV-1 and Other Retroviruses.

The host transmembrane protein SERINC5 is incorporated into viral particles and restricts infection by certain retroviruses. However, what motif of SERINC5 mediates this process remains elusive. By conducting mutagenesis analyses, we found that the substitution of phenylalanine with alanine at position 412 (F412A) resulted in a >75-fold reduction in SERINC5's restriction function. The F412A substitution also resulted in the loss of SERINC5's function to sensitize HIV-1 neutralization by antibodies recognizing the envelope's membrane proximal region. A series of biochemical analyses revealed that F412A showed steady-state protein expression, localization at the cellular membrane, and incorporation into secreted virus particles to a greater extent than in the wild type. Furthermore, introduction of several amino acid mutations at this position revealed that the aromatic side chains, including phenylalanine, tyrosine, and tryptophan, were required to maintain SERINC5 functions to impair the virus-cell fusion process and virion infectivity. Moreover, the wild-type SERINC5 restricted infection of lentiviruses pseudotyped with envelopes of murine leukemia viruses, simian immunodeficiency virus, and HIV-2, and F412A abrogated this function. Taken together, our results highlight the importance of the aromatic side chain at SERINC5 position 412 to maintain its restriction function against diverse retrovirus envelopes. IMPORTANCE The host protein SERINC5 is incorporated into progeny virions of certain retroviruses and restricts the infectivity of these viruses or sensitizes the envelope glycoprotein to a class of neutralizing antibodies. However, how and which part of SERINC5 engages with the diverse array of retroviral envelopes and exerts its antiretroviral functions remain elusive. During mutagenesis analyses, we eventually found that the single substitution of phenylalanine with alanine, but not with tyrosine or tryptophan, at position 412 (F412A) resulted in the loss of SERINC5's functions toward diverse retroviruses, whereas F412A showed steady-state protein expression, localization at the cellular membrane, and incorporation into progeny virions to a greater extent than the wild type. Results suggest that the aromatic side chain at position 412 of SERINC5 plays a critical role in mediating antiviral functions toward various retroviruses, thus providing additional important information regarding host and retrovirus interaction.

An Amino Acid Polymorphism within the HIV-1 Nef Dileucine Motif Functionally Uncouples Cell Surface CD4 and SERINC5 Downregulation.

Serine incorporator 5 (SERINC5) reduces the infectivity of progeny HIV-1 virions by incorporating into the outer host-derived viral membrane during egress. To counter SERINC5, the HIV-1 accessory protein Nef triggers SERINC5 internalization by engaging the adaptor protein 2 (AP-2) complex using the [D/E]xxxL[L/I]167 Nef dileucine motif. Nef also engages AP-2 via its dileucine motif to downregulate the CD4 receptor. Although these two Nef functions are related, the mechanisms governing SERINC5 downregulation are incompletely understood. Here, we demonstrate that two primary Nef isolates, referred to as 2410 and 2391 Nef, acquired from acutely HIV-1 infected women from Zimbabwe, both downregulate CD4 from the cell surface. However, only 2410 Nef retains the ability to downregulate cell surface SERINC5. Using a series of Nef chimeras, we mapped the region of 2391 Nef responsible for the functional uncoupling of these two antagonistic pathways to the dileucine motif. Modifications of the first and second x positions of the 2410 Nef dileucine motif to asparagine and aspartic acid residues, respectively (ND164), impaired cell surface SERINC5 downregulation, which resulted in reduced infectious virus yield in the presence of SERINC5. The ND164 mutation additionally partially impaired, but did not completely abrogate, Nef-mediated cell surface CD4 downregulation. Furthermore, the patient infected with HIV-1 encoding 2391 Nef had stable CD4+ T cell counts, whereas infection with HIV-1 encoding 2410 Nef resulted in CD4+ T cell decline and disease progression. IMPORTANCE A contributing factor to HIV-1 persistence is evasion of the host immune response. HIV-1 uses the Nef accessory protein to evade the antiviral roles of the adaptive and intrinsic innate immune responses. Nef targets SERINC5, a restriction factor which potently impairs HIV-1 infection by triggering SERINC5 removal from the cell surface. The molecular determinants underlying this Nef function remain incompletely understood. Recent studies have found a correlation between the extent of Nef-mediated SERINC5 downregulation and the rate of disease progression. Furthermore, single-residue polymorphisms outside the known Nef functional motifs can modulate SERINC5 downregulation. The identification of a naturally occurring Nef polymorphism impairing SERINC5 downregulation in this study supports a link between Nef downregulation of SERINC5 and the rate of plasma CD4+ T cell decline. Moreover, the observed functional impairments of this polymorphism could provide clues to further elucidate unknown aspects of the SERINC5 antagonistic pathway via Nef.

SERINC5 Can Enhance Proinflammatory Cytokine Production by Primary Human Myeloid Cells in Response to Challenge with HIV-1 Particles.

HIV-1 has to overcome physical barriers posed by host cell restriction factors (RFs) for efficient replication. Some RFs, including Trim5α and tetherin, trigger antiviral signaling in addition to directly impairing HIV replication. SERINC5 (S5) is an RF that is incorporated into HIV-1 particles to potently impair their infectivity and is efficiently antagonized by the viral pathogenesis factor Nef. Since effects of S5 on HIV-1 infectivity were mostly studied in reporter cell lines, we analyzed the effects of S5 during infection of primary HIV-1 target cells. In activated CD4+ T lymphocytes, virion incorporation of S5 only moderately impaired virion infectivity and was not associated with altered innate immune recognition. In contrast, in monocyte-derived macrophages, S5 virion incorporation potentiated the production of proinflammatory cytokines with very potent but donor-dependent effects on virion infectivity. Nef counteracted effects of S5 on both cytokine production and virion infectivity. Similar S5-induced cytokine production was observed in immature monocyte-derived dendritic cells. Notably, S5-mediated enhancement of cytokine production was not linked to the efficacy of productive infection and could be overcome by using vesicular stomatitis virus glycoprotein (VSV-G) but not infectivity restriction-insensitive HIV-1 Env for cell entry. Moreover, inhibiting entry of S5-negative HIV-1 ΔNef particles increased proinflammatory cytokine production comparably to virion incorporation of S5. Together, these results describe the sensitization of noninfectious HIV-1 particles to proinflammatory cytokine production by myeloid target cells as an additional and Nef-sensitive activity of S5. Moreover, the study reveals important cell-type and donor-dependent differences in the sensitivity of HIV target cells for antiviral effects of S5.IMPORTANCE SERINC5 (S5) is a host cell restriction factor (RF) that impairs the infectivity of HIV-1 particles in target cell lines. To assess the potential physiological relevance of this restriction, we assessed the effects of S5 on HIV-1 infection of relevant primary human target cells. We found that effects of S5 on infection of CD4+ T lymphocytes were negligible. In myeloid target cells, however, virion incorporation of S5 potently suppressed infectivity and promoted innate immune recognition of HIV-1 particles characterized by proinflammatory cytokine production. Both effects were not observed in cells of all donors analyzed, were exerted independently of one another, and were counteracted by the HIV-1 pathogenesis factor Nef. These results identify the sensitization of HIV-1 particles for innate immune recognition by myeloid target cells as a novel activity of S5 and emphasize the need to study RF function in the context of primary target cells and taking donor variabilities into account.

Nef inhibits HIV transcription and gene expression in astrocytes and HIV transmission from astrocytes to CD4+ T cells.

HIV infects astrocytes in a restricted manner but leads to abundant expression of Nef, a major viral factor for HIV replication and disease progression. However, the roles of Nef in HIV gene expression and replication in astrocytes and viral transfer from astrocytes to CD4+ T cells remain largely unclear. In this study, we attempted to address these issues by transfecting human primary astrocytes with HIV molecular clones with intact Nef and without Nef (a nonsense Nef mutant) and comparing gene expression and replication in astrocytes and viral transfer from astrocytes to CD4+ T cells MT4. First, we found that lack of Nef expression led to increased extracellular virus production from astrocytes and intracellular viral protein and RNA expression in astrocytes. Using a HIV LTR-driven luciferase reporter gene assay, we showed that ectopic Nef expression alone inhibited the HIV LTR promoter activity in astrocytes. Consistent with the previously established function of Nef, we showed that the infectivity of HIV derived from astrocytes with Nef expression was significantly higher than that with no Nef expression. Next, we performed the co-culture assay to determine HIV transfer from astrocytes transfected to MT4. We showed that lack of Nef expression led to significant increase in HIV transfer from astrocytes to MT4 using two HIV clones. We also used Nef-null HIV complemented with Nef in trans in the co-culture assay and demonstrated that Nef expression led to significantly decreased HIV transfer from astrocytes to MT4. Taken together, these findings support a negative role of Nef in HIV replication and pathogenesis in astrocytes.

Nef homodimers down-regulate SERINC5 by AP-2-mediated endocytosis to promote HIV-1 infectivity.

SERINC5 is a multipass intrinsic membrane protein that suppresses HIV-1 infectivity when incorporated into budding virions. The HIV-1 Nef virulence factor prevents viral incorporation of SERINC5 by triggering its down-regulation from the producer cell membrane through an AP-2-dependent endolysosomal pathway. However, the mechanistic basis for SERINC5 down-regulation by Nef remains elusive. Here we demonstrate that Nef homodimers are important for SERINC5 down-regulation, trafficking to late endosomes, and exclusion from newly synthesized viral particles. Based on previous X-ray crystal structures, we mutated three conserved residues in the Nef dimer interface (Leu112, Tyr115, and Phe121) and demonstrated attenuated homodimer formation in a cell-based fluorescence complementation assay. Point mutations at each position reduced the infectivity of HIV-1 produced from transfected 293T cells, the Jurkat TAg T-cell line, and donor mononuclear cells in a SERINC5-dependent manner. In SERINC5-transfected 293T cells, virion incorporation of SERINC5 was increased by dimerization-defective Nef mutants, whereas down-regulation of SERINC5 from the membrane of transfected Jurkat cells by these mutants was significantly reduced. Nef dimer interface mutants also failed to trigger internalization of SERINC5 and localization to Rab7+ late endosomes in T cells. Importantly, fluorescence complementation assays demonstrated that dimerization-defective Nef mutants retained interaction with both SERINC5 and AP-2. These results show that down-regulation of SERINC5 and subsequent enhancement of viral infectivity require Nef homodimers and support a mechanism by which the Nef dimer bridges SERINC5 to AP-2 for endocytosis. Pharmacological disruption of Nef homodimers may control HIV-1 infectivity and viral spread by enhancing virion incorporation of SERINC5.

Differential Pressures of SERINC5 and IFITM3 on HIV-1 Envelope Glycoprotein over the Course of HIV-1 Infection.

Infection of human immunodeficiency virus type 1 (HIV-1) is subject to restriction by cellular factors. Serine incorporator 5 (SERINC5) and interferon-inducible transmembrane 3 (IFITM3) proteins represent two of these restriction factors, which inhibit HIV-1 entry into target cells. Both proteins impede fusion of the viral membrane with the cellular membrane and the formation of a viral fusion pore, and both are countered by the HIV-1 envelope glycoprotein (Env). Given the immense and lasting pressure which Env endures from host adaptive immune responses, it is important to understand whether and how HIV-1 Env is able to maintain the resistance to SERINC5 and IFITM3 throughout the course of infection. We have thus examined a panel of HIV-1 Env clones that were isolated at different stages of viral infection-transmission, acute, and chronic. While HIV-1 Env clones from the transmission stage are resistant to both SERINC5 and IFITM3, as infection progresses into the acute and chronic stages, the resistance to IFITM3 but not to SERINC5 is gradually lost. We further discovered a significant correlation between the resistance of HIV-1 Env to soluble CD4 inhibition and the resistance to SERINC5 but not to IFITM3. Interestingly, the miniprotein CD4 mimetic M48U1 sensitizes HIV-1 Env to the inhibition by SERINC5 but not IFITM3. Together, these data indicate that SERINC5 and IFITM3 exert differential inhibitory pressures on HIV-1 Env over different stages of HIV-1 infection and that HIV-1 Env uses varied strategies to resist these two restriction factors.IMPORTANCE HIV-1 Env protein is exposed to the inhibition not only by humoral response, but also by host restriction factors, including serine incorporator 5 (SERINC5) and interferon-inducible transmembrane 3 (IFITM3). This study investigates how HIV-1 envelope glycoprotein (Env) manages to overcome the pressures from all these different host inhibition mechanisms over the long course of viral infection. HIV-1 Env preserves the resistance to SERINC5 but becomes sensitive to IFITM3 when infection progresses into the chronic stage. Our study also supports the possibility of using CD4 mimetic compounds to sensitize HIV-1 Env to the inhibition by SERINC5 as a potential therapeutic strategy.

SERINC5 Inhibits HIV-1 Infectivity by Altering the Conformation of gp120 on HIV-1 Particles.

SERINC5 is a 10-transmembrane-domain cellular protein that is incorporated into budding HIV-1 particles and reduces HIV-1 infectivity by inhibiting virus-cell fusion. HIV-1 susceptibility to SERINC5 is determined by sequences in the viral Env glycoprotein gp120, and the antiviral effect of SERINC5 is counteracted by the viral accessory protein Nef. While the precise mechanism by which SERINC5 inhibits HIV-1 infectivity is unclear, previous studies have suggested that SERINC5 affects Env conformation. To define the effects of SERINC5 on Env conformation, we quantified the binding of HIV-1 particles to immobilized Env-specific monoclonal antibodies. We observed that SERINC5 reduced the binding of HIV-1 particles bearing a SERINC5-susceptible Env to antibodies that recognize the V3 loop, a soluble CD4 (sCD4)-induced epitope, and an N-linked glycan. In contrast, SERINC5 did not alter the capture of HIV-1 particles bearing the SERINC5-resistant Env protein. Moreover, the effect of SERINC5 on antibody-dependent virus capture was abrogated by Nef expression. Our results indicate that SERINC5 inhibits HIV-1 infectivity by altering the conformation of gp120 on virions and/or physical masking of specific HIV-1 Env epitopes.IMPORTANCE SERINC5 is a host cell protein that inhibits the infectivity of HIV-1 by a novel and poorly understood mechanism. Here, we provide evidence that the SERINC5 protein alters the conformation of the HIV-1 Env proteins and that this action is correlated with SERINC5's ability to inhibit HIV-1 infectivity. Defining the specific effects of SERINC5 on the HIV-1 glycoprotein conformation may be useful for designing new antiviral strategies targeting Env.

Downregulation of SERINC5 expression in buffy coats of HIV-1-infected patients with detectable or undetectable viral load.

Among the host restriction factors against HIV, SERINC5 has been described in vitro, but the mRNA level of SERINC5 in vivo has been little studied. We compare SERINC5 expression in subjects with HIV-1 (highly active antiretroviral treatment (HAART) and HAART-naïve) with and without suppression of viral load. A cross-sectional study was performed with 107 individuals distributed as follows: 24 with HAART-naïve and detectable viral load (> 50 copies/mL), 13 with HAART and detectable viral load (> 50 copies/mL), 50 with HAART and undetectable viral load (≤ 50 copies/mL), and 20 without HIV-1. SERINC5 expression in buffy coats was determined using RT-qPCR. The viral load was determined using real-time PCR and the amount of CD4 + and CD8 + T-lymphocytes was measured using flow cytometry. The data were normalized with the Shapiro-Wilk test and the Kruskal-Wallis test was subsequently performed. The relative expression was compared with a T-test and the remaining data with the Mann-Whitney U-test. ANCOVA multiple linear regression analysis was performed between characteristics of patients with SERINC5 expression. The mean and SD of the SERINC5 expression in the three groups with HIV-1 was 0.9 ± 0.2 and without HIV-1 was 1.7 ± 0.14 (P < 0.001). Multiple linear regression did not show the participation of CD4 +, CD8 + , viral load, infection time, or treatment time. No differences in the SERINC5 expression were found among the studied groups of patients with HIV-1. When comparing the groups with and without HIV-1 infection, SERINC5 was downregulation in the HIV-1 groups.

The retroviral accessory proteins S2, Nef, and glycoMA use similar mechanisms for antagonizing the host restriction factor SERINC5.

Serine incorporator 5 (SERINC5) is a recently identified restriction factor that blocks virus entry but is antagonized by three unrelated retroviral accessory proteins. The S2 protein from equine infectious anemia virus (EIAV) has been reported to reduce SERINC5 expression at steady-state levels likely via the endocytic pathway; however, the precise mechanism is still unclear. Here, we investigated how EIAV S2 protein down-regulates SERINC5 compared with down-regulation induced by Nef from HIV-1 and glycoMA proteins from murine leukemia virus (MLV). Using bimolecular fluorescence complementation (BiFC) assay and immunoprecipitation (IP), we detected an interaction between S2 and SERINC5. We found that this interaction relies on the S2 myristoylation site, indicating that it may occur on the plasma membrane. S2 internalized SERINC5 via receptor-mediated endocytosis and targeted it to endosomes and lysosomes, resulting in a ubiquitination-dependent decrease in SERINC5 expression at steady-state levels. Both BiFC and IP detected a glycoMA-SERINC5 interaction, but a Nef-SERINC5 interaction was detected only by BiFC. Moreover, S2 and glycoMA down-regulated SERINC5 more effectively than did Nef. We further show that unlike Nef, both S2 and glycoMA effectively down-regulate SERINC2 and also SERINC5 from Xenopus tropicalis (xSERINC5). Moreover, we detected expression of the equine SERINC5 (eSERINC5) protein and observed that its expression is much weaker than expression levels of SERINC5 from other species. Nonetheless, eSERINC5 had a strong antiviral activity that was effectively counteracted by S2. We conclude that HIV-1, EIAV, and MLV share a similar mechanism to antagonize viral restriction by host SERINC5.

Longitudinal within-host evolution of HIV Nef-mediated CD4, HLA and SERINC5 downregulation activity: a case study.

The HIV accessory protein Nef downregulates the viral entry receptor CD4, the Human Leukocyte Antigen (HLA)-A and -B molecules, the Serine incorporator 5 (SERINC5) protein and other molecules from the infected cell surface, thereby promoting viral infectivity, replication and immune evasion. The nef locus also represents one of the most genetically variable regions in the HIV genome, and nef sequences undergo substantial evolution within a single individual over the course of infection. Few studies however have simultaneously characterized the impact of within-host nef sequence evolution on Nef protein function over prolonged timescales. Here, we isolated 50 unique Nef clones by single-genome amplification over an 11-year period from the plasma of an individual who was largely naïve to antiretroviral treatment during this time. Together, these clones harbored nonsynonymous substitutions at 13% of nef's codons. We assessed their ability to downregulate cell-surface CD4, HLA and SERINC5 and observed that all three Nef functions declined modestly over time, where the reductions in CD4 and HLA downregulation (an average of 0.6% and 2.0% per year, respectively) achieved statistical significance. The results from this case study support all three Nef activities as being important to maintain throughout untreated HIV infection, but nevertheless suggest that, despite nef's mutational plasticity, within-host viral evolution can compromise Nef function, albeit modestly, over prolonged periods.

CD4 Expression and Env Conformation Are Critical for HIV-1 Restriction by SERINC5.

Serine incorporator 5 (SERINC5) is a recently identified restriction factor that strongly blocks HIV-1 entry but is counteracted by Nef. Notably, tier 1 HIV-1 Env proteins are sensitive to SERINC5, whereas the majority of tier 2/3 Env proteins are resistant to SERINC5, when viruses are produced from CD4-negative cells and tested by a single-round replication assay. Here, we investigated the Env-dependent SERINC5 antiviral mechanism by comparing tier 1 NL Env with tier 3 AD8 Env proteins. We found that when NL and AD8 viruses were inoculated into CD4+ T cells and human peripheral blood mononuclear cells (PBMCs), the propagation of the two viruses was restricted to a similar level when Nef was not expressed. Using a bimolecular fluorescence complementation (BiFC) assay, we detected Env-Env association and Env-SERINC5 interactions. A much greater level of NL Env-SERINC5 interactions was detected than was AD8 Env-SERINC5 interactions, which was further validated by immunoprecipitation assays. In addition, SERINC5 dissociated the NL Env trimeric complex more effectively than the AD8 Env trimeric complex when CD4 was not expressed. However, when CD4 was expressed, SERINC5 became more capable of interacting with AD8 Env and dissociating its trimeric complex. Moreover, AD8 and several other tier 2/3 viruses produced in the presence of CD4 became sensitive to SERINC5 when measured by the single-round replication assay. Because tier 1 and tier 2/3 Env trimers have open and closed conformations, respectively, and CD4 opens the closed conformation, we conclude that SERINC5 selectively dissociates Env trimers with an open conformation to restrict HIV-1 replication.IMPORTANCE Restriction factors provide the first line of defense against retrovirus infection by posing several blocks to the viral replication cycle. SERINC5 is a novel restriction factor that strongly blocks HIV-1 entry, although it is counteracted by Nef. Currently, it is still unclear how HIV-1 entry is blocked by SERINC5. Notably, this entry block is dependent on viral Env proteins. Laboratory-adapted HIV-1 strains are sensitive, whereas primary isolates are highly resistant to SERINC5. Env proteins mediate virus entry via extensive conformational rearrangements from a closed ground state to a CD4-bound open state. We detected Env-Env associations and Env-SERINC5 interactions in live cells by a novel bimolecular fluorescence assay. We demonstrate that CD4 expression increases the Env sensitivity to SERINC5 and allows SERINC5 to dissociate the Env complex, suggesting that SERINC5 restriction is dependent on Env conformation. Our results provide new insights into the poorly defined Env-dependent SERINC5 antiviral mechanism.

Murine Leukemia Virus Glycosylated Gag Reduces Murine SERINC5 Protein Expression at Steady-State Levels via the Endosome/Lysosome Pathway to Counteract SERINC5 Antiretroviral Activity.

Glycosylated Gag (glycoGag) is an accessory protein expressed by most gammaretroviruses, including murine leukemia virus (MLV). MLV glycoGag not only enhances MLV replication and disease progression but also increases human immunodeficiency virus type 1 (HIV-1) infectivity as Nef does. Recently, SERINC5 (Ser5) was identified as the target for Nef, and the glycoGag Nef-like activity has been attributed to the Ser5 antagonism. Here, we investigated how glycoGag antagonizes Ser5 using MLV glycoMA and murine Ser5 proteins. We confirm previous observations that glycoMA relocalizes Ser5 from plasma membrane to perinuclear punctated compartments and the important role of its Y36XXL39 motif in this process. We find that glycoMA decreases Ser5 expression at steady-state levels and identify two other glycoGag crucial residues, P31 and R63, for the Ser5 downregulation. The glycoMA and Ser5 interaction is detected in live cells using a bimolecular fluorescence complementation assay. Ser5 is internalized via receptor-mediated endocytosis and relocalized to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes by glycoMA. Although glycoMA is not polyubiquitinated, the Ser5 downregulation requires Ser5 polyubiquitination via the K48- and K63-linkage, resulting in Ser5 destruction in lysosomes. Although P31, Y36, L39, and R63 are not required for glycoMA interaction with Ser5, they are required for Ser5 relocalization to lysosomes for destruction. In addition, although murine Ser1, Ser2, and Ser3 exhibit very poor antiviral activity, they are also targeted by glycoMA for lysosomal destruction. We conclude that glycoGag has a broad activity to downregulate SERINC proteins via the cellular endosome/lysosome pathway, which promotes viral replication.IMPORTANCE MLV glycoGag not only enhances MLV replication but also increases HIV-1 infectivity similarly as Nef. Recent studies have discovered that both glycoGag and Nef antagonize a novel host restriction factor Ser5 and promote viral replication. Compared to Nef, the glycoGag antagonism of Ser5 is still poorly understood. MLV glycoGag is a transmembrane version of the structural Gag protein with an extra 88-amino-acid leader region that determines its activity. We now show that glycoGag interacts with Ser5 in live cells and internalizes Ser5 via receptor-mediated endocytosis. Ser5 is polyubiquitinated and relocalized to endosomes and lysosomes for massive destruction. In addition to the previously identified tyrosine-based sorting signal, we find two more important residues for Ser5 relocalization and downregulation. We also find that the Ser5 sensitivity to glycoGag is conserved in the SERINC family. Together, our findings highlight the important role of endosome/lysosome pathway in the enhancement of viral replication by viral proteins.

Characterization of Endogenous SERINC5 Protein as Anti-HIV-1 Factor.

When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-α) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 Δnef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface.IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.

An N-Glycosylated Form of SERINC5 Is Specifically Incorporated into HIV-1 Virions.

SERINC5 is an inhibitor of retroviral infectivity that is counteracted by viral proteins, including HIV-1 Nef. Inhibition of infectivity by SERINC5 is associated with its incorporation into virions. Nef counteracts this inhibition, presumably by removing SERINC5 from sites of virion assembly at the plasma membrane. While evaluating the virion incorporation of SERINC5, we observed that a relatively high molecular weight form was preferentially present in virions. We used various glycosidases to establish that virion-associated SERINC5 is modified by N-linked, complex glycans, whereas the majority of SERINC5 in cells is of relatively low molecular weight and is modified by high-mannose glycans. Sequence alignment of SERINC family proteins led us to identify a conserved N-glycosylation site, N294, in SERINC5. We mutated this site to evaluate its effect on glycosylation, the restrictive activity of SERINC5, and the sensitivity of SERINC5 to antagonism by Nef. Our results demonstrate that N294 is the major site of N-glycosylation in SERINC5. Although N-glycosylation was required neither for restrictive activity nor for sensitivity to Nef per se, we observed a decrease in the steady-state expression of glycosylation-deficient SERINC5 (the N294A mutant) compared to the wild-type protein. Expression of this mutant was partly restored by treatment of cells with MG132 (a proteasome inhibitor) but not with bafilomycin A1 (a lysosomal inhibitor). We conclude that although not required for restrictive activity or Nef sensitivity, N-linked glycosylation is important for maintaining the steady-state expression of SERINC5 and that nonglycosylated SERINC5 is likely subjected to a quality control mechanism that induces its proteasomal degradation.IMPORTANCE SERINC5 is a member of a family of multipass transmembrane proteins that inhibit the infectivity of retroviruses, including HIV-1. These proteins are incorporated into virions and inhibit infection of target cells unless counteracted by viral antagonists such as HIV-1 Nef. The only other biological function with which these proteins have been associated is the formation of serine-containing membrane lipids. Here we show that SERINC5 is a glycosylated protein and that N-glycosylation is important for its steady-state expression. In the absence of N-glycosylation, SERINC5 is prone to proteasomal degradation. Nonetheless, N-glycosylation per se is required neither for the ability of SERINC5 to inhibit HIV-1 infectivity nor for its sensitivity to antagonism by Nef.

HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System.

The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH4Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction.IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.