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Natural HIV-1 Nef Polymorphisms Impair SERINC5 Downregulation Activity.

ABSTRACT: HIV-1 Nef enhances virion infectivity by counteracting host restriction factor SERINC5; however, the impact of natural Nef polymorphisms on this function is largely unknown. We characterize SERINC5 downregulation activity of 91 primary HIV-1 subtype B nef alleles, including isolates from 45 elite controllers and 46 chronic progressors. Controller-derived Nef clones display lower ability to downregulate SERINC5 (median 80% activity) compared with progressor-derived clones (median 96% activity) (p = 0.0005). We identify 18 Nef polymorphisms associated with differential function, including two CTL escape mutations that contribute to lower SERINC5 downregulation: K94E, driven by HLA-B?08, and H116N, driven by the protective allele HLA-B?57. HIV-1 strains encoding Nef K94E and/or H116N display lower infectivity and replication capacity in the presence of SERINC5. Our results demonstrate that natural polymorphisms in HIV-1 Nef can impair its ability to internalize SERINC5, indicating that variation in this recently described function may contribute to differences in viral pathogenesis.

SUBMITTER: Jin SW

PROVIDER: S-EPMC6925589 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Natural HIV-1 Nef Polymorphisms Impair SERINC5 Downregulation Activity.

Jin Steven W SW Alsahafi Nirmin N Kuang Xiaomei T XT Swann Shayda A SA Toyoda Mako M Göttlinger Heinrich H Walker Bruce D BD Ueno Takamasa T Finzi Andrés A Brumme Zabrina L ZL Brockman Mark A MA

Cell reports 20191101 6

HIV-1 Nef enhances virion infectivity by counteracting host restriction factor SERINC5; however, the impact of natural Nef polymorphisms on this function is largely unknown. We characterize SERINC5 downregulation activity of 91 primary HIV-1 subtype B nef alleles, including isolates from 45 elite controllers and 46 chronic progressors. Controller-derived Nef clones display lower ability to downregulate SERINC5 (median 80% activity) compared with progressor-derived clones (median 96% activity) (p ...[more]

PMID: 31693887

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Project description: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.

Project description:It has recently emerged that HIV-1 Nef counteracts the antiviral host proteins SERINC3 and SERINC5. In particular, SERINC5 inhibits the infectivity of progeny virions when incorporated. SERINC3 and SERINC5 are also counteracted by the unrelated murine leukemia virus glycosylated Gag (glycoGag) protein, which possesses a potent Nef-like activity on HIV-1 infectivity. We now report that a minimal glycoGag termed glycoMA can fully substitute for Nef in promoting HIV-1 replication in Jurkat T lymphoid cells, indicating that Nef enhances replication in these cells mainly by counteracting SERINCs. In contrast, the SERINC antagonist glycoMA was unable to substitute for Nef in MOLT-3 T lymphoid cells, in which HIV-1 replication was highly dependent on Nef, and remained so even in the absence of SERINC3 and SERINC5. As in MOLT-3 cells, glycoMA was unable to substitute for Nef in stimulating HIV-1 replication in primary human cells. Although the ability of Nef mutants to promote HIV-1 replication in MOLT-3 cells correlated with the ability to engage endocytic machinery and to downregulate CD4, Nef nevertheless rescued virus replication under conditions where CD4 downregulation did not occur. Taken together, our observations raise the possibility that Nef triggers the endocytosis of a novel antiviral factor that is active against both laboratory-adapted and primary HIV-1 strains.IMPORTANCE The Nef protein of HIV-1 and the unrelated glycoGag protein of a murine leukemia virus similarly prevent the uptake of antiviral host proteins called SERINC3 and SERINC5 into HIV-1 particles, which enhances their infectiousness. We now show that although both SERINC antagonists can in principle similarly enhance HIV-1 replication, glycoGag is unable to substitute for Nef in primary human cells and in a T cell line called MOLT-3. In MOLT-3 cells, Nef remained crucial for HIV-1 replication even in the absence of SERINC3 and SERINC5. The pronounced effect of Nef on HIV-1 spreading in MOLT-3 cells correlated with the ability of Nef to engage cellular endocytic machinery and to downregulate the HIV-1 receptor CD4 but nevertheless persisted in the absence of CD4 downregulation. Collectively, our results provide evidence for a potent novel restriction activity that affects even relatively SERINC-resistant HIV-1 isolates and is counteracted by Nef.

Dataset Information

Natural HIV-1 Nef Polymorphisms Impair SERINC5 Downregulation Activity.

Publications

Natural HIV-1 Nef Polymorphisms Impair SERINC5 Downregulation Activity.

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