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      Tetherin downmodulation by SIVmac Nef lost with the H196Q escape variant is restored by an upstream variant

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          Abstract

          The H 196 residue in SIVmac239 Nef is conserved across the majority of HIV and SIV isolates, lies immediately adjacent to the AP-2 (adaptor protein 2) binding di-leucine domain (ExxxLM 195), and is critical for several described AP-2 dependent Nef functions, including the downregulation of tetherin (BST-2/CD317), CD4, and others. Surprisingly, many stocks of the closely related SIVmac251 swarm virus harbor a nef allele encoding a Q 196. In SIVmac239, this variant is associated with loss of multiple AP-2 dependent functions. Publicly available sequences for SIVmac251 stocks were mined for variants linked to Q 196 that might compensate for functional defects associated with this residue. Variants were engineered into the SIVmac239 backbone and in Nef expression plasmids and flow cytometry was used to examine surface tetherin expression in primary CD4 T cells and surface CD4 expression in SupT1 cells engineered to express rhesus CD4. We found that SIVmac251 stocks that encode a Q 196 residue in Nef uniformly also encode an upstream R 191 residue. We show that R 191 restores the ability of Nef to downregulate tetherin in the presence of Q 196 and has a similar but less pronounced impact on CD4 expression. However, a published report showed Q 196 commonly evolves to H 196 in vivo, suggesting a fitness cost. R 191 may represent compensatory evolution to restore the ability to downregulate tetherin lost in viruses harboring Q 196.

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          Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding.

          The study of early events in the human immunodeficiency virus type 1 (HIV-1) life cycle can be limited by the relatively low numbers of cells that can be infected synchronously in vitro. Although the efficiency of HIV-1 infection can be substantially improved by centrifugal inoculation (spinoculation or shell vial methods), the underlying mechanism of enhancement has not been defined. To understand spinoculation in greater detail, we have used real-time PCR to quantitate viral particles in suspension, virions that associate with cells, and the ability of those virions to give rise to reverse transcripts. We report that centrifugation of HIV-1(IIIB) virions at 1,200 x g for 2 h at 25 degrees C increases the number of particles that bind to CEM-SS T-cell targets by approximately 40-fold relative to inoculation by simple virus-cell mixing. Following subsequent incubation at 37 degrees C for 5 h to allow membrane fusion and uncoating to occur, the number of reverse transcripts per target cell was similarly enhanced. Indeed, by culturing spinoculated samples for 24 h, approximately 100% of the target cells were reproducibly shown to be productively infected, as judged by the expression of p24(gag). Because the modest g forces employed in this procedure were found to be capable of sedimenting viral particles and because CD4-specific antibodies were effective at blocking virus binding, we propose that spinoculation works by depositing virions on the surfaces of target cells and that diffusion is the major rate-limiting step for viral adsorption under routine in vitro pulsing conditions. Thus, techniques that accelerate the binding of viruses to target cells not only promise to facilitate the experimental investigation of postentry steps of HIV-1 infection but should also help to enhance the efficacy of virus-based genetic therapies.
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            Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein.

            Like other pathogenic viruses, HIV-1 down-modulates surface expression of major histocompatibility complex class I (MHC-I) molecules in infected cells, thus impairing lysis by cytotoxic T lymphocytes. We have observed that this phenomenon depends on the expression of Nef. nef is an early gene of primate lentiviruses, which is necessary for maintaining high virus loads and inducing AIDS. Nef is not necessary for viral replication in vitro and stimulates the endocytosis of CD4. We show that the expression of MHC-I at the surface of lymphoid, monocytic and epithelial cells was reduced in the presence of Nef protein from various HIV-1 strains. Whereas MHC-I protein synthesis and transport through the endoplasmic reticulum and cis Golgi apparatus occurred normally in Nef(+) cells, surface MHC-I molecules were rapidly internalized, accumulated in endosomal vesicles and were degraded. The stimulation of MHC-I endocytosis by Nef represents a previously undocumented viral mechanism for evading the immune response.
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              Tetherin-driven adaptation of Vpu and Nef function and the evolution of pandemic and nonpandemic HIV-1 strains.

              Vpu proteins of pandemic HIV-1 M strains degrade the viral receptor CD4 and antagonize human tetherin to promote viral release and replication. We show that Vpus from SIVgsn, SIVmus, and SIVmon infecting Cercopithecus primate species also degrade CD4 and antagonize tetherin. In contrast, SIVcpz, the immediate precursor of HIV-1, whose Vpu shares a common ancestry with SIVgsn/mus/mon Vpu, uses Nef rather than Vpu to counteract chimpanzee tetherin. Human tetherin, however, is resistant to Nef and thus poses a significant barrier to zoonotic transmission of SIVcpz to humans. Remarkably, Vpus from nonpandemic HIV-1 O strains are poor tetherin antagonists, whereas those from the rare group N viruses do not degrade CD4. Thus, only HIV-1 M evolved a fully functional Vpu following the three independent cross-species transmissions that resulted in HIV-1 groups M, N, and O. This may explain why group M viruses are almost entirely responsible for the global HIV/AIDS pandemic.
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                Author and article information

                Contributors
                Role: Data curationRole: Formal analysisRole: MethodologyRole: Writing – review & editing
                Role: MethodologyRole: Resources
                Role: ConceptualizationRole: Funding acquisitionRole: Resources
                Role: ConceptualizationRole: Formal analysisRole: Writing – original draft
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                7 August 2020
                2020
                : 15
                : 8
                : e0225420
                Affiliations
                [1 ] Division of Microbiology, Tulane National Primate Research Center, Covington, LA, United States of America
                [2 ] Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America
                [3 ] Department of Microbiology and Immunology, School of Medicine, Tulane University Health Sciences Center, New Orleans, LA, United States of America
                University Hospital Tuebingen, GERMANY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-9602-2364
                http://orcid.org/0000-0002-9332-7891
                Article
                PONE-D-19-30708
                10.1371/journal.pone.0225420
                7413475
                32764749
                f71b52c3-5726-41ac-b0a1-10a097e0e944
                © 2020 Schouest et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 4 November 2019
                : 13 July 2020
                Page count
                Figures: 3, Tables: 0, Pages: 13
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;
                Award ID: R01 AI138782
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: P510 D011104
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: P30 AI045008
                Award Recipient :
                NIH provided funding for this study in the form of a base grant awarded to TNPRC (P51OD011104), a R01 grant awarded to JAH and NJM (AI138782), and a P30 grant awarded to JAH (AI045008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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