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The P-Selectin Ligand PSGL-1 (CD162) Is Efficiently Incorporated by 2

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The P-Selectin Ligand PSGL-1 (CD162) Is Efficiently Incorporated by 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.29.450454; this version posted June 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 The P-selectin ligand PSGL-1 (CD162) is efficiently incorporated by 2 primary HIV-1 isolates and can facilitate trans-infection. 3 4 1,2 1 1,2 3 Jonathan Burnie , Arvin Tejnarine Persaud , Laxshaginee Thaya , Qingbo Liu , Huiyi 5 3 1 1 3 4 Miao , Stephen Grabinsky , Vanessa Norouzi , Paolo Lusso , Vera A. Tang , Christina 6 1,2 Guzzo * 7 8 1 Department of Biological Sciences, University of Toronto Scarborough, 1265 Military Trail, 9 Toronto, Ontario, Canada 10 2 Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, 11 Ontario, Canada 12 3 Viral Pathogenesis Section, Laboratory of Immunoregulation (LIR), National Institute of 13 Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, 14 Maryland, USA. 15 4 Faculty of Medicine, Department of Biochemistry, Microbiology, and Immunology, Univer- 16 sity of Ottawa, Flow Cytometry and Virometry Core Facility, Ottawa, Ontario, Canada 17 * Corresponding author [email protected] 18 19 20 Short title: 21 Virion-incorporated PSGL-1 can facilitate HIV-1 infection. 22 23 24 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.29.450454; this version posted June 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 2 of 48 ABSTRACT 25 While P-selectin glycoprotein ligand-1 (PSGL-1/CD162) has been studied extensively for its 26 role in mediating leukocyte rolling through interactions with its receptor, P-selectin, recently, 27 it was identified as a novel HIV-1 host restriction factor. One key mechanism of HIV-1 28 restriction is the ability of PSGL-1 to be physically incorporated into the external viral envelope, 29 which effectively reduces infectivity by blocking virus attachment through the steric hindrance 30 caused by its large ectodomain. Importantly, a large portion of the literature demonstrating the 31 antiviral activity of PSGL-1 has utilized viruses produced in transfected cells which express 32 high levels of PSGL-1. However, herein we show that virion-incorporated PSGL-1 is far less 33 abundant on the surface of viruses produced via infection of physiologically relevant models 34 (T cell lines and primary cells) compared to transfection (overexpression) models. Unique to 35 this study, we show that PSGL-1 is incorporated in a broad range of HIV-1 and SIV isolates, 36 supporting the physiological relevance of this incorporation. We also report that high levels of 37 virion-incorporated PSGL-1 are detectable in plasma from viremic HIV-1 infected individuals, 38 further corroborating the clinical relevance of PSGL-1 in natural infection. Additionally, we 39 show that PSGL-1 on viruses is functionally active and can bind its cognate receptor, P-selectin, 40 and that virions captured via P-selectin can subsequently be transferred to HIV-permissive 41 bystander cells in a model of trans-infection. Taken together, our data suggest that PSGL-1 42 may have diverse roles in the physiology of HIV-1 infection, not restricted to the current 43 antiviral paradigm. 44 45 IMPORTANCE 46 PSGL-1 is an HIV-1 host restriction factor which reduces viral infectivity by physically 47 incorporating into the envelope of virions. While the antiviral effects of PSGL-1 in viruses 48 produced by transfection models is profound, HIV-1 continues to remain infectious when 49 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.29.450454; this version posted June 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 3 of 48 produced through natural infection, even when PSGL-1 is incorporated. To study this 50 discordance, we compared the differences in infectivity and PSGL-1 abundance in viruses 51 produced by transfection or infection. Viruses produced via transfection contained unnaturally 52 high levels of incorporated PSGL-1 compared to viruses from primary cells, and were much 53 less infectious. We also found PSGL-1 to be present on a broad range of HIV-1 isolates, 54 including those found in plasma from HIV-infected patients. Remarkably, we show that virion- 55 incorporated PSGL-1 facilitates virus capture and transfer to HIV-permissive host cells via 56 binding to P-selectin. These findings suggest that PSGL-1 may also work to enhance infection 57 in vivo. 58 59 KEYWORDS: P-selecin glycoprotein ligand-1 (PSGL-1/CD162), P-selectin (CD62P), 60 human immunodeficiency virus type 1 (HIV-1), HIV-1 infection, HIV-1 envelope (gp120), 61 HIV-1 restriction factors, virion-incorporated proteins, virion capture, calibrated flow 62 virometry 63 64 INTRODUCTION 65 P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is an adhesion molecule expressed on all 66 leukocytes that plays a critical role in the early stages of inflammation due to its ability to bind 67 P-, E- and L- selectins (1–5). In particular, PSGL-1 has been well-characterized for its 68 involvement in leukocyte recruitement (rolling and tethering) and extravasation into tissues 69 (reviewed in (1, 3, 4, 6, 7)). Stucturally, PSGL-1 is a highly glycosylated homodimeric 70 transmembrane protein, with an extracellular domain (ECD) of 50-60 nm in length that extends 71 far out from the cellular surface (1, 8, 9). In 2019, Liu et al. were the first to identify PSGL-1 72 as a novel HIV restriction factor in activated human CD4+ T cells, with inherent antiviral 73 activity through a variety of mechansims in host cells (10). Notably, using viruses produced 74 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.29.450454; this version posted June 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 4 of 48 through transfection, Liu et al. showed that when increasing amounts of PSGL-1 plasmid DNA 75 (pDNA) were co-transfected with an HIV-1 infectious molecular clone (IMC), virion 76 infectivity was reduced in a dose-dependent manner (10). Upon further investigation virions 77 were also shown to physically incorporate PSGL-1 using electron microscopy and Western 78 blotting techniques, and this virion incorporation of PSGL-1 was identified as an additional 79 mechanism to diminish HIV-1 infectivity (10). Experiments performed by Murakami et al. and 80 Fu et al. in 2020 also demonstrated through knock-down of PSGL-1 in Jurkat cells and 81 CRISPR-mediated KO of PSGL-1 in primary cells respectively, that physiological levels of 82 PSGL-1 on the cell surface can also result in a modest impairment of particle infectivity (11, 83 12). 84 Subsequent studies examining the antiviral effects of virion-incorporated PSGL-1 85 demonstrated that PGSL-1 could block the binding of virions to target cells (11, 12). More 86 specifically, it was shown that the large ECD of PSGL-1 was required for this inhibitory effect 87 and that PSGL-1 also has a broad spectrum antiviral effect when ectopically expressed in the 88 envelopes of other viruses, including murine leukemia virus, influenza A and SARS CoV-1 89 and -2 (11, 13, 14). Additional characterization of virion-incorporated PSGL-1 revealed a 90 concomitant reduction in the incorporation of the HIV envelope glycoprotein (Env) into virions 91 by sequestering gp41 at the cell membrane (15), providing another measure by which PSGL-1 92 can restrict virion infectivity. Research on other structurally similar proteins with large 93 extracellular domains has also shown similar inhibitory effects on viral infection and in line 94 with this concept, PSGL-1 was recently reported to be a part of a larger group of antiviral 95 proteins termed Surface-Hinged, Rigidly-Extended Killer (SHREK) proteins (14). 96 97 While much has been rapidly uncovered about the antiviral functions of PSGL-1 on HIV-1 and 98 other viruses since its identification as a host restriction factor in 2019 (10), many unanswered 99 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.29.450454; this version posted June 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 5 of 48 questions about the function of PSGL-1 in the physiology of HIV-1 infection remain. Indeed, 100 while the initial proteomic screen that identified PSGL-1 as a host restriction factor and limited 101 others employed activated primary CD4+ T cells (10–12), the majority of experiments 102 performed to characterize the antiviral functions of this protein have used viruses produced via 103 co-transfection of PSGL-1 and viral constructs in adherent cell lines. While transfection models 104 are commonly used to produce viruses and express specific host proteins, it is well recognized 105 that the cell type used to produce viruses can alter the composition of cellular proteins 106 incorporated into the virion surface (16–18).
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