P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

Abstract

Background P-selectin glycoprotein ligand-1 (PSGL-1/CD162) has been studied extensively for its role in mediating leukocyte rolling through interactions with its cognate receptor, P-selectin. Recently, PSGL-1 was identified as a novel HIV-1 host restriction factor, particularly when expressed at high levels in the HIV envelope. Importantly, while the potent antiviral activity of PSGL-1 has been clearly demonstrated in various complementary model systems, the breadth of PSGL-1 incorporation across genetically diverse viral isolates and clinical isolates has yet to be described. Additionally, the biological activity of virion-incorporated PSGL-1 has also yet to be shown. Results Herein we assessed the levels of PSGL-1 on viruses produced through transfection with various amounts of PSGL-1 plasmid DNA (0–250 ng), compared to levels of PSGL-1 on viruses produced through infection of T cell lines and primary PBMC. We found that very low levels of PSGL-1 plasmid DNA (< 2.5 ng/well) were necessary to generate virus models that could closely mirror the phenotype of viruses produced via infection of T cells and PBMC. Unique to this study, we show that PSGL-1 is incorporated in a broad range of HIV-1 and SIV isolates and that virions with incorporated PSGL-1 are detectable in plasma from viremic HIV-1-infected individuals, corroborating the relevance of PSGL-1 in natural infection. Additionally, we show that PSGL-1 on viruses can bind its cognate selectin receptors, P-, E-, and L-selectins. Finally, we show viruses with endogenous levels of PSGL-1 can be captured by P-selectin and transferred to HIV-permissive bystander cells, highlighting a novel role for PSGL-1 in HIV-1 infection. Notably, viruses which contained high levels of PSGL-1 were noninfectious in our hands, in line with previous findings reporting the potent antiviral activity of PSGL-1. Conclusions Our results indicate that levels of PSGL-1 incorporation into virions can vary widely among model systems tested, and that careful tailoring of plasmid levels is required to recapitulate physiological systems when using pseudovirus models. Taken together, our data suggest that PSGL-1 may play diverse roles in the physiology of HIV-1 infection, particularly due to the functionally active state of PSGL-1 on virion surfaces and the breadth of PSGL-1 incorporation among a wide range of viral isolates.

Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

“…Virion capture assays (VCA, Fig. 1A ) are routinely used by our group and others to characterize various virion-incorporated cellular proteins ( 5 , 25 , 36 ). As an initial test of the extent of CD14 incorporation into HIV virions, we screened a small panel of peripheral blood mononuclear cell (PBMC)-derived HIV-1 isolates (HIV-1 IIIB , HIV-1 NL4-3 , HIV-1 BaL , and HIV-1 SF162 ) by VCA ( Fig.…”

“…We next wanted to determine whether virion-incorporated CD14 confers additional biological functions to HIV-1 virions. Our recent work highlights the usefulness of pseudoviral particles as models for functional studies on virion-incorporated proteins ( 25 , 36 , 37 ), and we therefore turned to PV models of HIV-1 for this study. These PVs are produced by co-transfection of 293T cells with plasmids encoding the HIV-1 backbone (pSG3ΔEnv) and cellular CD14, as depicted in Fig.…”

“…For example, the incorporation of integrin α4β7 enables HIV-1 virions to transit more efficiently to gut-associated lymphoid tissues ( 11 ), which are coincidentally home to a large population of HIV target cells, CD4 + T cells ( 23 , 24 ). More recently, our work has also shown that virion-incorporation of CD162 (P-selectin glycoprotein ligand-1) can enable HIV-1 virions to be captured and transferred to susceptible cells by interaction with CD62P (P selectin), the cognate ligand of CD162 ( 25 ).…”

“…Additionally, the use of fluorescence reference beads in compliance with standardized frameworks enables a quantitative estimate of antigen abundance on the surface of virions and reports these data in calibrated units ( 34 , 35 ). Though nanoscale cytometry is extensively used in the extracellular vesicles (EVs) field, our lab has adapted these techniques for the study of cellular proteins incorporated into the HIV-1 envelope ( 25 , 36 , 37 ) and applied them here to investigate CD14 incorporation by HIV-1.…”

Single-step rapid chromatographic purification and characterization of clinical stage oncolytic VSV-GP

“…Moreover, the largest challenges arise not only due to their size, but also due to the shape and complexity of surface charges on the viruses ( Singh and Heldt, 2022 ). Furthermore, the surface charges on the virus particles are further modulated by incorporation of host cell proteins on the surface of virions ( Burnie, 2022 ).…”