Identification of CD38, CD97, and CD278 on the HIV surface using a novel flow virometry screening assay.

While numerous cellular proteins in the HIV envelope are known to alter virus infection, methodology to rapidly phenotype the virion surface in a high throughput, single virion manner is lacking. Thus, many human proteins may exist on the virion surface that remain undescribed. Herein, we developed a novel flow virometry screening assay to discover new proteins on the surface of HIV particles. By screening a CD4+ T cell line and its progeny virions, along with four HIV isolates produced in primary cells, we discovered 59 new candidate proteins in the HIV envelope that were consistently detected across diverse HIV isolates. Among these discoveries, CD38, CD97, and CD278 were consistently present at high levels on virions when using orthogonal techniques to corroborate flow virometry results. This study yields new discoveries about virus biology and demonstrates the utility and feasibility of a novel flow virometry assay to phenotype individual virions.

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.

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.

Human monocytes store and secrete preformed CCL5, independent of de novo protein synthesis.

Monocytes are a subset of circulating peripheral blood mononuclear cells with diverse roles in immunity, including sentinel roles in cytokine secretion. Conventionally, cytokines require an inductive stimulus for their expression and secretion, resulting in a time lag from the time of stimulation to when the proteins are packaged and secreted. Because cytokines are the main communicators in the immune system, their temporal expression is a key factor in coordinating responses to efficiently resolve infection. Herein, we identify that circulating human monocytes contain preformed cytokines that are stored intracellularly, in both resting and activated states. Having preformed cytokines bypasses the time lag associated with de novo synthesis, allowing monocytes to secrete immune mediators immediately upon activation or sensing of microbe-associated molecular patterns. We demonstrate here that, out of several cytokines evaluated, human monocytes contain a previously undescribed reservoir of the preformed chemokine CCL5. Furthermore, we showed that CCL5 could be secreted from monocytes treated with the protein synthesis inhibitor (cycloheximide) and Golgi blocker (brefeldin A). We examined the possibility for uptake of extracellular CCL5 from platelet aggregates and observed no significant levels of platelet binding to our enriched monocyte preparations, indicating that the source of preformed CCL5 was not from platelets. Preformed CCL5 was observed to be distributed throughout the cytoplasm and partially colocalized with CD63+ and Rab11A+ membranes, implicating endosomal compartments in the intracellular storage and trafficking of CCL5.