RESUMEN
The HIV envelope glycoprotein (Env) is a trimeric protein that facilitates viral binding and fusion with target cells. As the sole viral protein on the HIV surface, Env is important both for immune responses to HIV and in vaccine designs. Targeting Env in clinical applications is challenging due to its heavy glycosylation, high genetic variability, conformational camouflage, and its low abundance on virions. Thus, there is a critical need to better understand this protein. Flow virometry (FV) is a useful methodology for phenotyping the virion surface in a high-throughput, single virion manner. To demonstrate the utility of FV to characterize Env, we stained HIV virions with a panel of 85 monoclonal antibodies targeting different regions of Env. A broad range of antibodies yielded robust staining of Env, with V3 antibodies showing the highest quantitative staining. A subset of antibodies tested in parallel on viruses produced in CD4+ T cell lines, HEK293T cells, and primary cells showed that the cellular model of virus production can impact Env detection. Finally, in addition to being able to highlight Env heterogeneity on virions, we show FV can sensitively detect differences in Env conformation when soluble CD4 is added to virions before staining.
Asunto(s)
VIH-1 , Virión , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Humanos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , VIH-1/genética , VIH-1/fisiología , VIH-1/inmunología , Virión/metabolismo , Células HEK293 , Anticuerpos Anti-VIH/inmunología , Anticuerpos Monoclonales/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/virologíaRESUMEN
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.
Asunto(s)
Infecciones por VIH , VIH-1 , Selectina-P , Antivirales/metabolismo , ADN/metabolismo , VIH-1/genética , VIH-1/metabolismo , Humanos , Leucocitos Mononucleares , Glicoproteínas de Membrana , Selectina-P/metabolismoRESUMEN
HIV/AIDS continues to pose an enormous burden on global health. Current HIV therapeutics include inhibitors that target the enzymes HIV protease, reverse transcriptase, and integrase, along with viral entry inhibitors that block the initial steps of HIV infection by preventing membrane fusion or virus-coreceptor interactions. With regard to the latter, peptides derived from the HIV coreceptor CCR5 were previously shown to modestly inhibit entry of CCR5-tropic HIV strains, with a peptide containing residues 178-191 of the second extracellular loop (peptide 2C) showing the strongest inhibition. Here we use an iterative approach of amino acid scanning at positions shown to be important for binding the HIV envelope, and recombining favorable substitutions to greatly improve the potency of 2C. The most potent candidate peptides gain neutralization breadth and inhibit CXCR4 and CXCR4/CCR5-using viruses, rather than CCR5-tropic strains only. We found that gains in potency in the absence of toxicity were highly dependent on amino acid position and residue type. Using virion capture assays we show that 2C and the new peptides inhibit capture of CD4-bound HIV-1 particles by antibodies whose epitopes are located in or around variable loop 3 (V3) on gp120. Analysis of antibody binding data indicates that interactions between CCR5 ECL2-derived peptides and gp120 are localized around the base and stem of V3 more than the tip. In the absence of a high-resolution structure of gp120 bound to coreceptor CCR5, these findings may facilitate structural studies of CCR5 surrogates, design of peptidomimetics with increased potency, or use as functional probes for further study of HIV-1 gp120-coreceptor interactions.