Recombinant HIV type 1 (pPRB926_04.A9.4239) Glycoprotein 120 (a.a. 28-505) [His]

The aims of this study were twofold. The first was to determine if human immunodeficiency virus (HIV)-1 glycoprotein (gp) 120 and transactivator of transcription (Tat) stimulate the release of endothelial microvesicles (EMVs). The second was to determine whether viral protein-induced EMVs are deleterious to endothelial cell function (inducing endothelial cell inflammation, oxidative stress, senescence and increasing apoptotic susceptibility). Human aortic endothelial cells (HAECs) were treated with recombinant HIV-1 proteins Bal gp120 (R5), Lay gp120 (X4), or Tat. EMVs released in response to each viral protein were isolated and quantified. Fresh HAECs were treated with EMVs generated under control conditions and from each of the viral protein conditions for 24 h. EMV release was higher (P < 0.05) in HAECs treated with R5 (141 +/- 21 MV/mu l), X4 (132 +/- 20 MV/mu l), and Tat (130 +/- 20 MV/ mu l) compared with control (61 +/- 13 MV/mu l). Viral protein EMVs induced significantly higher endothelial cell release of proinflammatory cytokines and expression of cell adhesion molecules than control. Reactive oxygen species production was more pronounced (P < 0.05) in the R5-, X4- and Tat-EMV-treated cells. In addition, viral proteinstimulated EMVs significantly augmented endothelial cell senescence and apoptotic susceptibility. Concomitant with these functional changes. viral protein-stimulated EMVs disrupted cell expression of micro-RNAs 34a, 126, 146a, 181b, 221, and miR-Let-7a (P < 0.05). These results demonstrate that HIV-1 gp120 and Tat stimulate microvesicle release from endothelial cells, and these microvesicles confer pathological effects on endothelial cells by inducing inflammation. oxidative stress, and senescence as well as enhancing susceptibility to apoptosis. Viral protein-generated EMVs may contribute to the increased risk of vascular disease in patients with HIV-1. NEW & NOTEWORTHY Human immunodeficiency virus (HIV)1-related proteins glycoprotein (gp) 120 and transactivator of transcription (Tat)-mediated endothelial damage and dysfunction are poorly understood. Endothelial microvesicles (EMVs) serve as indicators and potent mediators of endothelial dysfunction. In the present study we determined if HIV-1 R5- and X4-tropic gp120 and Tat stimulate EMV release in vitro and if viral protein-induced EMVs are deleterious to endothelial cell function. gp120 and Tat induced a marked increase in EMV release. Viral protein-induced EMVs significantly increased endothelial cell inflammation, oxidative stress, senescence, and apoptotic susceptibility in vitro. gpl 20- and Tat-derived EMVs promote a proinflammatory, pro-oxidative. prosenescent, and proapoptotic endothelial phenotype and may contribute to the endothelial damage and dysfunction associated with gp120 and Tat.

The HIV-1 envelope (Env) glycoprotein is the primary target of the humoral immune response and a critical vaccine candidate. However, Env is densely glycosylated and thereby substantially protected from neutralisation. Importantly, glycan N301 shields V3 loop and CD4 binding site epitopes from neutralising antibodies. Here, we use molecular dynamics techniques to evaluate the structural rearrangements that maintain the protective qualities of the glycan shield after the loss of glycan N301. We examined a naturally occurring subtype C isolate and its N301A mutant; the mutant not only remained protected against neutralising antibodies targeting underlying epitopes, but also exhibited an increased resistance to the VRC01 class of broadly neutralising antibodies. Analysis of this mutant revealed several glycans that were responsible, independently or through synergy, for the neutralisation resistance of the mutant. These data provide detailed insight into the glycan shield's ability to compensate for the loss of a glycan, as well as the cascade of glycan movements on a protomer, starting at the point mutation, that affects the integrity of an antibody epitope located at the edge of the diminishing effect. These results present key, previously overlooked, considerations for HIV-1 Env glycan research and related vaccine studies.