Semen Exosomes Promote Transcriptional Silencing of HIV-1 by Disrupting NF-κB/Sp1/Tat Circuitry

Abstract

Exosomes play various roles in host responses to cancer and infective agents, and semen exosomes (SE) inhibit HIV-1 infection and transmission, although the mechanism(s) by which this occurs is unclear. Here, we show that SE block HIV-1 proviral transcription at multiple transcriptional check points including transcription factor recruitment to the LTR, and transcription initiation and elongation. Biochemical and functional studies show that SE inhibit HIV-1 long terminal repeat (HIV-LTR)-driven viral gene expression and virus replication. Through partitioning of the HIV-1 RNA, we found that SE reduced the optimal expression of various viral RNA species. CHIP-RT-qPCR and EMSA analysis of infected cells identified human transcription factors NF-kB and Sp1, as well as RNA Pol II and the viral protein Tat as targets of SE. Of interest, SE inhibited HIV-1 LTR activation mediated by HIV-1 or Tat, but not by the mitogen PMA or TNFα. SE inhibited the DNA binding activities of NF-kB and Sp1 and blocked the recruitment of these transcription factors and Pol II to the HIV LTR promoter. Importantly, SE directly blocked NF-kB, Sp1, and Pol II binding to the LTR, and inhibited the interactions of Tat/NF-kB and Tat/Sp1, suggesting that SE-mediated inhibition of the functional quadripartite complex-NF-kB-Sp1-Pol II-Tat, may be a novel mechanism of proviral transcription repression. These data provide a novel molecular basis for SE-mediated inhibition of HIV-1 and identify Tat as a potential target of SE. HIV is most commonly transmitted sexually, and semen is the primary vector. Despite progress in studies of HIV pathogenesis and the success of combination antiretroviral therapy in controlling viral replication, current therapy cannot completely control sexual transmission. Thus, there is need for identifying effective methods of controlling HIV replication and transmission. Recently, it was shown that human semen contains exosomes that protect against HIV infection in vitro. In this study, we identified a mechanism by which semen exosomes inhibited HIV-1 RNA expression. We found that semen exosomes inhibit recruitment of transcription factors NF-kB and Sp1, as well as RNA Pol II to the promoter region in the 5' long terminal repeat (LTR) of HIV-1. The HIV-1 early protein, transcriptional activator (Tat) was a target of semen exosomes, and semen exosomes inhibited the binding and recruitment of Tat to the HIV-1 LTR.

Semen exosomes inhibit HIV infection and HIV‐induced proinflammatory cytokine production independent of the activation state of primary lymphocytes

“…We conclude from these data that exposure of lymphocytes to the level of SE used in this study (100 µg·mL −1 ) does not alter basal or stimuli‐induced cell death. These results are consistent with observations in cell lines .…”

“…Prior studies have shown that SE do not alter basal activity of the HIV promoter but the effect of SE on activation of cellular programs is unknown. To evaluate the effect of SE on viral and cellular activation, we examined the response of primary PBMCs isolated from the whole blood of 11 HIV‐infected individuals that were receiving suppressive ART (PBMC ART ) and had suppressed viral loads (Table ).…”

Semen exosomes

“…These studies will determine if SE induce viral reactivation in lymphocytes isolated from HIV-infected ART-suppressed individuals. Of note, SE had no effect on viral reactivation in an in vitro cell model of HIV-1 latency [139].…”

“…Although the exact mechanism(s) by which SE inhibit HIV-1 infection is unknown, studies show that multiple steps in the HIV-1 lifecycle are affected by SE including: (1) conversion of ssRNA to dsDNA (reverse transcription), (2) copying of integrated proviral DNA, and (3) transcription of viral RNA from proviral DNA [63,139], as depicted in Figure 5. Since three of these replication steps are key points where HIV-1 mutations arise, SE may inhibit infection through multiple mechanisms [137].…”

“…It has been observed that SE target viral factors and specifically blocks HIV-1 lifecycle events even when viral binding and entry steps are bypassed [139]. SE inhibit Tat…”