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 checkpoints, including transcription factor recruitment to the long terminal repeat (LTR), transcription initiation, and elongation. Biochemical and functional studies show that SE inhibit HIV-1 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. Chromatin immunoprecipitation-real-time quantitative PCR (ChIP-RT-qPCR) and electrophoretic mobility shift assay (EMSA) analysis of infected cells identified the human transcription factors NF-κB and Sp1, as well as RNA polymerase (Pol) II and the viral protein transcriptional activator (Tat), as targets of SE. Of interest, SE inhibited HIV-1 LTR activation mediated by HIV-1 or Tat, but not by the mitogen phorbol myristate acetate (PMA) or tumor necrosis factor alpha (TNF-α). SE inhibited the DNA binding activities of NF-κB and Sp1 and blocked the recruitment of these transcription factors and Pol II to the HIV-1 LTR promoter. Importantly, SE directly blocked NF-κB, Sp1, and Pol II binding to the LTR and inhibited the interactions of Tat/NF-κB and Tat/Sp1, suggesting that SE-mediated inhibition of the functional quadripartite complex NF-κB-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.IMPORTANCE 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 a need to identify 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-κB 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.

Keywords: HIV-1; LTR; NF-κB; RNA Pol II; Sp1; Tat; exosomes; long terminal repeat; promoter; semen; transcription.