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.

Acellular particles (extracellular vesicles and membraneless condensates) have important research, drug discovery, and therapeutic implications. However, their isolation and retrieval have faced enormous challenges, impeding their use. Here, a novel size-guided particle purification liquid chromatography (PPLC) is integrated into a turbidimetry-enabled system for dye-free isolation, online characterization, and retrieval of intact acellular particles from biofluids. The chromatographic separation of particles from different biofluids—semen, blood, urine, milk, and cell culture supernatants—is achieved using a first-in-class gradient size exclusion column (gSEC). Purified particles are collected using a fraction collector. Online UV–Vis monitoring reveals biofluid-dependent particle spectral differences, with semen being the most complex. Turbidimetry provides the accurate physical characterization of seminal particle (Sp) lipid contents, sizes, and concentrations, validated by a nanoparticle tracking analysis, transmission electron microscopy, and naphthopyrene assay. Furthermore, different fractions of purified Sps contain distinct DNA, RNA species, and protein compositions. The integration of Sp physical and compositional properties identifies two archetypal membrane-encased seminal extracellular vesicles (SEV)—notably SEV large (SEVL), SEV small (SEVS), and a novel non-archetypal-membraneless Sps, herein named membraneless condensates (MCs). This study demonstrates a comprehensive yet affordable platform for isolating, collecting, and analyzing acellular particles to facilitate extracellular particle research and applications in drug delivery and therapeutics. Ongoing efforts focus on increased resolution by tailoring bead/column chemistry for each biofluid type.

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