Antiretroviral Drugs Alter the Content of Extracellular Vesicles from HIV-1-Infected Cells

Abstract

To date, the most effective treatment of HIV-1 is a combination antiretroviral therapy (cART), which reduces viral replication and reverses pathology. We investigated the effect of cART (RT and protease inhibitors) on the content of extracellular vesicles (EVs) released from HIV-1-infected cells. We have previously shown that EVs contain non-coding HIV-1 RNA, which can elicit responses in recipient cells. In this manuscript, we show that TAR RNA levels demonstrate little change with the addition of cART treatment in cell lines, primary macrophages, and patient biofluids. We determined possible mechanisms involved in the selective packaging of HIV-1 RNA into EVs, specifically an increase in EV-associated hnRNP A2/B1. More recent experiments have shown that several other FDA-approved drugs have the ability to alter the content of exosomes released from HIV-1-infected cells. These findings on cART-altered EV content can also be applied to general viral inhibitors (interferons) which are used to treat other chronic infections. Additionally, we describe unique mechanisms of ESCRT pathway manipulation by antivirals, specifically the targeting of VPS4. Collectively, these data imply that, despite antiretroviral therapy, EVs containing viral products are continually released and may cause neurocognitive and immunological dysfunction.

Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

“…Second, in fractions 14.4-15.6, CD63 was present in moderate amounts, whereas levels of CD9 and CD81 increased. This population was also consistent with an exosomal category containing viral particles demonstrated in previous publications [16,50,79], although, in our case, this group is likely to be exosomes that are larger in size or more dense than the smaller ones seen in fractions 8.4-12.0. Finally, the 16.8-18.0 fractions showed a distinct reduction of CD63 but a drastic increase in CD9, CD81, and Actin.…”

“…First, fractions 8.4-12.0 contained high levels of CD63 and moderate levels of CD9 and CD81. Previous publications have classified this group as true exosomes, distinct from viral particles and apoptotic bodies [16,50,79]. Second, in fractions 14.4-15.6, CD63 was present in moderate amounts, whereas levels of CD9 and CD81 increased.…”

“…Our laboratory has substantial experience in the isolation and preparation of exosomes and EVs for characterization and functional analysis, particularly in the context of viral infection [12,21,22,26,27,[50][51][52]. For the experiments used here, 293T cells and VP40 clones were grown in exosome-free DMEM containing 10% heat-inactivated FBS (ultracentrifuged at 100 000 ×g for 90 minutes to remove vesicles), 1% l-glutamine, and 1% streptomycin/penicillin (Quality Biological).…”

“…It should be noted that ZetaView analysis detects the total number of vesicles or large particles present, with exosomes comprising only a fraction of these. Therefore, to determine the distribution of true exosomes containing VP40, we used our previously published strategy of exosome characterization by density gradient ultracentrifugation [50]. Through this methodology, we have shown that it is possible to clearly separate out exosomes from potentially contaminating viral particles, thus making more detailed characterizations of specific EV populations possible.…”

Large‐scale, cross‐flow based isolation of highly pure and endocytosis‐competent extracellular vesicles

“…EVs were resuspended in proportional volumes of PBS (see methods). The tetraspanin molecules CD9 and CD63 [8,25,26,38,41], the ESCRT-I associated tumour suppressor gene 101 (TSG101) [19,20,25,42–44], and the late-endosome/lipid raft protein Flotillin-2 [7,45–47] were selected as markers. All were present in EV obtained by these three methods, but not the intracellular control Tubulin (Figure 2(a)).…”