Extracellular vesicles (EVs) or exosomes have been implicated in the pathophysiology of infections and cancer. The negative regulatory factor (Nef) encoded by simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) plays a critical role in the progression to AIDS and impairs endosomal trafficking. Whether HIV-1 Nef can be loaded into EVs has been the subject of controversy, and nothing is known about the connection between SIV Nef and EVs. We find that both SIV and HIV-1 Nef proteins are present in affinity-purified EVs derived from cultured cells, as well as in EVs from SIV-infected macaques. Nef-positive EVs were functional, i.e., capable of membrane fusion and depositing their content into recipient cells. The EVs were able to transfer Nef into recipient cells. This suggests that Nef readily enters the exosome biogenesis pathway, whereas HIV virions are assembled at the plasma membrane. It suggests a novel mechanism by which lentiviruses can influence uninfected and uninfectable, i.e., CD4-negative, cells. IMPORTANCE Extracellular vesicles (EVs) transfer biologically active materials from one cell to another, either within the adjacent microenvironment or further removed. EVs also package viral RNAs, microRNAs, and proteins, which contributes to the pathophysiology of infection. In this report, we show that both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) incorporate the virus-encoded Nef protein into EVs, including EVs circulating in the blood of SIV-infected macaques and that this presents a novel mechanism of Nef transfer to naive and even otherwise non-infectable cells. Nef is dispensable for viral replication but essential for AIDS progression in vivo . Demonstrating that Nef incorporation into EVs is conserved across species implicates EVs as novel mediators of the pathophysiology of HIV. It could help explain the biological effects that HIV has on CD4-negative cells and EVs could become biomarkers of disease progression.

Isolation of extracellular vesicles (EVs) from cell culture supernatant or plasma can be accomplished in a variety of ways. Common measures to quantify relative success are: concentration of the EVs, purity from non‐EVs associated protein, size homogeneity and functionality of the final product. Here, we present an industrial‐scale workflow for isolating highly pure and functional EVs using cross‐flow based filtration coupled with high‐molecular weight Capto Core size exclusion. Through this combination, EVs loss is kept to a minimum. It outperforms other isolation procedures based on a number of biochemical and biophysical assays. Moreover, EVs isolated through this method can be further concentrated down or directly immunopurified to obtain discreet populations of EVs. From our results, we propose that cross‐flow/Capto Core isolation is a robust method of purifying highly concentrated, homogenous, and functionally active EVs from industrial‐scale input volumes with few contaminants relative to other methods.

The transition from transcription initiation into elongation is controlled by transcription factors, which recruit positive transcription elongation factor b (P-TEFb) to promoters to phosphorylate RNA polymerase II. A fraction of P-TEFb is recruited as part of the inhibitory 7SK small nuclear ribonucleoprotein particle (snRNP), which inactivates the kinase and prevents elongation. However, it is unclear how P-TEFb is captured from the promoter-bound 7SK snRNP to activate elongation. Here, we describe a mechanism by which transcription factors mediate the enzymatic release of P-TEFb from the 7SK snRNP at promoters to trigger activation in a gene-specific manner. We demonstrate that Tat recruits PPM1G/PP2Cγ to locally disassemble P-TEFb from the 7SK snRNP at the HIV promoter via dephosphorylation of the kinase T loop. Similar to Tat, nuclear factor (NF)-κB recruits PPM1G in a stimulus-dependent manner to activate elongation at inflammatory-responsive genes. Recruitment of PPM1G to promoter-assembled 7SK snRNP provides a paradigm for rapid gene activation through transcriptional pause release.

HIV encodes Tat, a small protein that facilitates viral transcription by binding an RNA structure (trans-activating RNA [TAR]) formed on nascent viral pre-messenger RNAs. Besides this well-characterized mechanism, Tat appears to modulate cellular transcription, but the target genes and molecular mechanisms remain poorly understood. We report here that Tat uses unexpected regulatory mechanisms to reprogram target immune cells to promote viral replication and rewire pathways beneficial for the virus. Tat functions through master transcriptional regulators bound at promoters and enhancers, rather than through cellular ‘TAR-like’ motifs, to both activate and repress gene sets sharing common functional annotations. Despite the complexity of transcriptional regulatory mechanisms in the cell, Tat precisely controls RNA polymerase II recruitment and pause release to fine-tune the initiation and elongation steps in target genes. We propose that a virus with a limited coding capacity has optimized its genome by evolving a small but ‘multitasking’ protein to simultaneously control viral and cellular transcription.