3C). by the absence of Vpr, the transcriptional activity of the viral long terminal repeat (LTR) from Vpr-deficient proviruses was significantly reduced. Together, these results characterize a novel postintegration restriction of HIV-1 replication in MDDCs and show that the interaction of Vpr with the DCAF1/DDB1 E3 ubiquitin ligase complex and the yet-to-be-identified host factor might alleviate this restriction by inducing transcription from the viral LTR. Taken together, these findings identify a robust cell culture system that is amenable to addressing mechanisms underlying Vpr-mediated enhancement of HIV-1 replication. IMPORTANCE Despite decades of work, the function of the HIV-1 protein Vpr remains poorly understood, primarily due to the lack of an cell culture system that demonstrates a deficit in replication upon infection with viruses in the absence of Vpr. In this report, we describe a novel cell infection system that utilizes primary human dendritic cells, which display a robust decrease in viral replication upon infection with Vpr-deficient HIV-1. We show that this replication difference occurs in a single round of infection and is due to Axitinib decreased transcriptional output from the integrated viral genome. Viral transcription could be rescued by virion-associated Vpr. Using mutational analysis, we show that domains of Vpr involved in binding to the DCAF1/DDB1/E3 ubiquitin ligase complex and prevention of cell cycle progression into mitosis are required for LTR-mediated viral expression, suggesting that the evolutionarily conserved G2 cell cycle arrest function of Vpr is essential for HIV-1 replication. but are absolutely essential for replication (1). These proteins serve to counteract host restriction factors that would normally limit HIV-1 infection (1, 2). Of the accessory proteins encoded by HIV-1, Vpr is the only one whose function remains relatively unclear. Vpr is a small, 96-amino-acid, 14-kDa protein that is packaged into the budding virion through associations with the p6 region of Gag (3,C10). This association allows Vpr to be present in the cell at a relatively high quantity (200 to 300 molecules/virion) upon initial infection (11). Previous studies have extensively characterized the outcome of Vpr expression in various cell types. In cycling cells, Vpr expression results in G2/M cell cycle arrest, which Axitinib culminates in the induction of apoptosis (12,C14). It is well established that Vpr-mediated G2/M cell cycle arrest is mediated through its association with the Cul4A/DCAF/DDB1 E3 (CRL4DCAF1) ubiquitin ligase complex (15,C17). In addition, HIV-1 Vpr has been shown to recruit and degrade a number of DNA damage response (DDR) proteins, including the SLX4-SLX1/MUS81-EME1 structure-specific endonuclease complex (SLX4com), uracil DNA glycosylase 2 (UNG2), and helicase-like transcription factor (HLTF) (18,C21), via the CRL4DCAF1 complex, resulting in G2/M cell cycle arrest, although the role that this process plays during HIV-1 infection still remains unclear. Although a number of previous studies have examined the requirement Axitinib of Vpr for HIV-1 replication in various cell types, including primary CD4+ T cells and monocyte-derived macrophages (MDMs), differences in virus replication have not been consistently observed (18, 20, 22,C26). Vpr expression is dispensable for infection in activated CD4+ T cells (22,C25, 27), presumably due to Axitinib the well-characterized cytostatic and cytopathic functions of Vpr in cycling cells (12). In contrast, recent studies with MDMs suggest that Vpr is necessary for HIV-1 envelope (Env) expression, and the purported Rabbit Polyclonal to MOK consequence of infection of MDMs with Vpr-deficient viruses was reported to be decreased viral production and reduced cell-to-cell spread to CD4+ T cells (22, 28). Notably, there has been considerable heterogeneity in replication differences between wild-type (WT) and Vpr-deficient viruses and host responses to virus infection in MDMs, presumably due to donor and experimental variability between studies (12, 29, 30). Additionally, it has also been reported that Vpr expression in macrophages can both inhibit and induce type I interferon (IFN) responses (18, 28, 31,C34). Dendritic cells (DCs) are sentinel cells that bridge innate and Axitinib adaptive immunity (35). They actively patrol peripheral tissues, including mucosal sites of HIV-1 transmission, in search of foreign pathogens. Because of this, monocyte-derived DCs (MDDCs) are among the first cells to interact with HIV-1 upon sexual transmission of the virus (36,C40). While MDDCs are less susceptible to infection than activated CD4+ T cells and macrophages, they are still able to be infected at a low but consistent level (41,C44). In contrast to work with MDMs and CD4+ T cells, there have been isolated descriptions of the effects of Vpr on the.

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