br Results br Discussion Cell contact mediated spread

Results
Discussion Cell-contact-mediated spread of HIV-1 has been estimated to be at least an order of magnitude more efficient compared to passive dissemination of particles through the extracellular milieu. Thus, this form of viral transmission can have significant effects on the pathogenesis of HIV. First, this mode of transmission can mediate the spread of HIV-1 within T-cell-dense anatomical compartments (Law et al., 2016, Murooka et al., 2012, Sewald et al., 2012, Sewald et al., 2015). Second, the ability of cell-to-cell transmission to generate infected folate analogue with multiple proviruses could facilitate recombination, thus increasing viral diversity (Dang et al., 2004, Del Portillo et al., 2011, Dixit and Perelson, 2004, Jung et al., 2002). Third, HIV-1 cell-to-cell transmission could be an important mechanism for the massive depletion of CD4+ T cells in untreated infected patients (Doitsh et al., 2010). Our present study proposes that an additional role of HIV-1 cell-to-cell transmission in vivo is the generation of latently infected cells by direct infection of resting CD4+ T cells. Latently infected resting CD4+ T cells represent the most important source of rebounding virus when treatment is interrupted (Ruelas and Greene, 2013). The pool of latently infected cells is comprised of a phenotypically heterogeneous population of cells (Churchill et al., 2016). Therefore, a deep understanding of how these cells are generated and maintained and how proviral latency is regulated is essential for the development of any curative approach. The most widely accepted mechanism for the generation of latently infected resting CD4+ T cells is the return of fully or intermediately activated cells to a resting state after infection with HIV-1 (Shan et al., 2017). Based on this mechanism, several cell line and primary cell in vitro models have been developed over the years to study HIV-1 transcription and latency (Pace et al., 2011). These models have been instrumental in the characterization of fundamental processes that regulate HIV-1 transcription and latency. However, these models are limited by the use of cells that may not fully represent the natural pool of latently infected cells in vivo and may be biased toward chromatin-dependent repression. Models of HIV-1 latency based on direct infection of resting CD4+ T cells have been less popular, partly because of the relative resistance of these cells to HIV-1 infection. These cells have been shown to express some restriction factors that limit HIV infection (Baldauf et al., 2012, Zack et al., 2013). Despite the presence of these factors, it has been demonstrated that resting cells can still be infected with HIV directly and at a sufficient frequency to allow the study of HIV latency in this context (Agosto et al., 2007, Lassen et al., 2012, Plesa et al., 2007, Swiggard et al., 2005, Vatakis et al., 2007). Studying HIV latency in the context of direct infection of resting CD4+ T cells can overcome some of the shortcomings of models involving activated T cells and cell lines. In vitro models of direct infection of primary resting CD4+ T cells preserve the natural diversity of cellular phenotypes because artificial manipulations are minimized. Resting CD4+ T cells are already at a state predisposed to proviral latency; hence, studying HIV latency in this context could reveal previously unrecognized mechanisms for regulating HIV transcription. The in vitro model presented in this study has the added benefit of incorporating T cell-T cell interactions as part of the process of generating latent infection. With this model of HIV-1 latency, we demonstrate that productively infected activated CD4+ T cells directly transmit HIV-1 to uninfected resting CD4+ T cells, leading to the generation of pools of latently infected resting CD4+ T cells. This mechanism for the generation of latently infected cells has been underappreciated and likely represents an important outcome of HIV-1 spread in vivo before the initiation of effective therapy.