T-Cell Subsets That Harbor Human Immunodeficiency Virus (HIV) In Vivo: Implications for HIV Pathogenesis. homogeneous pool of target cells. Instead, individual subsets of CD4+ T cells and myeloid cells are thought to be differentially infected by the virus than resting cells (Alexaki et al., 2008). One explanation for limited infectivity of resting cells, compared to activated and dividing cells, is low intracellular concentrations of nucleotides within resting cells (Goldstone et al., 2012). In resting cells nucleotides are hydrolyzed by the host protein SAM domain and HD domain-containing protein 1 (SAMHD1) (Goldstone et al., 2012). The activity of SAMHD1 is thought to involve its phosphorylation and is active in resting CD4+ T cells and myeloid cells, and its expression and activity are thought to limit infection of these cells by HIV/SIV (Baldauf et al., 2012; Laguette et al., 2011). Recent studies have implicated viral protein x Rabbit polyclonal to LRRIQ3 (Vpx), a viral accessory protein expressed by some strains of SIV and by HIV-2, in binding to SAMHD1 leading to its proteasomal degradation (Laguette et al., 2011). SIVs used to experimentally infect Asian macaques and HIV-2 originate from SIVsmm, which is a virus that naturally infects sooty mangabeys in western Africa and expresses the viral accessory protein Vpx. HIV-1 and other immunodeficiency lentiviruses, like SIVagm, do not express Vpx Ropinirole HCl (Fregoso et al., 2013). Given the differential expression of Vpx by HIVs and SIVs one prediction might be that these viruses differ in their proclivity to infect resting CD4+ T cells and myeloid cells (Figure 1C). It was therefore possible to examine the proclivity of viruses with and without Vpx to infect different cellular targets. We hypothesized that viruses encoding Vpx would Ropinirole HCl infect CD28+ memory CD4+ T cells and myeloid cells more efficiently than viruses without Vpx. Open in a separate window Figure 1 Memory CD4+ T cells and Ropinirole HCl myeloid cells express SAMHD1SAMHD1 mRNA in na?ve CD4+ Ropinirole HCl T cells, CD28+ memory CD4+ T cells, CD28? memory CD4+ T cells, and myeloid cells in peripheral blood of SIV-uninfected (A) and SIV-infected (B) rhesus macaques. Expression relative to -actin mRNA. (C) Total Ropinirole HCl and phosphorylated SAMHD1 protein in na?ve CD4+ T cells, CD28+ memory CD4+ T cells, CD28? memory CD4+ T cells, and myeloid cells in peripheral blood of SIV-uninfected animals. Forty g of primary cell extract or 20 g of THP-1 cell extract were separated by SDS-PAGE and Western blotted using antibodies against SAMHD1, phosphorylated SAMHD1 or -actin. Horizontal lines indicate the median. Western blots are representative of three experiments. Myeloid cells contain little, if any, viral DNA in mucosal sites Given that mucosal sites have been shown to be massively depleted of CD4+ T cells during the acute phase of infection and throughout the chronic phase of infection (Brenchley et al., 2004b; Mattapallil et al., 2005a; Picker et al., 2004; Veazey et al., 1998), we hypothesized that without preferred CD4+ T cell targets, viruses expressing Vpx would more efficiently infect myeloid cells at mucosal sites. Therefore, we flow cytometrically sorted the few memory CD28+, CD28? memory CD4+ T cells when possible, and myeloid cells from small intestine, large intestine, liver, and BAL of SIV-infected Asian macaques (Figure 2). The myeloid cells were sorted as to include all myeloid cell types, including macrophages, monocytes, and the various subsets of dendritic cells (gating strategy in Figure S1). Each subset of CD4+ T cells was not equally abundant at each anatomical site. For example, na?ve CD4+ T cells and differentiated CD28? memory CD4+ T cells were not abundant in the liver or within the GI tract (Figure 2A-C). Thus we were unable to sort sufficient numbers of cells corresponding to each CD4+ T cell subset. However, it was possible to amplify viral DNA from CD28+ memory CD4+ T cells from all four mucosal sites of every animal we examined. Moreover, we successfully amplified viral DNA from na?ve CD4+ T cells from the small and large intestines of approximately 50% of the animals. There were very low frequencies of na?ve CD4+ T cells in the liver of all animals, but we were able to obtain sufficient numbers of liver na?ve CD4+ T cells from two animals in our cohorts to amplify.