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  • SAMHD emerges as a ubiquitous and potent barrier to

    2018-10-20

    SAMHD1 emerges as a ubiquitous and potent barrier to productive HIV-1 infection in DCs, myeloid cells, and resting CD4+ T cells. Thus, it appeared likely that SAMHD1 might also be effective when transducing HSPCs with lentiviral vectors. Efficient transduction of HSPCs requires prestimulation with cytokines. We were surprised to find that SAMHD1 levels were very low or even absent in freshly isolated HSPCs, while SAMHD1 was already highly expressed in HSPCs 2h after the start of culturing. Therefore, we hypothesized that Vpx-mediated SAMHD1 degradation would result in a significant increase in the transduction rate of HSPCs with lentiviral vectors. Surprisingly, this was not the case. We even tested MOIs of 1 and 10 to exclude that the conventionally used high MOI of 100 masked the effects of Vpx+ VLP pretreatment (data not shown). To validate the results we obtained by monitoring GFP expression, we also quantified the number of chromosomally integrated proviral DNA: results were identical with a lack of increase of proviral DNA in HSPCs, while there was a substantial increase in MDMs after pretreatment with Vpx+ viruses. We also quantified the viral DNA intermediates during reverse transcription in HSPCs and MDMs pretreated with Vpx. We observed no increase in HSPCs and a vigorous one in MDMs. Thus, overall, Vpx+ viruses seemed to have no role in relieving the block in lentiviral transduction in HSPCs. We wondered if Vpx+ viruses participate in the degradation of SAMHD1 in HSPCs. Indeed, that was the case: Vpx degraded SAMHD1 in HSPCs, but residual levels tended to be higher in HSPCs than in MDMs. Considering the data so far, we expected that Vpx had a more modest effect in HSPCs than MDMs and thus a one-sided statistical test can be justified; using a two-sided statistical test gave no significant result and thus we prefer to define it more as a trend than as a significant difference. We further verified to what extent Vpx affected the activity of SAMHD1 or p-SAMHD1. p-SAMHD1 has no anti-HIV activity while still being able to deplete the dNTP pool (White et al., 2013; Cribier et al., 2013). Indeed, dpn Supplier of SAMHD1 negatively regulates SAMHD1\'s RNase activity and impedes HIV-1 restriction (Ryoo et al., 2014). Taking into account our data so far, we were not surprised that HSPCs had a much higher p-SAMHD1 protein expression than MDMs, and that Vpx had only a minor effect on decreasing p-SAMHD1 in HSPCs. These results might explain that despite the overall substantial Vpx-mediated decrease of SAMHD1 in HSPCs, we did not observe any clear change in the transduction rate. While the mechanism of SAMHD1\'s anti-HIV activity (i.e., dNTPase activity vs RNase activity) is controversial, we investigated whether Vpx-mediated degradation of SAMHD1 affects the dNTP pool in HSPCs. Vpx-mediated degradation of SAMHD1 only slightly increased the dNTP pool in HSPCs, but greatly in MDMs, as reported (Lahouassa et al., 2012). This negative result might be explained by the lack of degradation of p-SAMHD1. We observed substantial increases of the dNTP pools when adding dNs and less prominent ones when adding dNTPs to the two cell types; again, it was much more pronounced in MDMs. Lentiviral reverse transcriptases uniquely remain functional even at the low dNTP concentrations found in non-dividing cells (Amie et al., 2013). Thus, the regulation of the dNTP pools by SAMHD1 may be an epiphenomenon of studying SAMHD1\'s anti-HIV activity rather than being causally related. However, we cannot exclude that changes in the dNTP pool may contribute to a minor extent to the reverse transcriptase activity. The assays used above might confound a minor role of Vpx-mediated degradation of SAMHD1 since the consecutive treatment first with viruses carrying Vpx or silencing SAMHD1 with shRNA and the adding reporter viruses may not necessarily target the same cells. In contrast, this is the case when using viruses carrying Vpx and encoding GFP, and thus, these viruses may even reveal minor effects of Vpx on lentiviral transduction rate (Lahouassa et al., 2012; Baldauf et al., 2012). Indeed, by this approach, we found a minor but significant increase of GFP+ cells going along with a decrease in SAMHD1. Moreover, the residual cell population that expresses high amounts of SAMHD1 remained refractory to lentiviral transduction.