Extended Data Fig. 6: Principles of X4 HIV-1 fusion and infection assays in resting CD4+ T cells.
Two different types of conditions for HIV-1 challenge were used depending on the scientific question, that is the target protein of interest and the step of the HIV replication cycle under investigation. a, First, to characterize the effect of KOs of either host dependency factors (CXCR4, CD4) or restriction factors (PSGL-1) for HIV binding and entry, we employed the well-established HIV fusion assay without spinoculation. Here, cells were exposed to the X4 HIV-1 BlaM-Vpr inoculum for 4 hours at 37 °C. Cells with either CXCR4 KO or nucleofected with NTC-gRNA were used. Representative dot plots of the flow cytometric detection of the CCF2 substrate cleavage by BlaM in viable cells after virion fusion are shown. As specificity controls, cells were pretreated with either the HIV-1 fusion inhibitor T20 or an anti-CD4 mAb. One representative experiment is shown (n = 7). b, Second, to characterize the role of potential cellular restriction factors (SAMHD1, MX2) or host dependency factors (CPSF6) at post-entry steps of the replication cycle (for example at reverse transcription, nuclear import, integration) we sought to efficiently overcome the natural restriction at virion entry and allow a high-level of virus delivery (see Fig. 3 and Fig. 5). In this context, we applied spinoculation at 650 g for 150 min at 37 °C. To prove that this is optimal for high-level virion delivery into resting CD4+ T cells, we performed a quantitative HIV-1 fusion assay. This spinoculation condition allowed X4 HIV-1 to enter into virtually every resting CD4+ T cell that expresses the co-receptor CXCR4, that is typically around 95% of the cell population. As specificity control, the small molecule inhibitor AMD3100, which blocks CXCR4-dependent HIV-1 fusion, was used. Means ± s.e.m. are shown (n = 4).
