PMC:3320587 / 24124-26916 JSONTXT

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The HIV-1Lai/Bal-Env infectious clone is isogenic for the entire sequence of the parental HIV-1Lai infectious clone except for the substitution of the HIV-1Bal envelope co-receptor binding region in place of the HIV-1Lai envelope co-receptor binding region. We note that we confirmed that HIV-1Lai/Bal-Env grew in PBMC at a similar rate to wild-type HIV-1Bal, indicating proper co-receptor engagement and internalization of this infectious clone (data not shown).\nNext, we knocked down NFAT5 mRNA levels in MDM using an siRNA that suppresses both NFAT5 mRNA and NFAT5 protein levels [31]. As shown in Figure 2, transfection of the siRNA specific for NFAT5 reduces NFAT5 mRNA levels in both MTb-uninfected (p = 0.048) and MTb-infected (p = 0.021) MDM as compared to transfection of control GFP siRNA into MTb-uninfected or -infected MDM (Figure 2B). We note that although siRNA normally is effective for 48–72 hours in cell lines that divide rapidly, in human MDM, which are non-dividing cells, siRNA to host factors remains detectable and functional up to at least 15 days post-transfection [47].\nMDM in which NFAT5 expression had been inhibited with NFAT5 siRNA or that were transfected with control GFP siRNA were then infected with 1000 TCID50 of HIV-1Lai/Bal-env. After overnight virus infection, the cells were then co-infected with the MTb clinical strain CDC1551. Free virus levels were then measured in culture supernatants from MDM transfected with NFAT5-specific siRNA or GFP control siRNA at days 6, 9 and 12 post-HIV-1 infection to measure the impact of NFAT5 inhibition on MTb-induced HIV replication.\nAs shown in Figure 2C, HIV-1 replication was suppressed at days 6 and 9 post-infection in the NFAT5 siRNA-treated cells as compared to cells treated with control siRNA, and it was significantly inhibited by day 12 post-infection (p\u003c0.05) (Figure 2C). Thus, knock down of NFAT5 expression significantly impairs HIV-1 subtype B replication in MDM co-infected with 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