NFAT5 is important for enhancement of HIV-1 subtype C replication in response to MTb infection
As shown in Figure 1, the subtype C LTR was the most active of the HIV-1 LTR subtypes in the reporter assays. Subtype C LTRs generally have three functional NF-κB sites in their LTRs, and subtype C is the predominant viral subtype in the African and Asian HIV-1 epidemics where MTb co-infection is extremely common [1], [2], [28], [52]. We thus next extended our analysis of the role of NFAT5 in MTb/HIV-1 co-infection to a subtype C isolate. We first constructed an infectious molecular clone of the HIV-1 subtype C primary isolate HIV-198IN22, which has two NFAT5 binding sites in its LTR and three NF-κB sites (Figure 6A). We disrupted the two NFAT5 binding sites by changing the TT of each site to CC to create a mutant virus that we named HIV-198IN22-N5-Mut. Bulk PBMC from four normal donors were infected overnight with 1000 TCID50 of wild-type HIV-198IN22 or HIV-198IN22-N5-Mut and the cultures were then co-infected with MTb CDC1551 or left infected with virus alone. At day 11 post-viral infection, wild-type HIV-198IN22 replication was greater, but not significantly so, as compared to replication of HIV-198IN22 NFAT5-Mut (Figure 6B). However, in the context of MTb co-infection, wild-type HIV-198IN22 replication was significantly increased (p<0.05) at day 11 over HIV-198IN22 NFAT5-Mut replication (Figure 6C), indicating that the absence of NFAT5 binding sites was particularly detrimental to virus replication in the presence of MTb co-infection. Thus, even when three functional NF-κB binding sites are present in the LTR, as in the HIV-1 subtype C infectious clone studied here, disruption of NFAT5 binding to the LTR impairs virus replication in response to MTb co-infection.
10.1371/journal.ppat.1002620.g006 Figure 6  Disruption of the NFAT5 site in HIV-1 subtype C significantly impairs MTb-induced viral replication.
(A) Mutation of the NFAT5 binding site in an HIV-1 subtype C infectious molecular clone. NFAT5 binding site mutations were introduced into the LTR of subtype C HIV-198IN22-WT. The NFAT5 binding site mutant (HIV-198IN22 N5-Mut) LTR sequence is shown alongside that of HIV-198IN22-WT. The HIV-198IN22 isolate analyzed here contains three NF-κB and two NFAT5 binding sites. The unique 3′ terminal adenine, which is important for NFAT5 binding to its site, is shown in blocks. Mutations were introduced into the 3′ LTR of the HIV-198IN22-WT proviral sequence. PBMC from four normal donors were infected with 1000 TCID50 of HIV-198IN22–WT or HIV-198IN22-N5-Mut. Cells were then (B) left infected with virus alone or (C) co-infected with MTb strain CDC1551 (10∶1 cells∶bacilli). Culture supernatants were collected at days 3, 7 and 11 post-infection and viral p24 levels were measured. Replication of HIV-198IN22-N5-Mut was significantly reduced at day 11 post-infection in comparison to HIV-198IN22–WT (*, p<0.05) in the presence of MTb co-infection.