To perform these experiments, we constructed a panel of infectious HIV-1Lai/Bal-env molecular clones where either the NFAT5 site was specifically disrupted or the two NF-κB binding sites were either individually or dually disrupted (Figure 3A). Specifically, we constructed a clone in which the NFAT5 binding site was mutated (named HIV-1Lai/Bal-env-N5-Mut) by changing the CC dinucleotide to TT in the core NFAT5 binding site and introduced substitution mutations into the NF-κB/NFAT5 shared binding element that we predicted would disrupt NF-κB binding but preserve NFAT5 binding. Specifically, we mutated two guanines (GG) in the κB I site and changed them to thymine and adenine (TA) (HIV-1Lai/Bal-env-κB I-mut) and also introduced the same GG to TA change in the second, more distal NF-κB site (κB II) in HIV-1Lai/Bal-env (HIV-1Lai/Bal-env-κB II-Mut). We also created a double NF-κB site mutant virus (HIV-1Lai/Bal-env-κB I+II-Mut) in order to test the impact of complete disruption of NF-κB binding to the HIV-1 LTR on MTb regulation.