HIV has proven to be a virus capable of evading both the immune system and antiviral therapy with relative ease. RNA interference (RNAi) offers a promising mechanism to exploit for antiviral therapy; however, previous studies using anti-HIV RNAi have observed viral escape by mutation of the target sequence. Consequently, target sequence conservation is now the primary consideration used when designing antiviral RNAi strategies. We have constructed a computational and experimental system to design and test anti-HIV-1 RNAi targets. We observed potent inhibition of viral replication due to a novel shRNA directed against the highly conserved TAR region; however, viral replication eventually recovered. Upon sequencing the escaped virus, we found that when targeting a highly conserved viral sequence, RNAi-escaped viruses have mutations in regions involved in the regulation of gene expression and not in the target sequence itself. HIV gene expression is controlled by a balance of positive (e.g. Tat/TAR, NF-kB p50/p65, Sp1) and negative (e.g. YY-1, NF-kB p50/p50, HDACs) regulators. We therefore hypothesize that targeting different loci of a viral gene regulatory network, or a viral life cycle, can readily be balanced by compensatory mutations, such that the virus can potentially manipulate any of the multiple loci in its genome to find the optimal escape mechanism from RNAi therapy. Such novel viral escape mechanisms further highlight the need to design combination RNAi therapies that simultaneously block multiple compensatory functions within the HIV gene regulatory network.