Transcriptional gene silencing (TGS) of mammalian genes can be induced by specific short interfering RNA (siRNA) targeting promoter regions. We have previously reported potent TGS of HIV-1 by siRNA (PromA), which targets tandem NF-kB motifs within the viral 5’LTR promoter. In this study, we screened a panel of siRNA with the aim of identifying novel 5’LTR targets, to provide multiplexing potential with enhanced viral silencing and application for gene therapy development. Systematic examination identified a novel siRNA target, si143, which was confirmed to silence HIV through the induction of TGS. The observed silencing effect was prolonged with virus suppression >15 days, despite a limited ability to induce post-TGS. Epigenetic changes associated with silencing were suggested by partial reversal by histone deacetylase inhibitors and confirmed by Chromatin IP analyses, which showed induction of H3K27me3 and H3K9me3, reduction in H3K9Ac and recruitment of Argonaute-1, all characteristic marks of heterochromatin and TGS. Together these epigenetic changes mimic those associated with HIV-1 latency. Further, robust resistance to viral reactivation induced by increasing concentrations of SAHA and/or TNF was observed in the J-Lat 9.2 cell latency model, when transduced with shPromA and/or sh143. Importantly, the combination shRNA PromA/143 transduced J-Lat 9.2 cells showed limited reactivation, even at supra-physiological concentrations, compared to controls. These data further support si/shRNA-mediated TGS approaches to HIV-1 and provides the opportunity to combine si/shRNA targets to pursue a functional cure, whereby the viral reservoir is locked in latency following antiretroviral therapy cessation.