Tax Induces the Recruitment of NF-κB to Unintegrated HIV-1 DNA To Rescue Viral Gene Expression and Replication

Abstract

Integrase-deficient expression vectors based on HIV-1 are becoming increasingly popular as tools for gene therapy in vivo due to their inability to cause insertional mutagenesis. However, many IN− lentiviral vectors are able to achieve only low levels of gene expression, and methods to increase this low level have not been extensively explored. ABSTRACT We previously reported that the normally essential step of integration of the HIV-1 proviral DNA intermediate into the host cell genome becomes dispensable in T cells that express the human T cell leukemia virus 1 (HTLV-1) Tax protein, a known activator of cellular NF-κB. The rescue of integrase (IN)-deficient HIV-1 replication by Tax results from the strong activation of transcription from the long terminal repeat (LTR) promoter on episomal HIV-1 DNA, an effect that is closely correlated with the recruitment of activating epigenetic marks, such as H3Ac, and depletion of repressive epigenetic marks, such as H3K9me3, from chromatinized unintegrated proviruses. In addition, activation of transcription from unintegrated HIV-1 DNA coincides with the recruitment of NF-κB to the two NF-κB binding sites found in the HIV-1 LTR enhancer. Here, we report that the recruitment of NF-κB to unintegrated viral DNA precedes, and is a prerequisite for, Tax-induced changes in epigenetic marks, so that an IN− HIV-1 mutant lacking both LTR NF-κB sites is entirely nonresponsive to Tax and fails to undergo the epigenetic changes listed above. Interestingly, we found that induction of Tax expression at 24\u2009h postinfection, when unintegrated HIV-1 DNA is already fully repressed by inhibitory chromatin modifications, is able to effectively reverse the epigenetic silencing of that DNA and rescue viral gene expression. Finally, we report that heterologous promoters introduced into IN-deficient HIV-1-based vectors are transcriptionally active even in the absence of Tax and do not increase their activity when the HIV-1 promoter and enhancer, located in the LTR U3 region, are deleted, as has been recently proposed. IMPORTANCE Integrase-deficient expression vectors based on HIV-1 are becoming increasingly popular as tools for gene therapy in vivo due to their inability to cause insertional mutagenesis. However, many IN− lentiviral vectors are able to achieve only low levels of gene expression, and methods to increase this low level have not been extensively explored. Here, we analyzed how the HTLV-1 Tax protein is able to rescue the replication of IN− HIV-1 in T cells, and we describe IN− lentiviral vectors, lacking any inserted origin of replication, that are able to express a heterologous gene effectively.