HIV-1 latency in resting CD4 + T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART). We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4 + T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS) HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4 + T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4 + T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB) was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4 + T cells. Overexpression of PTB in resting CD4 + T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4 + T cells. Virus culture experiments showed that overexpression of PTB in resting CD4 + T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.
HIV-1 has the ability to establish a state of latent infection in resting memory CD4 + T cells. These latently infected cells represent a stable reservoir for the virus that is a major barrier to viral eradication. Understanding how this reservoir is established, maintained, and reactivated is essential for developing methods to target and eliminate these cells. Currently, many proposed mechanisms of HIV-1 latency involve a dramatic reduction in ongoing HIV-1 transcription. However, some HIV-1 mRNAs are made, and it has been unclear why the cells are unable to produce virus. This study describes the surprising observation that mRNAs encoding the viral regulatory proteins Tat and Rev are retained in the nucleus of infected resting CD4 + T cells. A cellular HIV-1 RNA-binding protein called polypyrimidine tract binding protein was shown to reverse latency when overexpressed in resting CD4 + T cells. This overexpression of polypyrimidine tract binding protein was sufficient to allow release of replication-competent HIV-1 from latently infected cells without inducing cellular stimulation. These experiments suggest that multiple factors contribute to the maintenance of HIV-1 latency in vivo; however, perturbation of the level of a specific cellular protein is sufficient to overcome these blocks and allow for virus production.