Background: We have previously described a model of direct infection of resting CD4+ T cells and contrasted it with models of activated CD4+ T cell infection. We found that infected resting CD4 cells express low levels of viral protein without releasing infectious virus, raising the possibility that reservoirs may express HIV proteins in vivo and be visible to the immune system. Unspliced RNA (usRNA) encoding gag was the predominant HIV RNA form detected in infected resting cells. We designed experiments to ask if nascent transcription occurs in resting CD4+ T cells or if the Gag signal detected is due to an artifact such as read-through transcription or incoming virus.
Methods: To address the contribution of incoming virus to Gag signal, we first sorted Gag+ and Gag-negative cells from cultures infected in vitro and measured levels of HIV DNA in both populations, similar to an approach we used in vivo. RT-PCR and FACS analysis were used to determine whether other viral proteins (made from spliced RNA forms) were present in cells infected in vitro and in CD4+ T cells from ART patients. In addition, given that tat/rev is present at very low levels in vivo in patients on ART, we asked if tat/rev is required for LTR driven expression.
Results: We found that Gag+ cells were strongly (more than 100-fold) enriched for HIV DNA compared to Gag-negative cells in infected cultures. In addition to spliced HIV RNA forms, further evidence of nascent transcription included direct and indirect evidence of new synthesis of multiple HIV proteins by FACS. Read-through transcripts were detectable but present at low levels compared to gag RNA in both cells infected in vitro and in CD4 cells from ART patients. Stimuli such as IL-7 and Romidepsin preferentially induced gag usRNA over read-through transcripts. In contrast, SAHA induced both read-through and gag usRNA transcription two-fold. Notably, we show that low-level protein expression can occur in the absence of tat/rev using a viral vector with a deletion of tat/rev gene expression.
Conclusions: Nascent LTR transcription occurs in HIV-infected resting CD4+ T cells. In vitro and in vivo data suggest that Gag is the predominant transcript (usRNA) and protein expressed in HIV infected individuals on ART. The relative contributions of replication competent and defective proviruses to viral protein expression in vivo remain undefined.