RNA processing mechanisms that support Tat expression in HIV-1 latency. (#139)
Integrated human immunodeficiency virus type 1 (HIV-1) provirus sustains a latent infection in resting CD4+ memory T-cells due to multiple restrictions that prevent viral gene expression. These restrictions include transcriptional interference, where an upstream cellular promoter eclipses the viral promoter, driving transcription of a cellular gene that encases the HIV-1 provirus integrated within an intron. Alternative RNA-splicing may form chimeric cellular-viral mRNAs that include tat exon-2. We identified these chimeric cell-tat mRNAs in latently infected T-cells. Cellular RNA splicing factors SRSF1, hnRNPA1 and PTB were differentially expressed and localised in resting and activated T-cells and was altered further following HIV-1 infection. We tested the importance of the chimeric cell-tat RNAs by creating plasmid vectors that expressed such chimeric spliced RNAs. Tat exon-2, with native upstream stop codons, was placed in various exonic contexts within the human growth hormone (hGH) gene. We transfected TZMbl reporter cells with tat-hGH plasmid constructs and in vitro transcribed RNAs that lacked a functional 7-methylguanosine (m7G) cap. All chimeric tat-hGH plasmids typically expressed Tat protein at >15% of the positive control, irrespective of the context of the tat reading frame, its start codon or any upstream stop codons. To test if Tat protein could be expressed through an internal ribosome entry site (IRES) translation-control mechanism we performed transfection with in vitro transcribed uncapped and 7-methyladenosine (m7A)-capped RNAs and confirmed efficient IRES-mediated Tat expression, at 10 fold over the negative control. Our data shows that RNA splicing events that include Tat exon-2 within a cellular mRNA allows functional Tat protein expression independently of a proximal cellular m7G cap structure and proceeds irrespective of the context of adjacent overlapping cellular reading frames. Our data suggests that tat exon-2 contains an IRES that provides a novel pathway for Tat expression from latent HIV provirus and may support an ongoing expression of Tat that participates in immune activation and residual pathogenesis during treated HIV infection.