The retrovirus human immunodeficiency virus type 1 (HIV-1) requires various cofactors for transcriptional and post-transcriptional regulation. In previous research, it was determined that Tat-specific Factor 1 (Tat-SF1), a human protein, aids in post transcriptional regulation of the different size classes of HIV-1 RNAs. However, the molecular mechanism behind this is unknown but data would be consistent with roles in RNA splicing, export, and/or stability. We hypothesized that if Tat-SF1 has a role in HIV RNA export, then knocking it down will result in changes to the nuclear and cytoplasmic HIV RNA levels. We also used these cells to better understand the role Tat-SF1 may play in RNA stability, examining the persistence of RNA levels over time. To do this, we first transfected HeLa cells with an shRNA-expressing plasmid targeting Tat-SF1 to knockdown the gene, followed by a transfection with pSG3Δenv, a non-infectious HIV-1 plasmid. Treating the transfected HeLa cells with actinomycin D to halt transcription allowed us to analyze viral RNA stability. In addition, fractions containing the cytoplasm and nucleus were purified from the HeLa cells to examine nuclear export. Lastly, reverse transcription quantitative PCR was performed to compare the levels of the three HIV-1 RNA size classes in the knockdown vs. control samples. Our results show that Tat-SF1 selectively stabilizes some of the HIV RNAs. Preliminary results suggest that Tat-SF1 plays a role in exporting the unspliced HIV RNAs to the cytoplasm. Future works will involve quantifying the other two HIV RNA size classes and how they are exported.

Analyzing the role of Tat-SF1 in HIV-1 RNA stability and export

2019 Spring National Meeting

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