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Human Tat‐specific Factor 1 Binds and Exports HIV‐1 RNA to the Cytoplasm
Author(s) -
Miller Heather
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.460.9
Subject(s) - rna , electrophoretic mobility shift assay , biology , rna splicing , gene knockdown , microbiology and biotechnology , cytoplasm , rna binding protein , transcription (linguistics) , transfection , virology , gene , transcription factor , genetics , linguistics , philosophy
Human immunodeficiency virus type 1 (HIV‐1) requires many human cofactors for propagation. Multiple groups have shown that human Tat‐specific factor 1 (Tat‐SF1) is an HIV‐1 dependency factor. This protein's knockdown does not affect the overall levels of viral RNA, however it does regulate the relative size classes (unspliced, singly spliced, and fully spliced). The molecular mechanism behind this is unknown, but data is consistent with roles in enhancing splicing, export, and/or stability of the viral RNA. We hypothesized that if Tat‐SF1 plays any one of these roles, then we should be able to demonstrate binding of the protein to the RNA genome. Furthermore, if Tat‐SF1 is critical for nuclear export of the viral RNAs, then knocking down Tat‐SF1 should result in more nuclear retention of the viral genome. Fragments of the HIV‐1 genome were synthesized by in vitro transcription and end‐labelled with fluorescein. These RNA probes were used in both electrophoretic mobility shift assays (EMSA) and florescence polarization (FP) assays. To analyze RNA export, HeLa cells with an shRNA‐expressing plasmid targeting Tat‐SF1 were also transfected with pSG3Δenv, a non‐infectious HIV‐1 plasmid. RNA from nuclear and cytoplasmic fractions was purified, followed by RT‐qPCR analysis. Our results suggest that Tat‐SF1 plays a role in binding the HIV‐1 genome and exporting the unspliced transcripts to the cytoplasm. This work provides additional molecular details on how Tat‐SF1 acts as an HIV‐1 dependency factor. Support or Funding Information High Point University This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .