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Human Tat-specific factor 1 binds the HIV-1 genome and selectively transports HIV-1 RNAs

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Abstract

Human immunodeficiency virus type 1 (HIV-1) propagation requires many human cofactors. Multiple groups have demonstrated that Tat-specific factor 1 (Tat-SF1) is an HIV-1 dependency factor. Depletion of this protein lowers HIV-1 infectivity, however, it does not affect the overall levels of viral RNA. Rather, Tat-SF1 regulates the relative levels of each RNA size class. This would be consistent with roles in splicing, transport, and/or stability of viral RNAs. We hypothesized that if Tat-SF1 plays any of these roles, then we should detect binding of the protein to the RNA genome. Furthermore, knocking down Tat-SF1 should result in altered RNA stability and/or localization in human cells. Fragments of the HIV-1 genome were used as RNA probes in electrophoretic mobility shift assays and fluorescence correlation spectroscopy experiments. Our results show that Tat-SF1 can form a complex with TAR RNA in vitro, independent of Tat. This factor interacts with at least one additional location in the 5’ end of the HIV-1 genome. Tat seems to enhance the formation of this complex. To analyze HIV-1 RNA localization, HeLa cells with Tat-SF1 knocked down were also transfected with a proviral clone. RNA from nuclear and cytoplasmic fractions was purified, followed by RT-qPCR analysis. Tat-SF1 likely binds the HIV-1 RNA genome at TAR and potentially other locations and selectively transports HIV-1 RNAs, facilitating the unspliced RNA’s nuclear export while retaining singly spliced RNAs in the nucleus. This is a novel role for this HIV-1 dependency factor.

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Acknowledgements

The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pSG3∆env from Drs. John C. Kappes and Xiaoyun Wu and HIV-1 IIIB Tat recombinant protein. This work was fully funded by High Point University.

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Author notes

  1. Molly J. Hulver and Julia P. Trautman have contributed equally to this work and share first authorship.

Authors and Affiliations

  1. Biochemistry and Molecular Biology Program, University of Rochester Medical Center, Rochester, NY, 14627, USA

    Molly J. Hulver

  2. Department of Biology, High Point University, High Point, NC, 27268, USA

    Julia P. Trautman & Sebastian K. Roszczenko

  3. Creighton School of Medicine, Omaha, NE, 68178, USA

    Amanda P. Goodwin

  4. Department of Chemistry, High Point University, High Point, NC, 27268, USA

    Keir H. Fogarty & Heather B. Miller

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  1. Molly J. Hulver
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Hulver, M.J., Trautman, J.P., Goodwin, A.P. et al. Human Tat-specific factor 1 binds the HIV-1 genome and selectively transports HIV-1 RNAs. Mol Biol Rep 47, 1759–1772 (2020). https://doi.org/10.1007/s11033-020-05267-z

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