Biochemistry and Molecular Biology Program, University of Rochester Medical Center, Rochester, NY, 14627, USA.
Department of Biology, High Point University, High Point, NC, 27268, USA.
Mol Biol Rep. 2020 Mar;47(3):1759-1772. doi: 10.1007/s11033-020-05267-z. Epub 2020 Feb 3.
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.
人类免疫缺陷病毒 1 型（HIV-1）的传播需要多种人类辅助因子。多个研究小组已经证明，Tat 特异性因子 1（Tat-SF1）是 HIV-1 的依赖因子。该蛋白的耗竭降低了 HIV-1 的感染性，但不会影响病毒 RNA 的总水平。相反，Tat-SF1 调节每个 RNA 大小类别的相对水平。这与 RNA 的剪接、运输和/或稳定性有关。我们假设，如果 Tat-SF1 发挥这些作用中的任何一种作用，那么我们应该检测到该蛋白与 RNA 基因组的结合。此外，敲低 Tat-SF1 应该导致 RNA 稳定性和/或在人类细胞中的定位改变。使用 HIV-1 基因组的片段作为 RNA 探针进行电泳迁移率变动分析和荧光相关光谱实验。我们的结果表明，Tat-SF1 可以在体外与 TAR RNA 形成复合物，而无需 Tat。该因子与 HIV-1 基因组 5' 端的至少另一个位置相互作用。 Tat 似乎增强了该复合物的形成。为了分析 HIV-1 RNA 的定位，用 Tat-SF1 敲低的 HeLa 细胞也转染了前病毒克隆。纯化核和细胞质部分的 RNA，然后进行 RT-qPCR 分析。Tat-SF1 可能在 TAR 及其他潜在位置结合 HIV-1 RNA 基因组，并选择性地转运 HIV-1 RNA，促进未剪接 RNA 的核输出，同时将单剪接 RNA 保留在核内。这是这种 HIV-1 依赖性因子的新作用。
