Department of Medicine, Penn State College of Medicine, 500 University Drive, Hershey, PA, 17033, USA.
Department of Microbiology and Immunology, Penn State College of Medicine, 500 University Drive, Hershey, PA, 17033, USA.
Retrovirology. 2024 Jun 19;21(1):13. doi: 10.1186/s12977-024-00645-y.
Retroviruses exploit host proteins to assemble and release virions from infected cells. Previously, most studies focused on interacting partners of retroviral Gag proteins that localize to the cytoplasm or plasma membrane. Given that several full-length Gag proteins have been found in the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings involving previously unknown host processes. Here we systematically compared nuclear factors identified in published HIV-1 proteomic studies and performed our own mass spectrometry analysis using affinity-tagged HIV-1 and RSV Gag proteins mixed with nuclear extracts. We identified 57 nuclear proteins in common between HIV-1 and RSV Gag, and a set of nuclear proteins present in our analysis and ≥ 1 of the published HIV-1 datasets. Many proteins were associated with nuclear processes which could have functional consequences for viral replication, including transcription initiation/elongation/termination, RNA processing, splicing, and chromatin remodeling. Examples include facilitating chromatin remodeling to expose the integrated provirus, promoting expression of viral genes, repressing the transcription of antagonistic cellular genes, preventing splicing of viral RNA, altering splicing of cellular RNAs, or influencing viral or host RNA folding or RNA nuclear export. Many proteins in our pulldowns common to RSV and HIV-1 Gag are critical for transcription, including PolR2B, the second largest subunit of RNA polymerase II (RNAPII), and LEO1, a PAF1C complex member that regulates transcriptional elongation, supporting the possibility that Gag influences the host transcription profile to aid the virus. Through the interaction of RSV and HIV-1 Gag with splicing-related proteins CBLL1, HNRNPH3, TRA2B, PTBP1 and U2AF1, we speculate that Gag could enhance unspliced viral RNA production for translation and packaging. To validate one putative hit, we demonstrated an interaction of RSV Gag with Mediator complex member Med26, required for RNA polymerase II-mediated transcription. Although 57 host proteins interacted with both Gag proteins, unique host proteins belonging to each interactome dataset were identified. These results provide a strong premise for future functional studies to investigate roles for these nuclear host factors that may have shared functions in the biology of both retroviruses, as well as functions specific to RSV and HIV-1, given their distinctive hosts and molecular pathology.
逆转录病毒利用宿主蛋白组装并从受感染的细胞中释放病毒粒子。以前，大多数研究都集中在定位在细胞质或质膜上的逆转录病毒 Gag 蛋白的相互作用伙伴上。鉴于已经在细胞核中发现了几种全长 Gag 蛋白，因此鉴定 Gag-核相互作用组对于涉及以前未知的宿主过程的新发现具有很高的潜力。在这里，我们系统地比较了已发表的 HIV-1 蛋白质组学研究中鉴定的核因子，并使用亲和标记的 HIV-1 和 RSV Gag 蛋白与核提取物混合进行了我们自己的质谱分析。我们在 HIV-1 和 RSV Gag 之间鉴定出 57 种共同的核蛋白，并且在我们的分析中存在一组核蛋白和≥1 个已发表的 HIV-1 数据集。许多蛋白与核过程相关联，这可能对病毒复制具有功能影响，包括转录起始/延伸/终止、RNA 处理、剪接和染色质重塑。例如，促进染色质重塑以暴露整合的前病毒、促进病毒基因的表达、抑制拮抗细胞基因的转录、防止病毒 RNA 的剪接、改变细胞 RNA 的剪接、或影响病毒或宿主 RNA 折叠或 RNA 核输出。我们在 RSV 和 HIV-1 Gag 下拉实验中共同鉴定出的许多蛋白对转录至关重要，包括 PolR2B，即 RNA 聚合酶 II（RNAPII）的第二大亚基，以及 LEO1，它是 PAF1C 复合物成员，调节转录延伸，支持 Gag 影响宿主转录谱以辅助病毒的可能性。通过 RSV 和 HIV-1 Gag 与剪接相关蛋白 CBLL1、HNRNPH3、TRA2B、PTBP1 和 U2AF1 的相互作用，我们推测 Gag 可以增强未剪接的病毒 RNA 产生，以进行翻译和包装。为了验证一个假定的命中，我们证明了 RSV Gag 与 Mediator 复合物成员 Med26 的相互作用，该复合物对于 RNA 聚合酶 II 介导的转录是必需的。尽管 57 种宿主蛋白与两种 Gag 蛋白相互作用，但每个互作组数据集都鉴定出了独特的宿主蛋白。这些结果为未来的功能研究提供了强有力的前提，以研究这些核宿主因子在逆转录病毒生物学中的作用，以及它们在 RSV 和 HIV-1 中的特定作用，因为它们的宿主和分子病理学是独特的。
