逆转录病毒利用宿主蛋白组装并从感染的细胞释放病毒粒子。以前,大多数研究集中在定位于细胞质或质膜的逆转录病毒Gag蛋白的相互作用伙伴。鉴于已经在细胞核中发现了几种全长Gag蛋白,鉴定Gag-核相互作用组对于涉及先前未知的宿主过程的新发现具有很高的潜力。在这里,我们系统地比较了在已发表的HIV-1蛋白质组学研究中鉴定的核因子,并使用与核提取物混合的亲和标记的HIV-1和RSVGag蛋白进行了我们自己的质谱分析。我们鉴定了HIV-1和RSVGag之间共有的57种核蛋白,和一组核蛋白存在于我们的分析和发表的HIV-1数据集≥1。许多蛋白质与核过程有关,这些过程可能对病毒复制产生功能性影响,包括转录起始/延伸/终止,RNA加工,拼接,和染色质重塑。例子包括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。RSV和HIV-1Gag常见的许多蛋白质对转录至关重要,包括PolR2B,RNA聚合酶II(RNAPII)的第二大亚基,和LEO1,一个调节转录延伸的PAF1C复合物成员,支持Gag影响宿主转录谱以帮助病毒的可能性。通过RSV和HIV-1Gag与剪接相关蛋白CBLL1、HNRNPH3、TRA2B、PTBP1和U2AF1,我们推测Gag可以增强未剪接的病毒RNA的产生,用于翻译和包装。为了验证一个假定的命中,我们证明了RSVGag与RNA聚合酶II介导的转录所需的Mediator复合物成员Med26的相互作用。尽管57种宿主蛋白与两种Gag蛋白相互作用,鉴定了属于每个相互作用组数据集的独特宿主蛋白。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两种逆转录病毒生物学中可能具有共同功能的作用。以及RSV和HIV-1特有的功能,鉴于其独特的宿主和分子病理学。
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.