Chang Jordan, Parent Leslie J
bioRxiv. 2023 Feb 25:2023.02.24.529990. doi: 10.1101/2023.02.24.529990.
The retroviral Gag protein of human immunodeficiency virus type 1 (HIV-1) plays a central role in the selection of unspliced viral genomic RNA for packaging into new virions. Previously, we demonstrated that full-length HIV-1 Gag undergoes nuclear trafficking where it associates with unspliced viral RNA (vRNA) at transcription sites. To further explore the kinetics of HIV-1 Gag nuclear localization, we used biochemical and imaging techniques to examine the timing of HIV-1 entry into the nucleus. We also aimed to determine more precisely Gag's subnuclear distribution to test the hypothesis that Gag would be associated with euchromatin, the transcriptionally active region of the nucleus. We observed that HIV-1 Gag localized to the nucleus shortly after its synthesis in the cytoplasm, suggesting that nuclear trafficking was not strictly concentration-dependent. Furthermore, we found that HIV-1 Gag preferentially localized to the transcriptionally active euchromatin fraction compared to the heterochromatin-rich region in a latently-infected CD4+ T cell line (J-Lat 10.6) treated with latency-reversal agents. Interestingly, HIV-1 Gag was more closely associated with transcriptionally-active histone markers near the nuclear periphery, where the HIV-1 provirus was previously shown to integrate. Although the precise function of Gag's association with histones in transcriptionally-active chromatin remains uncertain, together with previous reports, this finding is consistent with a potential role for euchromatin-associated Gag molecules to select newly transcribed unspliced vRNA during the initial stage of virion assembly.
The traditional view of retroviral assembly posits that HIV-1 Gag selection of unspliced vRNA begins in the cytoplasm. However, our previous studies demonstrated that HIV-1 Gag enters the nucleus and binds to unspliced HIV-1 RNA at transcription sites, suggesting that genomic RNA selection may occur in the nucleus. In the present study, we observed nuclear entry of HIV-1 Gag and co-localization with unspliced viral RNA within 8 hours post-expression. In CD4+ T cells (J-Lat 10.6) treated with latency reversal agents, as well as a HeLa cell line stably expressing an inducible Rev-dependent provirus, we found that HIV-1 Gag preferentially localized with histone marks associated with enhancer and promoter regions of transcriptionally active euchromatin near the nuclear periphery, which favors HIV-1 proviral integration sites. These observations support the hypothesis that HIV-1 Gag hijacks euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized genomic RNA for packaging.
人类免疫缺陷病毒1型（HIV-1）的逆转录病毒Gag蛋白在选择未剪接的病毒基因组RNA以包装到新病毒颗粒中起着核心作用。此前，我们证明全长HIV-1 Gag会进行核运输，在转录位点与未剪接的病毒RNA（vRNA）结合。为了进一步探究HIV-1 Gag核定位的动力学，我们使用生化和成像技术来检查HIV-1进入细胞核的时间。我们还旨在更精确地确定Gag在细胞核内的分布，以验证Gag会与常染色质（细胞核的转录活性区域）相关的假设。我们观察到HIV-1 Gag在细胞质中合成后不久就定位于细胞核，这表明核运输并非严格依赖浓度。此外，我们发现，在用潜伏期逆转剂处理的潜伏感染CD4+ T细胞系（J-Lat 10.6）中，与富含异染色质的区域相比，HIV-1 Gag优先定位于转录活性常染色质部分。有趣的是，HIV-1 Gag与核周附近转录活性组蛋白标记物的联系更为紧密，此前已证明HIV-1前病毒在此处整合。尽管Gag与转录活性染色质中的组蛋白结合的确切功能仍不确定，但结合之前的报道，这一发现与常染色质相关的Gag分子在病毒颗粒组装初始阶段选择新转录的未剪接vRNA的潜在作用是一致的。
逆转录病毒组装的传统观点认为，HIV-1 Gag对未剪接vRNA的选择始于细胞质。然而，我们之前的研究表明，HIV-1 Gag进入细胞核并在转录位点与未剪接的HIV-1 RNA结合，这表明基因组RNA的选择可能发生在细胞核中。在本研究中，我们观察到HIV-1 Gag在表达后8小时内进入细胞核并与未剪接的病毒RNA共定位。在用潜伏期逆转剂处理的CD4+ T细胞（J-Lat 10.6）以及稳定表达诱导型Rev依赖性前病毒的HeLa细胞系中，我们发现HIV-1 Gag优先与核周附近转录活性常染色质增强子和启动子区域相关的组蛋白标记物共定位，这有利于HIV-1前病毒整合位点。这些观察结果支持了以下假设：HIV-1 Gag劫持与常染色质相关的组蛋白定位于活跃转录位点，促进捕获新合成的基因组RNA进行包装。
