Rohlfes Nicholas, Radhakrishnan Rajalingam, Singh Parmit K, Bedwell Gregory J, Engelman Alan N, Dharan Adarsh, Campbell Edward M
Integrative Cell Biology Graduate Program, Loyola University Chicago, Maywood, IL, USA.
Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
bioRxiv. 2024 Jun 24:2024.06.20.599834. doi: 10.1101/2024.06.20.599834.
The early stages of HIV-1 infection include the trafficking of the viral core into the nucleus of infected cells. However, much remains to be understood about how HIV-1 accomplishes nuclear import and the consequences of the import pathways utilized on nuclear events. The host factor cleavage and polyadenylation specificity factor 6 (CPSF6) assists HIV-1 nuclear localization and post-entry integration targeting. Here, we used a CPSF6 truncation mutant lacking a functional nuclear localization signal (NLS), CPSF6-358, and appended heterologous NLSs to rescue nuclear localization. We show that some, but not all, NLSs drive CPSF6-358 into the nucleus. Interestingly, we found that some nuclear localized CPSF6-NLS chimeras supported inefficient HIV-1 infection. We found that HIV-1 still enters the nucleus in these cell lines but fails to traffic to speckle-associated domains (SPADs). Additionally, we show that HIV-1 fails to efficiently integrate in these cell lines. Collectively, our results demonstrate that the NLS of CPSF6 facilitates steps of HIV-1 infection subsequent to nuclear import and additionally identify the ability of canonical NLS sequences to influence cargo localization in the nucleus following nuclear import.
HIV-1感染的早期阶段包括病毒核心转运至被感染细胞的细胞核。然而，关于HIV-1如何完成核输入以及所利用的输入途径对核事件的影响，仍有许多有待了解之处。宿主因子切割与聚腺苷酸化特异性因子6（CPSF6）协助HIV-1的核定位及进入后整合靶向。在此，我们使用了一种缺乏功能性核定位信号（NLS）的CPSF6截短突变体CPSF6-358，并附加异源NLS以挽救核定位。我们发现，部分而非全部NLS可驱动CPSF6-358进入细胞核。有趣的是，我们发现一些核定位的CPSF6-NLS嵌合体支持低效的HIV-1感染。我们发现HIV-1在这些细胞系中仍可进入细胞核，但无法转运至斑点相关结构域（SPADs）。此外，我们表明HIV-1在这些细胞系中无法有效整合。总体而言，我们的结果表明CPSF6的NLS促进了HIV-1核输入后的感染步骤，并额外确定了典型NLS序列在核输入后影响货物在细胞核中定位的能力。
