Duchon Alice A, St Gelais Corine, Titkemeier Nathan, Hatterschide Joshua, Wu Li, Musier-Forsyth Karin
Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, USA.
Ohio State Biochemistry Program, Ohio State University, Columbus, Ohio, USA.
J Virol. 2017 Oct 13;91(21). doi: 10.1128/JVI.01240-17. Print 2017 Nov 1.
A hallmark of retroviruses such as human immunodeficiency virus type 1 (HIV-1) is reverse transcription of genomic RNA to DNA, a process that is primed by cellular tRNAs. HIV-1 recruits human tRNA to serve as the reverse transcription primer via an interaction between lysyl-tRNA synthetase (LysRS) and the HIV-1 Gag polyprotein. LysRS is normally sequestered in a multi-aminoacyl-tRNA synthetase complex (MSC). Previous studies demonstrated that components of the MSC can be mobilized in response to certain cellular stimuli, but how LysRS is redirected from the MSC to viral particles for packaging is unknown. Here, we show that upon HIV-1 infection, a free pool of non-MSC-associated LysRS is observed and partially relocalized to the nucleus. Heat inactivation of HIV-1 blocks nuclear localization of LysRS, but treatment with a reverse transcriptase inhibitor does not, suggesting that the trigger for relocalization occurs prior to reverse transcription. A reduction in HIV-1 infection is observed upon treatment with an inhibitor to mitogen-activated protein kinase that prevents phosphorylation of LysRS on Ser207, release of LysRS from the MSC, and nuclear localization. A phosphomimetic mutant of LysRS (S207D) that lacked the capability to aminoacylate tRNA localized to the nucleus, rescued HIV-1 infectivity, and was packaged into virions. In contrast, a phosphoablative mutant (S207A) remained cytosolic and maintained full aminoacylation activity but failed to rescue infectivity and was not packaged. These findings suggest that HIV-1 takes advantage of the dynamic nature of the MSC to redirect and coopt cellular translation factors to enhance viral replication. Human tRNA, the primer for reverse transcription, and LysRS are essential host factors packaged into HIV-1 virions. Previous studies found that tRNA packaging depends on interactions between LysRS and HIV-1 Gag; however, many details regarding the mechanism of tRNA and LysRS packaging remain unknown. LysRS is normally sequestered in a high-molecular-weight multi-aminoacyl-tRNA synthetase complex (MSC), restricting the pool of free LysRS-tRNA Mounting evidence suggests that LysRS is released under a variety of stimuli to perform alternative functions within the cell. Here, we show that HIV-1 infection results in a free pool of LysRS that is relocalized to the nucleus of target cells. Blocking this pathway in HIV-1-producing cells resulted in less infectious progeny virions. Understanding the mechanism by which LysRS is recruited into the viral assembly pathway can be exploited for the development of specific and effective therapeutics targeting this nontranslational function.
诸如1型人类免疫缺陷病毒（HIV-1）之类的逆转录病毒的一个标志是将基因组RNA逆转录为DNA，这一过程由细胞tRNA引发。HIV-1通过赖氨酰-tRNA合成酶（LysRS）与HIV-1 Gag多蛋白之间的相互作用招募人类tRNA作为逆转录引物。LysRS通常被隔离在多氨酰-tRNA合成酶复合物（MSC）中。先前的研究表明，MSC的成分可响应某些细胞刺激而被动员起来，但LysRS如何从MSC重新定向到病毒颗粒进行包装尚不清楚。在这里，我们表明，在HIV-1感染后，观察到一个与MSC无关的游离LysRS池，并且部分重新定位到细胞核。HIV-1的热灭活会阻止LysRS的核定位，但用逆转录酶抑制剂处理则不会，这表明重新定位的触发发生在逆转录之前。用丝裂原活化蛋白激酶抑制剂处理可防止LysRS在Ser207处磷酸化、从MSC释放以及核定位，从而导致HIV-1感染减少。缺乏氨酰化tRNA能力的LysRS的拟磷酸化突变体（S207D）定位于细胞核，挽救了HIV-1的感染性，并被包装到病毒颗粒中。相反，磷酸化缺失突变体（S207A）仍位于细胞质中并保持完全的氨酰化活性，但未能挽救感染性且未被包装。这些发现表明，HIV-1利用MSC的动态特性来重新定向和选择细胞翻译因子以增强病毒复制。人类tRNA，即逆转录引物，以及LysRS是包装到HIV-1病毒颗粒中的必需宿主因子。先前的研究发现，tRNA的包装取决于LysRS与HIV-1 Gag之间的相互作用；然而，关于tRNA和LysRS包装机制的许多细节仍然未知。LysRS通常被隔离在高分子量的多氨酰-tRNA合成酶复合物（MSC）中，限制了游离LysRS-tRNA池。越来越多的证据表明，LysRS在多种刺激下会被释放，以在细胞内执行其他功能。在这里，我们表明，HIV-1感染会导致LysRS的游离池重新定位到靶细胞的细胞核。在产生HIV-1的细胞中阻断这一途径会导致产生的子代病毒颗粒的感染性降低。了解LysRS被招募到病毒组装途径中的机制可用于开发针对这种非翻译功能的特异性和有效疗法。
