Institut für Virologie, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, 01307 Dresden, Germany.
J Virol. 2011 Sep;85(18):9276-85. doi: 10.1128/JVI.00663-11. Epub 2011 Jun 29.
Gag nuclear localization has long been recognized as a hallmark of foamy virus (FV) infection. Two required motifs, a chromatin-binding site (CBS) and a nuclear localization signal (NLS), both located in glycine-arginine-rich box II (GRII), have been described. However, the underlying mechanisms of Gag nuclear translocation are largely unknown. We analyzed prototype FV (PFV) Gag nuclear localization using a novel live-cell fluorescence microscopy assay. Furthermore, we characterized the nuclear localization route of Gag mutants tagged with the simian vacuolating virus 40-NLS (SV40-NLS) and also dissected the respective contributions of the CBS and the NLS. We found that PFV Gag does not translocate to the nucleus of interphase cells by NLS-mediated nuclear import and does not possess a functional NLS. PFV Gag nuclear localization occurred only by tethering to chromatin during mitosis. This mechanism was found for endogenously expressed Gag as well as for Gag delivered by infecting viral particles. Thereby, the CBS was absolutely essential, while the NLS was dispensable. Gag CBS-dependent nuclear localization was neither essential for infectivity nor necessary for Pol encapsidation. Interestingly, Gag localization was independent of the presence of Pol, Env, and viral RNA. The addition of a heterologous SV40-NLS resulted in the nuclear import of PFV Gag in interphase cells, rescued the nuclear localization deficiency but not the infectivity defect of a PFV Gag ΔGRII mutant, and did not enhance FV's ability to infect G(1)/S-phase-arrested cells. Thus, PFV Gag nuclear localization follows a novel pathway among orthoretroviral Gag proteins.
Gag 的核定位一直被认为是泡沫病毒（FV）感染的一个标志。两个必需的基序，一个染色质结合位点（CBS）和一个核定位信号（NLS），都位于甘氨酸-精氨酸丰富盒 II（GRII）中，已经被描述过了。然而，Gag 核转位的潜在机制在很大程度上仍是未知的。我们使用一种新的活细胞荧光显微镜检测法分析了原型 FV（PFV）Gag 的核定位。此外，我们还对标记有猴空泡病毒 40-NLS（SV40-NLS）的 Gag 突变体的核定位途径进行了特征描述，还剖析了 CBS 和 NLS 的各自贡献。我们发现，PFV Gag 不会通过 NLS 介导的核输入转移到间期细胞的核内，也没有功能性的 NLS。PFV Gag 的核定位仅发生在有丝分裂时与染色质的连接。这种机制适用于内源性表达的 Gag 以及感染病毒颗粒所传递的 Gag。为此，CBS 是绝对必需的，而 NLS 是可有可无的。Gag CBS 依赖性核定位对于感染性既不是必需的，也不是 Pol 包装所必需的。有趣的是，Gag 的定位与 Pol、Env 和病毒 RNA 的存在无关。添加一个异源的 SV40-NLS 会导致 PFV Gag 在间期细胞中的核输入，挽救了 PFV Gag ΔGRII 突变体的核定位缺陷，但没有挽救其感染缺陷，也没有增强 FV 感染 G1/S 期停滞细胞的能力。因此，PFV Gag 的核定位遵循一种在正逆转录病毒 Gag 蛋白中独特的途径。
