Department of Medicine, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
Department of Microbiology and Immunology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
Viruses. 2020 Mar 27;12(4):370. doi: 10.3390/v12040370.
The Rous sarcoma virus Gag polyprotein transiently traffics through the nucleus, which is required for efficient incorporation of the viral genomic RNA (gRNA) into virus particles. Packaging of gRNA is mediated by two zinc knuckles and basic residues located in the nucleocapsid (NC) domain in Gag. To further examine the role of basic residues located downstream of the zinc knuckles in gRNA encapsidation, we used a gain-of-function approach. We replaced a basic residue cluster essential for gRNA packaging with heterologous basic residue motif (BR) with RNA-binding activity from either the HIV-1 Rev protein (Rev BR) or the HSV ICP27 protein (ICP27 BR). Compared to wild-type Gag, the mutant ICP27 BR and Rev BR Gag proteins were much more strongly localized to the nucleus and released significantly lower levels of virus particles. Surprisingly, both the ICP27 BR and Rev BR mutants packaged normal levels of gRNA per virus particle when examined in the context of a proviral vector, yet both mutants were noninfectious. These results support the hypothesis that basic residues located in the C-terminal region of NC are required for selective gRNA packaging, potentially by binding non-specifically to RNA via electrostatic interactions.
劳斯肉瘤病毒 Gag 多聚蛋白瞬时穿过细胞核，这对于将病毒基因组 RNA（gRNA）有效包装到病毒颗粒中是必需的。gRNA 的包装由位于 Gag 核衣壳（NC）结构域中的两个锌指和碱性残基介导。为了进一步研究锌指下游碱性残基在 gRNA 包装中的作用，我们采用了一种功能获得的方法。我们用来自 HIV-1 Rev 蛋白（Rev BR）或 HSV ICP27 蛋白（ICP27 BR）的具有 RNA 结合活性的异源碱性残基模体（BR）取代了 gRNA 包装所必需的碱性残基簇。与野生型 Gag 相比，突变的 ICP27 BR 和 Rev BR Gag 蛋白更强烈地定位于细胞核，并释放出显著较低水平的病毒颗粒。令人惊讶的是，当在前病毒载体的背景下检测时，这两种突变体的 gRNA 包装水平均正常，但均无感染性。这些结果支持了这样的假设，即在 NC 结构域的 C 末端区域中的碱性残基通过静电相互作用通过非特异性地与 RNA 结合来包装 gRNA。
