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RNA-Binding Domains of Heterologous Viral Proteins Substituted for Basic Residues in the RSV Gag NC Domain Restore Specific Packaging of Genomic RNA.
Viruses ( IF 3.8 ) Pub Date : 2020-03-27 , DOI: 10.3390/v12040370
Breanna L Rice 1 , Timothy L Lochmann 1 , Leslie J Parent 1, 2
Affiliation  

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.

中文翻译:

替代RSV Gag NC域中基本残基的异源病毒蛋白的RNA结合域可恢复基因组RNA的特定包装。

劳斯肉瘤病毒Gag多蛋白瞬时通过核运输,这是将病毒基因组RNA(gRNA)有效掺入病毒颗粒所必需的。gRNA的包装由两个锌指和位于Gag核衣壳(NC)域中的碱性残基介导。为了进一步研究位于gRNA衣壳化过程中位于锌指下游的碱性残基的作用,我们使用了功能获得法。我们用具有HIV-1 Rev蛋白(Rev BR)或HSV ICP27蛋白(ICP27 BR)的RNA结合活性的异源基本残基基序(BR)替换了gRNA包装所必需的基本残基簇。与野生型Gag相比,突变型ICP27 BR和Rev BR Gag蛋白更强地定位于细胞核,并释放出明显更低水平的病毒颗粒。出奇,在原病毒载体的背景下进行检测时,ICP27 BR和Rev BR突变体均包装了每个病毒颗粒正常水平的gRNA,但两个突变体均无感染性。这些结果支持这样的假设:选择性gRNA包装可能需要位于NC C端区域的碱性残基,可能是通过静电相互作用通过非特异性结合RNA来实现的。
更新日期:2020-04-20
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