An Interaction between the Nucleocapsid Protein and a Component of the Replicase-Transcriptase Complex Is Crucial for the Infectivity of Coronavirus Genomic RNA
An Interaction between the Nucleocapsid Protein and a Component of the Replicase-Transcriptase Complex Is Crucial for the Infectivity of Coronavirus Genomic RNA
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DOI：
10.1128/jvi.01287-10
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发表时间：
2010-10-01
影响因子：
5.4
通讯作者：
Masters, Paul S.
中科院分区：
文献类型：
--
作者：
Hurst, Kelley R.;Ye, Rong;Masters, Paul S.
冠状病毒核衣壳(N)蛋白通过与大的正链RNA病毒基因组和膜蛋白(M)的羧基末端内域相互作用，在病毒粒子组装中发挥重要作用。为了了解冠状病毒小鼠肝炎病毒(MHV)N蛋白结构域的功能，我们用密切相关的牛冠状病毒(BCOV)的N基因替换了MHV的N基因。由此产生的病毒突变是严重缺陷的，即使负责N-RNA、N-M或N-N相互作用的N蛋白的个别结构域在BCOV和MHV之间完全可以互换。BCOV N替换突变体中的损伤可以通过恢复N蛋白的中央富含丝氨酸和精氨酸(SR)结构域的突变来补偿。令人惊讶的是，第二类逆转突变被映射到复制酶亚单位非结构蛋白3(NSP3)的氨基末端。一个带有严重急性呼吸综合征冠状病毒N蛋白SR区插入的类似缺陷的MHV N突变体被相同的两类逆转突变拯救。我们的遗传结果证实了NSP3氨基末端片段的表达选择性地与感染细胞中的N蛋白结合，这种相互作用不依赖于RNA。此外，我们还发现N-NSP3的相互作用与N蛋白刺激MHV基因组RNA(GRNA)的感染力之间存在直接的相关性。我们的结果表明，这种以前未知的N-NSP3相互作用在感染早期基因组RNA到复制酶复合体的定位中起到了作用。
The coronavirus nucleocapsid (N) protein plays an essential role in virion assembly via interactions with the large, positive-strand RNA viral genome and the carboxy-terminal endodomain of the membrane protein (M). To learn about the functions of N protein domains in the coronavirus mouse hepatitis virus (MHV), we replaced the MHV N gene with its counterpart from the closely related bovine coronavirus (BCoV). The resulting viral mutant was severely defective, even though individual domains of the N protein responsible for N-RNA, N-M, or N-N interactions were completely interchangeable between BCoV and MHV. The lesion in the BCoV N substitution mutant could be compensated for by reverting mutations in the central, serine-and arginine-rich (SR) domain of the N protein. Surprisingly, a second class of reverting mutations were mapped to the amino terminus of a replicase subunit, nonstructural protein 3 (nsp3). A similarly defective MHV N mutant bearing an insertion of the SR region from the severe acute respiratory syndrome coronavirus N protein was rescued by the same two classes of reverting mutations. Our genetic results were corroborated by the demonstration that the expressed amino-terminal segment of nsp3 bound selectively to N protein from infected cells, and this interaction was RNA independent. Moreover, we found a direct correlation between the N-nsp3 interaction and the ability of N protein to stimulate the infectivity of transfected MHV genomic RNA (gRNA). Our results suggest a role for this previously unknown N-nsp3 interaction in the localization of genomic RNA to the replicase complex at an early stage of infection.