An Amino Acid of Human Parainfluenza Virus Type 3 Nucleoprotein Is Critical for Template Function and Cytoplasmic Inclusion Body Formation
An Amino Acid of Human Parainfluenza Virus Type 3 Nucleoprotein Is Critical for Template Function and Cytoplasmic Inclusion Body Formation
复制标题
人副流感病毒3型核蛋白的氨基酸对于模板功能和细胞质包涵体形成至关重要
DOI：
10.1128/jvi.01565-13
复制
发表时间：
2013-11-01
影响因子：
5.4
通讯作者：
Chen, Mingzhou
中科院分区：
文献类型：
--
作者：
Zhang, Shengwei;Chen, Longyun;Chen, Mingzhou
非节段负链RNA病毒的核蛋白（N）和磷蛋白（P）相互作用对于病毒复制至关重要；这包括N-0-P（N-0，不含RNA）相互作用以及N-RNA与P的相互作用。然而，N内介导N-P相互作用的精确位点以及详细的调节机制还不太清楚。使用人类副流感病毒 3 型 (HPIV3) 小基因组测定，我们发现 N 突变体 (N-L478A) 不支持报告基因表达。利用体内和体外免疫共沉淀，我们发现 N-L478A 保持了形成 N-L478A (0)-P、自组装和形成 N-L478A-RNA 的能力，但 N-L478A-RNA 不与 P 相互作用。利用免疫荧光测定，我们发现 N-P 相互作用为形成细胞质包涵体提供了最低要求，细胞质包涵体含有病毒 RNA、N、P 和HPIV3 感染细胞中的聚合酶。 N-L478A与P共表达时无法形成包涵体，但N的存在挽救了N-L478A形成包涵体的能力和N-L478A的转录功能，从而表明N和N-L478A形成的异源寡聚体具有功能并且能够形成包涵体。此外，我们发现N-L478A在病毒生长方面也存在缺陷。据我们所知，我们是第一个使用副粘病毒来鉴定 N 内的精确氨基酸，该氨基酸对于 N-RNA 和 P 相互作用至关重要，但对于包涵体形成的 N-0-P 相互作用不至关重要，包涵体似乎是 RNA 合成的真正位点。
The nucleoprotein (N) and phosphoprotein (P) interaction of nonsegmented negative-strand RNA viruses is essential for viral replication; this includes N-0-P (N-0, free of RNA) interaction and the interaction of N-RNA with P. The precise site(s) within N that mediates the N-P interaction and the detailed regulating mechanism, however, are less clear. Using a human parainfluenza virus type 3 (HPIV3) minigenome assay, we found that an N mutant (N-L478A) did not support reporter gene expression. Using in vivo and in vitro coimmunoprecipitation, we found that N-L478A maintains the ability to form N-L478A (0)-P, to self-assemble, and to form N-L478A-RNA but that N-L478A-RNA does not interact with P. Using an immunofluorescence assay, we found that N-P interaction provides the minimal requirement for the formation of cytoplasmic inclusion bodies, which contain viral RNA, N, P, and polymerase in HPIV3-infected cells. N-L478A was unable to form inclusion bodies when coexpressed with P, but the presence of N rescued the ability of N-L478A to form inclusion bodies and the transcriptional function of N-L478A, thereby suggesting that hetero-oligomers formed by N and N-L478A are functional and competent to form inclusion bodies. Furthermore, we found that N-L478A is also defective in virus growth. To our knowledge, we are the first to use a paramyxovirus to identify a precise amino acid within N that is critical for N-RNA and P interaction but not for N-0-P interaction for the formation of inclusion bodies, which appear to be bona fide sites of RNA synthesis.