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An Amino Acid of Human Parainfluenza Virus Type 3 Nucleoprotein Is Critical for Template Function and Cytoplasmic Inclusion Body Formation

机译:人副流感病毒3型核酸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

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摘要

The nucleoprotein (N) and phosphoprotein (P) interaction of nonsegmented negative-strand RNA viruses is essential for viral replication; this includes N0-P (N0, 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 (NL478A) did not support reporter gene expression. Using in vivo and in vitro coimmunoprecipitation, we found that NL478A maintains the ability to form NL478A0-P, to self-assemble, and to form NL478A-RNA but that NL478A-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. NL478A was unable to form inclusion bodies when coexpressed with P, but the presence of N rescued the ability of NL478A to form inclusion bodies and the transcriptional function of NL478A, thereby suggesting that hetero-oligomers formed by N and NL478A are functional and competent to form inclusion bodies. Furthermore, we found that NL478A 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 N0-P interaction for the formation of inclusion bodies, which appear to be bona fide sites of RNA synthesis.
机译:非分段负链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。其中包括N 0 -P(N 0 ,不含RNA)相互作用以及N-RNA与P的相互作用。N中的精确位点介导了然而,NP相互作用和详细的调节机制尚不清楚。使用人类3型副流感病毒(HPIV3)微型基因组测定,我们发现N突变体(NL478A)不支持报告基因表达。使用体内和体外共免疫沉淀,我们发现NL478A保持形成NL478A 0 -P,自组装和形成NL478A-RNA的能力,但是NL478A-RNA不与P相互作用使用免疫荧光测定法,我们发现NP相互作用为形成细胞质包涵体提供了最低限度的要求,其中包含HPIV3感染细胞中的病毒RNA,N,P和聚合酶。 NL478A与P共表达时无法形成包涵体,但N的存在挽救了NL478A形成包涵体的能力和NL478A的转录功能,从而表明N和NL478A形成的杂合寡聚体具有功能并能够形成包涵体。此外,我们发现NL478A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中对N-RNA和P相互作用至关重要但对N 0 -P相互作用至关重要的精确氨基酸,以形成包涵体似乎是RNA合成的真正位点。

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