title: An amino acid of human parainfluenza virus type 3 nucleoprotein is critical for template function and cytoplasmic inclusion body formation.
abstract: 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 (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.
authors.affiliation: State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.
authors.forename: Shengwei
authors.initials: S
authors.lastname: Zhang
authors.forename: Longyun
authors.initials: L
authors.lastname: Chen
authors.forename: Guangyuan
authors.initials: G
authors.lastname: Zhang
authors.forename: Qin
authors.initials: Q
authors.lastname: Yan
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Yang
authors.forename: Binbin
authors.initials: B
authors.lastname: Ding
authors.forename: Qiaopeng
authors.initials: Q
authors.lastname: Tang
authors.forename: Shengjun
authors.initials: S
authors.lastname: Sun
authors.forename: Zhulong
authors.initials: Z
authors.lastname: Hu
authors.forename: Mingzhou
authors.initials: M
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism / Q000821:virology; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000235:genetics / Q000378:metabolism*; D010224:Parainfluenza Virus 3, Human / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D010253:Respirovirus Infections / Q000378:metabolism / Q000821:virology; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014779:Virus Replication
chemical_list: D009698:Nucleoproteins; D010750:Phosphoproteins; D012367:RNA, Viral
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