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

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.