A Point Mutation in the RNA-Binding Domain of Human Parainfluenza Virus Type 2 Nucleoprotein Elicits Abnormally Enhanced Polymerase Activity.
The genome RNA of human parainfluenza virus type 2 (hPIV2) that acts as the template for the polymerase complex is entirely encapsidated by the nucleoprotein (NP). Recently, the crystal structure of NP of PIV5, a virus closely related to hPIV2, was resolved in association with RNA. Ten amino acids that contact the bound RNA were identified and are strictly conserved between PIV5 and hPIV2 NP. Mutation of hPIV2 NP Q202 (which contacts a base rather than the RNA backbone) to various amino acids resulted in an over 30-fold increase of polymerase activity as evidenced by a minireplicon assay, even though the RNA-binding affinity was unaltered. Using various modified minireplicons, we found that the enhanced reporter gene expression could be accounted for by increased minigenome replication, whereas mRNA synthesis itself was not affected by Q202 mutation. Moreover, the enhanced activities were still observed in minigenomes partially lacking the leader sequence and which were not of hexamer genome length. Unexpectedly, recombinant hPIV2 possessing the NP Q202A mutation could not be recovered from cDNA.IMPORTANCEWe examined the importance of amino acids in the putative RNA-binding domain of hPIV2 NP for polymerase activity using minireplicons. Abnormally enhanced genome replication was observed upon substitution mutation of the NP Q202 position to various amino acids. Surprisingly, this mutation enabled polymerase to use minigenomes that were partially lacking the leader sequence and not of hexamer genome length. This mutation does not affect fundamental properties of NP, e.g., recognition of gene junctional and editing signals. However, the strongly enhanced polymerase activity may not be viable for the infectious life cycle. This report highlights the potential of the polymerase complex with point mutations in NP and helps our detailed understanding of the molecular basis of gene expression.
Department of Microbiology, School of Medicine, Wakayama Medical University, Wakayama, Japan ymatsu@wakayama-med.ac.jp.
Yusuke
Y
Matsumoto
Department of Microbiology, School of Medicine, Wakayama Medical University, Wakayama, Japan.
Keisuke
K
Ohta
Department of Microbiology and Molecular Medicine, University of Geneva School of Medicine, Geneva, Switzerland.
Daniel
D
Kolakofsky
Department of Microbiology, School of Medicine, Wakayama Medical University, Wakayama, Japan.
Machiko
M
Nishio
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D016679:Genome, Viral / Q000235:genetics; D006801:Humans; D019251:Nucleocapsid / Q000378:metabolism*; D009698:Nucleoproteins / Q000235:genetics*; D010223:Parainfluenza Virus 2, Human / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D000071376:RNA-Binding Motifs / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics
D009698:Nucleoproteins; D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase
encapsidation; human parainfluenza virus type 2; nucleoprotein; polymerase