title: Role of NH(2)- and COOH-terminal domains of the P protein of human parainfluenza virus type 3 in transcription and replication.
abstract: The phosphoproteins (P proteins) of paramyxoviruses play a central role in transcription and replication of the viruses by forming the RNA polymerase complex L-P and encapsidation complex (N-P) with nucleocapsid protein (N) and binding to N protein-encapsidated genome RNA template (N-RNA template). We have analyzed the human parainfluenza virus type 3 (HPIV3) P protein and deletion mutants thereof in an in vitro transcription and in vivo replication system. The in vitro system utilizes purified N-RNA template and cell extract containing L and P proteins coexpressed via plasmids using a recombinant vaccinia virus expression system. The in vivo system takes advantage of minigenome replication, which measures luciferase reporter gene expression from HPIV3 minigenomes by viral proteins in a recombinant vaccinia virus expression system. These studies revealed that the C-terminal 20-amino-acid region of P is absolutely required for transcription in vitro and luciferase expression in vivo, suggesting its critical role in viral RNA synthesis. The N-terminal 40-amino-acid region, on the other hand, is essential for luciferase expression but dispensable for transcription in vitro. Consistent with these findings, the C-terminal domain is required for binding of P protein to the N-RNA template involved in both transcription and replication, whereas the N-terminal domain is required for the formation of soluble N-P complex involved in encapsidation of nascent RNA chains during replication. Coimmunoprecipitation analysis showed that the P protein forms a stable homooligomer (perhaps a trimer) that is present in L-P and N-P complexes in the higher oligomeric forms (at least a pentamer). Interestingly, coexpression of a large excess of N- or C-terminally deleted P with wild-type P had no effect on minigenome replication in vivo, notwithstanding the formation of heterooligomeric complexes. These data indicate that P protein with a deleted terminal domain can function normally within the P heterooligomeric complex to carry out transcription and replication in vivo.
authors.affiliation: Department of Virology, Lerner Research Institute, The Cleveland Clinic Foundation, OH 44195, USA.
authors.forename: B P
authors.initials: BP
authors.lastname: De
authors.forename: M A
authors.initials: MA
authors.lastname: Hoffman
authors.forename: S
authors.initials: S
authors.lastname: Choudhary
authors.forename: C C
authors.initials: CC
authors.lastname: Huntley
authors.forename: A K
authors.initials: AK
authors.lastname: Banerjee
mesh_terms: D002460:Cell Line; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D016296:Mutagenesis; D010224:Parainfluenza Virus 3, Human / Q000235:genetics*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger; D012367:RNA, Viral; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014779:Virus Replication
chemical_list: C095185:P protein, Human parainfluenza virus 3; D010750:Phosphoproteins; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; C056831:parainfluenza 3 virus, L Protein