Reexamination of the Sendai virus P protein domains required for RNA synthesis: a possible supplemental role for the P protein.
The Sendai virus P protein plays a central role in viral genome amplification and expression, forming complexes with the viral L protein to generate the polymerase (P-L) and unassembled N (P-N(o)). This latter complex prevents N from self-assembling illegitimately, i.e., independently of the concurrent assembly of a nascent viral genome, and is thought to represent the functional form of N in nucleocapsid assembly. Based upon earlier functional studies using an in vitro transcription/ replication system in which the P, L, and N proteins were coexpressed, we identified two regions of the P protein required for RNA synthesis, namely, the C-terminal 40% of the protein, and a second, apparently redundant domain near the N-terminus (either amino acids 1-77 or 78-145). The lack of sequence conservation in this second region, apart from overall negative charge, was reminiscent of the acidic activation domains of cellular transcription factors. However, we recently mapped a chaperone domain at the N-terminal of P (aa 33-41), which is required for stable complex formation with unassembled N(o) and, thus, for assembly and genome replication. In this present study we show that coexpression of N protein with P deletion mutants lacking this region (e.g., P delta 1-324) results in the sequestration of the mutant P by the illegitimately assembled form of N. As a consequence, P protein is unavailable for RNA synthesis from bona fide templates. We also find that in the absence of coexpressed N protein, the entire N-terminal 60% of the P protein is not required for mRNA synthesis. During these studies, a supplemental role for the P protein in viral RNA synthesis, independent of stable complex formation with L, was observed. This function involves, at least in part, the binding of additional copies of P to the N:RNA template.
Curran
J
J
Department of Genetics and Microbiology, University of Geneva Medical School, Switzerland. curran@cmu.unige.ch
D001665:Binding Sites; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases; D010222:Parainfluenza Virus 1, Human / Q000235:genetics* / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis*; D014763:Viral Matrix Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
C097020:M protein, Sendai virus; C055859:P protein, Sendai virus; D010750:Phosphoproteins; D012333:RNA, Messenger; D012367:RNA, Viral; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C064365:L protein, Sendai virus; D012321:DNA-Directed RNA Polymerases