The Sendai Virus V Protein Interacts with the NP Protein to Regulate Viral Genome RNA Replication

The interactions of Sendai virus proteins required for viral RNA synthesis have been characterized both by the yeast two-hybrid system and through the use of glutathioneS-transferase (gst)–viral fusion proteins synthesized in mammalian cells. Using the two-hybrid system we have confirmed the previously identified P–L (RNA polymerase), NP0-P (encapsidation substrate), and P–P complexes and now demonstrate NP–NP and NP0–V protein interactions. Expression of gstP and P proteins and binding to glutathione–Sepharose beads as a measure of complex formation confirmed the P–P interaction. The P–gstP binding occurred only on expression of the proteins in the same cell and was mapped to amino acids 345–411. We also show that full-length and deletion gstV and gstW proteins bound NP0protein when these sets of proteins were coexpressed and have identified one required region from amino acids 78–316. Neither gstV nor gstW bound NP assembled into nucleocapsids. Furthermore, both V and W proteins lacking the N-terminal 77 amino acids inhibited DI-H genome replicationin vitro,showing the biological relevance of the remaining region. We propose that the specific inhibition of genome replication by V and W proteins occurs through interference with either the formation or the use of the NP0– P encapsidation substrate.