Mutations in conserved domains IV and VI of the large (L) subunit of the sendai virus RNA polymerase give a spectrum of defective RNA synthesis phenotypes.

The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein. When aligned with amino acid sequences of L proteins from other negative-sense RNA viruses, the Sendai L protein contains six regions of good conservation, designated domains I-VI, which have been postulated to be important for the various enzymatic activities of the polymerase. To directly address the roles of domains IV and VI, 14 site-directed mutations were constructed either by changing clustered charged amino acids to ala or by substituting selected Sendai L amino acids with the corresponding sequence from measles virus L.

Mutations in conserved domain II of the large (L) subunit of the Sendai virus RNA polymerase abolish RNA synthesis.

The large (L) protein of Sendai virus complexes with the phosphoprotein (P) to form the active RNA-dependent RNA polymerase. The L protein is believed to be responsible for all of the catalytic activities of the polymerase associated with transcription and replication. Sequence alignment of the L proteins of negative-strand RNA viruses has revealed six conserved domains (I-VI) thought to be responsible for the enzymatic activities.

The Sendai virus C protein binds the L polymerase protein to inhibit viral RNA synthesis.

The Sendai virus nested set of C proteins which are expressed in an alternative open reading frame from the P mRNA has been shown to downregulate viral RNA synthesis. Utilizing a glutathione S-transferase (gst) C fusion protein (gstC), we have shown that C protein forms a complex with the L, but not the P, subunit of the viral RNA polymerase. When P, L, and gstC are coexpressed, an oligomer of P, through its interaction with L, is also bound to beads.

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 glutathione S-transferase (gst)-viral fusion proteins synthesized in mammalian cells. Using the two-hybrid system we have confirmed the previously identified P-L (RNA polymerase), NPo-P (encapsidation substrate), and P-P complexes and now demonstrate NP-NP and NPo-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.

Domains of the measles virus N protein required for binding to P protein and self-assembly.

The nucleocapsid protein (N, 525 amino acids) of measles virus plays a central role in the replication of the viral genomic RNA. Its functions require interactions with itself and with other viral components. The N protein encapsidates genomic RNA, a function reflected in its ability to self-assemble into nucleocapsid-like particles in the absence of other viral proteins.