Measles virus encodes an RNA-dependent RNA polymerase composed of the L and P proteins. Recent studies have shown that the L proteins of both Sendai virus and parainfluenza virus 3 form an L-L complex [Cevik, B., Smallwood, S., Moyer, S.A., 2003. The oligomerization domain resides at the very Nterminus of the Sendai virus L RNA polymerase protein. Virology 313, 525-536.; Smallwood, S., Moyer, S.A., 2004. The L polymerase protein of parainfluenza virus 3 forms anoligomer and can interact with the heterologous Sendai virus L, P and C proteins. Virology 318, 439-450.; Smallwood, S., Cevik, B., Moyer, S.A., 2002. Intragenic complementation and oligomerization of the L subunit of the Sendai virus RNA polymerase. Virology 304, 235-245.]. Using differentially tagged L proteins, we show here that measles L also forms an oligomer and the L-L binding site resides in the N-terminal 408 amino acids overlapping the P binding site in the same region of L. To identify amino acids important for binding P and L, site-directed mutagenesis of the L-408 protein was performed. Seven of twelve mutants in L-408 were unable to form a complex with measles P while the remainder did bind at least some P. In contrast, all of the mutants retained the ability to form the L-L complex, so different amino acids are involved in the L and P binding sites on L. Four of the 408 mutations defective in P binding were inserted into the full-length measles L protein and all retained L-L complex formation, but did not bind P. Full-length L mutants that did not bind P were also inactive in viral RNA synthesis, showing a direct correlation between P-L complex formation and activity.

The yeast retrotransposon, Ty1, produces a macromolecular structure known as a virus-like particle (VLP) as an essential part of its replication cycle. The Ty1 Gag-like structural protein TYA, p1-440, alone is capable of directing assembly of the VLP. In order to determine the TYA sequences required for assembly, we have produced a series of truncated and deleted TYA forms and assessed their ability to assemble into particles. Removal of 100 amino acids from the C-terminus renders the TYA protein, p1-340, incapable of particle assembly; however, p1-363 with 77 residues missing from the C-terminus is capable of assembly. Removal of 40 amino acids from the N-terminus (p41-440 and p41-381) does not affect particle formation but more severely N-truncated forms, p71-381 and p100-381, are present as large aggregates within the cells and are therefore either incapable of or unavailable for VLP formation. Analysis of an internally deleted TYA, p1-381 delta 62-114, has identified this as a possible region of the TYA protein important for subunit:subunit interactions during the particle assembly.

Sendai virus encodes an RNA-dependent RNA polymerase which is composed of the L and P proteins. Site-directed mutagenesis of the N terminus of L has identified amino acids important for binding P. Seven of nine mutants in amino acids 1 to 350 of Sendai L lost the ability to bind to Sendai P, although they were still able to bind the viral C protein. Loss of P binding correlated with the loss of all RNA synthesis activities. Two L mutants gave limited P-L complex formation and limited viral transcription and replication.The viruses belonging to the Paramyxovirinae family contain a negative-sense (Ϫ), unsegmented RNA genome of about 15 kb, which is encapsidated by the nucleocapsid protein (NP) in a helical nucleocapsid that serves as the template for all viral RNA synthesis (12). The virion-associated phosphoprotein (P) and the L protein are the two subunits of the viral RNAdependent RNA polymerase. Transcription initiates at the 3Ј end of the genome RNA, giving the sequential synthesis of (ϩ) strand leader (leϩ) RNA and then the NP, P/C/V, M, F, HN, and L mRNAs. During genome replication the synthesis of full-length viral RNA is coupled to its encapsidation by NP protein, utilizing an NP 0 -P protein complex as the source of NP. For Sendai virus, formation of the RNA polymerase requires the cotranslation of the P and L proteins, in which P binding stabilizes the L protein, probably by facilitating the correct folding of L (8, 9). The Sendai virus P protein is a tetramer forming a coiled coil (18,19), and the L binding site on P mapped to amino acids (aa) 412 to 479 (6, 17).The L protein is thought to contain all the catalytic functions required for RNA synthesis. Alignment of the L proteins of viruses of the order Mononegevirales showed six domains of relatively high conservation, designated I to VI from the N terminus to the C terminus of the protein, which were proposed to specify the essential activities common to all L proteins (14, 15). Recent characterization of Sendai virus L mutants in each of the six domains suggests that multiple domains contribute to the different steps in viral RNA synthesis, since mutants in different domains gave the same defective phenotypes (2,3,7,11,16). Viral RNA synthesis is downregulated by the C proteins encoded from the P gene (12). In the case of Sendai virus, the C proteins were shown to bind to the L polymerase subunit to inhibit RNA synthesis (9).The L proteins of paramyxoviruses are all over 2,000 aa, and studies to begin mapping the P binding site on L showed a general location in the N-terminal quarter or half of the protein (2, 10, 13). Of the mutants characterized in the various domains in the N-terminal half of Sendai virus L only one, S368R in domain I, abolished binding to Sendai virus P (2, 7, 16). Together these data suggested that the P binding site may reside from aa 1 to 400 of L encompassing the N terminus and domain I. In these studies site-directed mutagenesis identified residues within the N-terminal 350 aa of the L protein of Sendai virus that mediate b...