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The RNA-dependent RNA polymerases (RdRp) from Citrus tristeza virus (CTV) were tagged with HA and FLAG epitopes. Differentially tagged proteins were expressed either individually or concomitantly in Escherichia coli. Immunoprecipitation of the expressed proteins with anti-FLAG antibody followed by Western blot with anti-HA antibody demonstrated that molecules of RdRp from CTV interact to form oligomers. Yeast two-hybrid assays showed that molecules of RdRp interact in eukaryotic cells. Co-immunoprecipitation with anti-FLAG antibody of truncated HA-tagged RdRps (RdRpΔ1-166-HA, RdRpΔ1-390-HA, RdRp1-169-HA) co-expressed with full-length RdRp-FLAG showed that only RdRp1-169-HA interacted with the full-length FLAG-RdRp. Yeast two-hybrid assays with truncated RdRp constructs confirmed that the oligomerization site resides in the N-terminal region and that the first 169 aa of CTV RdRp are necessary and sufficient for oligomerization both in bacterial and yeast cells. Development of control strategies targeting viral RdRp oligomer formation may inhibit virus replication and prove useful in control of CTV., (© 2013 Elsevier Inc. All rights reserved.)

The RNA dependent RNA polymerase of Sendai virus consists of a complex of the large (L) and phosphoprotein (P) subunits where L is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. We previously showed that the L protein forms an oligomer [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] and mapped the L oligomerization domain between amino acids 1 and 174 of the protein [Cevik, B., Smallwood, S., Moyer, S.A., 2003. The oligomerization domain resides at the very N-terminus of the Sendai virus L RNA polymerase protein. Virology 313, 525-536]. An internal deletion encompassing amino acids 20 to 178 of the L protein lost polymerase activity but still formed an L-L oligomer. The first 25 amino acids of paramyxovirus L proteins are highly conserved and site-directed mutagenesis within this region eliminated the biological activity of the L protein but did not have any effect on P-L or L-L interactions. Moreover deletion of amino acids 2-18 in L abolished biological activity, but again the L-L binding was normal demonstrating that the oligomerization domain of L protein resides in two N-terminal regions of the protein. Therefore, sequences between both aa 2-19 and aa 20-178 can independently mediate Sendai L oligomerization, however, both are required for the activity of the protein.

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.

To catalyze RNA synthesis, the Sendai virus P-L RNA polymerase complex first binds the viral nucleocapsid (NC) template through an interaction of the P subunit with NP assembled with the genome RNA. For replication, the polymerase utilizes an NP(0)-P complex as the substrate for the encapsidation of newly synthesized RNA which involves both NP-RNA and NP-NP interactions. Previous studies showed that the C-terminal 124 amino acids of NP (aa 401-524) contain the P-NC binding site. To further delineate the amino acids important for this interaction, C-terminal truncations and site-directed mutations in NP were characterized for their replication activity and protein-protein interactions. This C-terminal region was found in fact to be necessary for several different protein interactions. The C-terminal 492-524 aa were nonessential for the complete activity of the protein. Deletion of amino acids 472-491, however, abolished replication activity due to a specific defect in the formation of the NP(0)-P complex. Binding of the P protein of the polymerase complex to NC required aa 462-471 of NP, while self-assembly of NP into NC required aa 440-461. Site-directed mutations from aa 435 to 491 showed, however, that the charged amino acids in this region were not essential for these defects.

The Sendai virus RNA-dependent RNA polymerase is composed of the L and P proteins. We previously showed that the L protein gives intragenic complementation and forms an oligomer where the L-L interaction site mapped to the N-terminal half of the protein (S. Smallwood et al., 2002, Virology, 00, 000-000). We now show that L oligomerization does not depend on P protein and progressively smaller N-terminal fragments of L from amino acids (aa) 1-1146 through aa 1-174 all bind wild-type L. C-terminal truncations up to aa 424, which bind L, can complement the transcription defect in an L mutant altered at aa 379, although these L truncation mutants do not bind P. The fragment of L comprising aa 1-895, furthermore, acts as a dominant-negative mutant to inhibit transcription of wild-type L. N-terminal deletions of aa 1-189 and aa 1-734 have lost the ability to form the L-L complex as well as the L-P complex, although they still bind C protein. These data are consistent with the L-L interaction site residing in aa 1-174. Site-directed mutations in the N-terminal 347 aa, of L which abolish P binding, do not affect L-L complex formation, so while the L and P binding sites on L are overlapping they are mediated by different amino acids. The N-terminal portions of L with aa 1-424, aa 1-381, and to a lesser extent aa 1-174, can complement the transcription defect in an L mutant altered at aa 77-81, showing their L-L interaction is functional.

The RNA-dependent RNA polymerase of Sendai virus consists of two subunits, the L and P proteins, where L is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. Sequence alignment of the L proteins of a variety of negative-stranded RNA viruses revealed six regions of good conservation, designated domains I-VI, which are thought to correspond to functional domains of the protein. Analysis of a number of site-directed mutants within the six domains of L allowed us to conclude that the activities of the polymerase are not simply compartmentalized and that each domain contributes to multiple steps in viral RNA synthesis. Nevertheless these domains can function in trans since we demonstrate here that intragenic complementation between pairs of coexpressed inactive L mutants can restore viral RNA synthesis on an added template. Although intragenic complementation is typically very inefficient, complementation to restore leader RNA synthesis was surprisingly very efficient for some pairs and complementation of mRNA synthesis and genome replication was less, but still significant. Complementation occurred with L mutants in five of the six domains, the exception being a domain III mutant, and required the cotranslation of the two L mutants. C-terminal truncations deleting up to half of L were capable of restoring transcription of an inactive domain I L mutant at amino acid 379. Oligomerization of L in the polymerase complex was demonstrated directly by the co-immunoprecipitation of differentially epitope-tagged full-length and truncated L proteins. These data are consistent with L protein being an oligomer with multiple independent domains each of which exhibits several functions., (Copyright 2002 Elsevier Science (USA))