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The respiratory syncytial virus (RSV) phosphoprotein (P) is a major polymerase co-factor that interacts with both the large polymerase fragment (L) and the nucleoprotein (N). The N-binding domain of RSV P has been investigated by co-expression of RSV P and N proteins in Escherichia coli. Pull-down assays performed with a series of truncated forms of P fused to glutathione S-transferase (GST) revealed that the region comprising the last nine C-terminal amino acid residues of P (233-DNDLSLEDF-241) is sufficient for efficient binding to N. Site-directed mutagenesis shows that the last four residues of this peptide are crucial for binding and must be present at the end of a flexible C-terminal tail. The presence of the P oligomerization domain (residues 100–160) was an important stabilizing factor for the interaction. The tetrameric full-length P fused to GST was able to pull down both helical and ring structures, whereas a monomeric C-terminal fragment of P (residues 161–241) fused to GST pulled down exclusively RNA–N rings. Electron-microscopy analysis of the purified rings showed the presence of two types of complex: undecamers (11N) and decamers (10N). Mass-spectrometry analysis of the RNA extracted from rings after RNase A treatment showed two peaks of 22 900 and 24 820 Da, corresponding to a mean RNA length of 67 and 73 bases, respectively. These results suggest strongly that each N subunit contacts 6 nt, with an extra three or four bases further protected from nuclease digestion by the ring structure at both the 5′ and 3′ ends.

cDNA expression vectors of Potato leafroll virus (PLRV) were used to analyse specific mutations in the proteinase and replicase domains of the proteins encoded by ORF1 and ORF2. Agrobacterium-mediated DNA transfer was used to introduce a PLRV RNA expression unit, controlled by the 35S promoter of Cauliflower mosaic virus, into potato leaf cells. Expression of unmodified PLRV cDNA led to the replication of viral genomic and subgenomic RNAs and accumulation of the viral capsid protein, whereas alteration of amino acids GDD513–515 of the replicase to VHD abolished PLRV replication. Mutations in the presumed H-D-S catalytic triad of the viral proteinase abolished the formation of viral genomic and subgenomic RNAs as well as synthesis of the viral capsid protein. Co-agroinoculation of the GDD mutant along with any of the proteinase mutants restored virus replication in leaf discs, showing that these mutants are able to complement each other. Moreover, mutation of the postulated serine residue of the catalytic triad of the proteinase altered the pattern of proteins synthesized in vitro in comparison to wild-type, further supporting the relevance of the H-D-S motif.