LOCALIZATION OF FUNCTIONAL REGIONS OF THE CUCUMBER MOSAIC-VIRUS RNA REPLICASE USING MONOCLONAL AND POLYCLONAL ANTIBODIES

Monoclonal antibodies were produced using a purified cucumber mosaic v irus (CMV) replicase complex, and Escherichia coli-expressed CMV 1a an d 2a proteins, as immunogens. Five out of eight monoclonal antibodies, which bound to the 1a and 2a proteins in immunoblots, inhibited the R NA-dependent RNA polymerase (RdRp) activity of the purified replicase complex in vitro. Epitope mapping showed that two of the inhibitory an tibodies interacted with regions of the la protein containing putative helicase and methyltransferase domains respectively. Two other inhibi tory antibodies mapped to a region of the 2a protein containing the GD D motif which is highly conserved in RdRps. Prior interaction of the l atter antibodies with a peptide containing the GDD motif prevented the antibody-mediated inhibition of the replicase. Polyclonal antibodies which inhibited the RdRp activity of the replicase complex were also p roduced using peptides corresponding to conserved helicase and polymer ase motifs in the 1a and 2a proteins. The greatest inhibition was show n by antibodies to a peptide containing the GDD motif. These results d emonstrate the functional importance of the identified sequence motifs in CMV RNA replication and indicate that the motifs are located in th e replicase complex at positions accessible to antibodies, consistent with roles in interacting with the RNA template, RNA primer and enzyme substrates.