Localization of Functional Regions of the Cucumber Mosaic Virus RNA Replicase Using Monoclonal and Polyclonal Antibodies

Abstract

Monoclonal antibodies were produced using a purified cucumber mosaic virus (CMV) replicase complex, and Escherichia coli-expressed CMV la and 2a proteins, as immunogens. Five out of eight monoclonal antibodies, which bound to the la and 2a proteins in immunoblots, inhibited the RNA-dependent RNA polymerase (RdRp) activity of the purified replicase complex in vitro. Epitope mapping showed that two of the inhibitory antibodies interacted with regions of the la protein containing putative helicase and methyltransferase domains respectively. Two other inhibitory antibodies mapped to a region of the 2a protein containing the GDD motif which is highly conserved in RdRps. Prior interaction of the latter 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 produced using peptides corresponding to conserved helicase and polymerase motifs in the la and 2a proteins. The greatest inhibition was shown by antibodies to a peptide containing the GDD motif. These results demonstrate the functional importance of the identified sequence motifs in CMV RNA replication and indicate that the motifs are located in the replicase complex at positions accessible to antibodies, consistent with roles in interacting with the RNA template, RNA primer and enzyme substrates.

Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus

“…The inhibition of the template-dependent RCNMV RNA polymerase by antibodies to a peptide containing the GDD motif shows that the region of the polymerase containing this motif is accessible to the antibodies. A similar finding was made with an RNA-dependent RNA polymerase of cucumber mosaic virus (CMV), the activity of which was inhibited by antibodies to a peptide containing the GDD motif of the CMV 2a protein (Hayes et al, 1994). On the basis of conservation of this or equivalent motifs in DNA-and RNA-dependent RNA and DNA polymerases, Delarue et al (1990) suggested that the basic structure of all these polymerases may be similar.…”

The tobacco mosaic virus RNA polymerase complex contains a plant protein related to the RNA-binding subunit of yeast eIF-3

“…In some experiments, the RNA polymerase was incubated with purified MBP-fusion protein or MBP (10 g) at 0°C for 30 min prior to the addition of the antibodies. Control experiments to check that the antibodies were free from ribonuclease activity were carried out as described previously (2,20).…”

“…The sodium taurodeoxycholatesolubilized polymerase was used for further purification. No attempt was made to remove the endogenous bound RNA template at this stage, since viral RNA polymerases bound to their templates have been found to be more stable through subsequent purification stages (2,20).…”

Monoclonal antibodies specific for Semliki Forest virus replicase protein nsP2.

“…For example, antibody-mediated inhibition of proteolytic processing of poliovirus polypeptide P3-7c (Hanecak et al, 1982), and inhibition of RNA-dependent RNA polymerase in a purified replicase complex of cucumber mosaic virus (Hayes et al, 1994). Our previous results have shown that recombinant nsP2 and its N-terminal fragment possess NTPase activity.…”