Proteomics

Studying the biochemistry of viral RNA-dependent RNA polymerases

The RNA-dependent RNA polymerase (RdRp) is the key enzyme for the replication of all positive-strand RNA viruses. The "palm domain" containing motifs A, B, C, and D is found in many polymerases whereas the E motif is unique to RdRps and reverse transcriptases. The motif E is located at the junction of the palm and thumb subdomains and is suggested to control the flexibility of the thumb during DNA polymerization We created E motif mutants in an infectious TBSV clone to render it deficient for replication. Upon infection of these mutants on transgenic Nicotiana benthamiana plants expressing a functional recombinant TBSV RdRp we could detect virus symptoms suggesting (partial) reversion of the mutation and restoration of infectivity. Revertants were isolated and the genetic changes in their RdRps were analysed. The transgenic-trans-complementation system we developed proved to be useful to determine essential amino acids residues within the RdRp motif E and other motifs. Understanding the role of different amoni acid residues in RdRp motifs will provide valuable information to develop anti-viral drugs.

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