Mitchell R. Lunn
Howard Hughes Medical Institute Medical Fellow

B.S. Biology and French, Tufts University, 2004.
D312 Fairchild
299 Campus Drive
Stanford CA 94305-5124
Research Summary

Positive-strand RNA viruses use virally encoded RNA-dependent RNA polymerases for genome replication. The high error rate of these polymerases effectively ensures that viruses resistant to any given antiviral compound will arise within a population. However, such a drug-resistant genome must be replicated, packaged, and secreted from a cell that is mixedly infected with its wild-type, drug-sensitive siblings. If the drug-sensitive viral genomes present in the cell dominantly interfere with any of the processes required for the growth of the drug-resistant virus, it will be unable to spread. Targeting viral proteins which, when defective or inhibited, have dominant negative effects on viral growth would be an advantageous antiviral approach; such proteins are herein defined as dominant drug targets.

Using poliovirus as a model system, the laboratory of Karla Kirkegaard, Ph.D. has discovered that mutations in poliovirus 2A protease are dominant and inhibitory to growth of any viruses in the same cell (Crowder S and Kirkegaard K, Nature Genetics 37:701-709). Protease 2A is a monomeric, soluble enzyme with several host protein substrates. It also cleaves the poliovirus polyprotein between VP1 and the 2A protease itself via an intramolecular event. If the VP1-2A cleavage were exclusively intramolecular, polyproteins with a defective 2A protease would accumulate even if functional 2A proteases were encoded by other genomes. This accumulation would inhibit coinfecting genomes when the accumulated VP1-2A protein coassembles with and contaminates wild-type viral capsids. The Kirkegaard laboratory showed that mutations in the 2A protease itself or the cleavage site produce dominant toxic genomes. This result is highly consistent with the hypothesis that the VP1-2A cleavage is exclusively intramolecular and the VP1-2A precursor is toxic, and defines poliovirus 2A as a dominant drug target.

Many positive-strand RNA viruses express proteins through proteolytic cleavage of a folded polyprotein. For a viral protease to be a dominant drug target, like poliovirus 2A, it must act via an exclusively intramolecular mechanism and the uncleaved product must be toxic to viral growth. All dengue virus proteins are expressed from a single open reading frame as a polyprotein and are ordered 5’-C-prM-E-NS1-NS2A·NS2B·NS3·NS4A-NS4B·NS5-3’. Using PCR-mediated mutagenesis and coinfection with wild-type dengue virus, I will test proteins in the Dengue genome to determine if any are dominant drug targets.

Copyright 2006 - 2007. The Laboratory of Karla Kirkegaard, Ph.D. All rights reserved.