A mutant of Sindbis virus that is resistant to pyrazofurin encodes an altered RNA polymerase

Pyrazofurin (PZF), a cytidine analog and an inhibitor of orotate monophosph ate decarboxylase, has been shown to decrease the levels of UTP and CTP in treated cells. When Sindbis Virus (SV)-infected Aedes albopictus cells were treated with PZF, the yield of Virus was reduced 100- to 1000-fold. By ser ial passage of our standard SVSTD in Ae. albopictus cells in the presence o f increasing concentrations of PZF, a mutant, SVPZF was derived, which was not inhibited by PZF. SVPZF is also resistant to adenosine, guanosine, and phosphono-acetyl-N-aspartate, all of which have been shown to decrease leve ls of UTP and CTP. Analysis of chimeric Viruses containing sequences from t he SVPZF and parental genomes showed that the sequence between nt 5262 and 7999 conferred the PZF-resistant phenotype. Sequencing of this region ident ified four mutations (nt 5750, 6627 7543, and 7593), which are predicted to lead to amino acid changes: opal550L in nsP3 and M287L, K592I, and P609T i n nsP4. Characterization of viruses containing one or more of these mutatio ns demonstrated that all three mutations in the nsP4 coding region are requ ired to produce full resistance to PZF. Using a molecular model of nsP4 bas ed on the structure of HIV reverse transcriptase, we located amino acid cha nge M287L at the tip of the fingers domain and K592I and P609T at the base of the thumb domain of the viral RNA polymerase. We suggest that these thre e amino acid changes in nsP4 alter the geometry of the NTP binding pocket s o as to increase the affinity of the enzyme for CTP and UTP, (C) 2000 Acade mic Press.