IDENTIFICATION OF MUTATIONS IN A SINDBIS VIRUS VARIANT ABLE TO ESTABLISH PERSISTENT INFECTION IN BHK CELLS - THE IMPORTANCE OF A MUTATION IN THE NSP2 GENE

Sindbis virus is a positive strand RNA virus that has provided a valua ble model for studying virus structure and replication. It is also bei ng developed as a vector for the expression of heterologous proteins. Many studies with this virus are carried out in cultured BHK cells whe re infection is usually highly cytopathic and within 1 or 2 days after infection all of the cells are dead. Weiss et al. had established a p ersistently infected culture of BHK cells by infecting the cells with a virus preparation highly enriched in defective interfering (DI) part icles and had isolated an attenuated virus, SIN-1 virus, from the cult ure [Weiss et al. (1980) J. Virol. 33, 463-474]. SIN-1 virus, free of DI particles, was able to establish a persistent infection in BHK cell s. We initiated studies to determine what changes in the genome of the virus were responsible for this phenotype. We describe here the cDNA cloning and sequencing of the 5' terminus and the four nonstructural p rotein genes from SIN-1 virus. A single coding mutation in the nsP2 ge ne (a predicted change of Pro-726 --> Ser) produced a virus that was a ble to establish persistent infection in BHK cells. Additional mutatio ns in the other genes were required to decrease the synthesis of viral RNA to a level similar to that found in cells infected with SIN-1 vir us. Incorporation of the nsP2 mutation into a Sindbis virus expression vector led to a higher level of synthesis of the reporter protein, be ta-galactosidase, than that obtained with the original Sindbis virus r eplicon. (C) 1997 Academic Press.