Selection of RNA replicons capable of persistent noncytopathic replicationin mammalian cells

The natural life cycle of alphaviruses, a group of plus-strand RNA viruses, involves; transmission to vertebrate hosts via mosquitoes. Chronic infecti ons are established in mosquitoes (and usually in mosquito cell cultures), but infection of susceptible vertebrate cells typically results in rapid sh utoff of host mRNA translation and cell death. Using engineered Sindbis vir us RNA replicons expressing puromycin acetyltransferase as a dominant selec table marker, we identified mutations allowing persistent, noncytopathic re plication in BHK-21 cells. Two of these adaptive mutations involved single- amino-acid substitutions in the C-terminal portion of nsP2, the viral helic ase-protease. At one of these loci, nsP2 position 726, numerous substitutio n mutations were created and characterized in the context of RNA replicons and infectious virus. Our results suggest a direct correlation between the level of viral RNA replication and cytopathogenicity, This work also provid es a series of alphavirus replicons for noncytopathic gene expression studi es (E, V, Agapov, I. Frolov, B. D, Lindenbach, B, M, Pragai, S, Schlesinger , and C, M. Rice, Proc Natl, Acad, Sci, USA 95:12989-12991, 1998) and a gen eral strategy for selecting RNA viral mutants adapted to different cellular environments.