A new strategy in design of (+)RNA virus infectious clones enabling their stable propagation in E-coil

Infectious clone methodology is a valuable tool of modern experimental viro logy. However, its use is often constrained by the instability of infectiou s clone constructs during propagation in E. coli. To circumvent this proble m, we have devised a strategy that could be suitable for design of (+)RNA v irus molecular clones in general. An infectious clone is assembled as "infe ctious DNA," and expression of problem regions present in the viral cDNA is prevented during propagation in E. coli by insertion of short introns. To demonstrate the feasibility of this approach, a highly unstable Japanese en cephalitis flavivirus infectious clone has been successfully converted into a remarkably stable infectious DNA construct with the specific infectivity of 10(6) pfu/mug in cell culture. The proposed strategy may be useful in t he design of self-amplifying gene therapy vectors and development of new im munization methodologies, and could facilitate creation of molecular reposi tories of existing viral vaccines. (C) 2001 Academic Press.