GENESIS OF SINDBIS VIRUS BY IN-VIVO RECOMBINATION OF NONREPLICATIVE RNA PRECURSORS

Genetically engineered RNA transcripts coding for various Sindbis viru s (SIN) genes were used to study structure and sequence requirements o f RNA recombination in BHK cells. Three different groups of RNA transc ripts were made: (i) RNAs which retain the ability to replicate and wh ich carry sequences coding for either viral polymerase or viral struct ural proteins; (ii) RNAs which lack the complete 3' end of the SIN gen ome and thus are incapable of replicating; and (iii) RNAs which lack t he complete 5' end of the SIN genome and also are incapable of replica ting. BHK cells were transfected with specific combinations of these p recursor RNAs, and virus production and RNA synthetic abilities of the released virus were determined. We demonstrate in vivo generation of infectious SIN by fusion of (i) replicative RNAs to nonreplicative RNA s and (ii) two nonreplicative RNA precursors. Both homologous and nonh omologous types of recombinations were observed. In the homologous typ e of recombination, a 694-nucleotide overlap at the crossover region o f the first pair of precursors resulted in the addition of an A residu e converting the UAG stop codon of nonstructural protein P4 to a UAA s top codon. In the nonhomologous type of recombination, the crossover s ites contained deletion of up to 76 nucleotides from one of the precur sors and complete preservation of junction sequence from the other pre cursor. This is also the first report that a cytoplasmic RNA virus can be generated from biologically nonreplicative RNA precursors. These r esults have implications for initiation of viral RNA synthesis and rec ombination between RNA viral genomes in general. We favor template swi tching as a mechanism for the fusion events described here and suggest inclusion of polymerase scanning of diverse nonreplicative RNAs as an inherent feature of the copy choice model of RNA recombination. Very importantly, the facile nature of RNA recombination occurring between nonreplicative RNA precursors should speed up the production and analy sis of targeted mutants of SIN and possibly other RNA viruses.