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.