NONHOMOLOGOUS RNA-RNA RECOMBINATION EVENTS AT THE 3' NONTRANSLATED REGION OF THE SINDBIS-VIRUS GENOME - HOT-SPOTS AND UTILIZATION OF NONVIRAL SEQUENCES

The mechanism of RNA-RNA recombination at the 3' nontranslated region (3'NTR) of the Sindbis virus (SIN) genome was studied by using nonrepl icative RNA precursors. The 11.7-kb SIN genome was transcribed in vitr o as two nonoverlapping RNA fragments. RNA-1 contained the entire 11.4 -kb protein coding sequence of SIN and also carried an additional 1.8- kb nonviral sequence at its 3' end. RNA-2 carried the remaining 0.26 o r 0.3 kb of the SIN genome containing the 3'NTR. Transfection of these two fragments into BHK cells resulted in vivo RNA-RNA recombination a nd release of infectious SIN recombinants. Eighteen plaque-purified re combinant viruses were sequenced to precisely map the RNA-RNA crossove r sites at the 3'NTR. Sixteen of the 18 recombinants were found to be genetically heterogeneous at the 3'NTR. Two major clustered sites with in the 3'NTR of RNA-2 were found to be fused to multiple locations on the nonviral sequence of RNA-1, resulting in insertions of 10 to 1,085 nucleotides at the 3'NTR Sequence analysis of crossover sites suggest ed only limited homology and heteroduplex-forming capability between s ubstrate RNAs. Analysis of additional 23 recombinant viruses generated by mutagenized donor and acceptor templates supports the occurrence o f recombination hot spots on donor templates. Introduction of a 17-nuc leotide rudimentary replicase recognition signal in the acceptor templ ate alone did not induce the polymerase to reinitiate at the 17-nucleo tide signal. Interestingly, deletion of a 24-nucleotide hot spot locus on the donor template abolished crossover events at one of the two si tes and allowed the polymerase to reinitiate at the 17-nucleotide repl icase recognition signal inserted at the acceptor template. The possib le roles of RNA-protein and RNA-RNA interactions in the differential r egulation of apparent pausing, template selection, and reinitiation ar e discussed.