NONHOMOLOGOUS RNA-RNA RECOMBINATION EVENTS AT THE 3' NONTRANSLATED REGION OF THE SINDBIS-VIRUS GENOME - HOT-SPOTS AND UTILIZATION OF NONVIRAL SEQUENCES
M. Hajjou et al., NONHOMOLOGOUS RNA-RNA RECOMBINATION EVENTS AT THE 3' NONTRANSLATED REGION OF THE SINDBIS-VIRUS GENOME - HOT-SPOTS AND UTILIZATION OF NONVIRAL SEQUENCES, Journal of virology, 70(8), 1996, pp. 5153-5164
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.