GENERATION OF CORONAVIRUS SPIKE DELETION VARIANTS BY HIGH-FREQUENCY RECOMBINATION AT REGIONS OF PREDICTED RNA SECONDARY STRUCTURE

Coronavirus RNA evolves in the central nervous systems (CNS) of mice d uring persistent infection. This evolution can be monitored by detecti on of a viral quasispecies of spike deletion variants (SDVs) (C. L. Ro we, S. C. Baker, M. J. Nathan, and J. O. Fleming, J. Virol. 71:2959-29 69, 1997). We and others have Pound that the deletions cluster in the region from 1,200 to 1,800 nucleotides from the 5' end of the spike ge ne sequence, termed the ''hypervariable'' region. To address how SDVs might arise, we generated the predicted folding structures of the posi tive- and negative-strand senses of the entire 4,139-nt spike RNA sequ ence. We found that a prominent, isolated stem-loop structure is coinc ident with the hypervariable region in each structure, To determine if this predicted stem-loop is a ''hot spot'' for RNA recombination, Re assessed whether this region of the spike is more frequently deleted t han three other selected regions of the spike sequence in a population of viral sequences isolated from the CNS of acutely and persistently infected mice. Using differential colony hybridization of cloned spike reverse transcription-PCR products, we detected SDVs in which the hot spot was deleted but did not detect SDVs in which other regions of th e spike sequence were exclusively deleted. Furthermore, sequence analy sis and mapping of the crossover sites of 25 distinct patterns of SDVs showed that the majority of crossover sites clustered to two regions at the base of the isolated stem-loop, which we designated as high-fre quency recombination sites 1 and 2. Interestingly, the majority of the left and right crossover sites of the SDVs were directly across from or proximal to one another, suggesting that these SDVs are likely gene rated by intramolecular recombination. Overall, our results are consis tent with there being an important role for the spike RNA secondary st ructure as a contributing factor in the generation of SDVs during pers istent infection.