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1、 Recombination in large RNA viruses: Coronaviruses Michael M. C. Lai Coronaviruses contain a very large RNA genome, which undergoes recombination at a very high frequency of nearly 25% for the entire genome. Recombination has been demonstrated to occur between viral genomes and between defective-int
2、erfering (DI) RNAs and viral RNA. It provides an evolutionary tool for both viral RNAs and DI RNA and may account for the diversity in the genomic structure of coronaviruses. The capacity of coronaviruses to undergo recombination may be related to its mRNA transcription mechanism, which involves dis
3、continuous RNA synthesis, suggesting the nonprocessive nature of the viral polymerase. Recombination is used as a tool for the mutagenesis of viral genomic RNA. Key words: defective-interfering RNA / mouse hepatitis virus / RNA evolution / RNA recombination 1996 Academic Press Ltd CORONAVIRUSES CONT
4、AINan extraordinarily large RNA genome (2731kb). This large RNA size imposes a severe burden on the virus because such an RNA can be expected to accumulate a large number of errors during RNA replication, assuming that the error frequency of coronaviral RNA polymerase is compara- ble to that of othe
5、r RNA viruses. Thus, coronaviruses must develop genetic mechanisms to counter the potentially deleterious effects of the errors. RNA recombination is one such mechanism. The discovery of RNA recombination in coro- naviruses1was made at a time when only picorna- viruses, but no other RNA viruses, had
6、 been demon- strated to be capable of RNA recombination. And it came with a vengeance, as murine coronaviruses were quickly shown to recombine at very high frequency under a variety of natural and experimental condi- tions. The capacity to recombine has now been demonstrated in several different cor
7、onaviruses. Recombination is an important mechanism contribut- ing to both the genetic stability and diversity of coronaviruses in nature. Characteristics of coronavirus RNA and its synthesis The coronavirus RNA genome is a single species of single-stranded, positive-sensed RNA 2731kb in length (see
8、 Lai 1990).2It consists of seven to 10 genes, one of which (gene 1) encodes a precursor of RNA polymerase of approximately 750800kDa. Gene composition and arrangement vary among the differ- ent coronaviruses (Figure 1). The enormous size (22kb) of the polymerase gene suggests that the polymerase has
9、 multiple functions. Each gene is expressed through one of the mRNAs, which are 3-coterminal and have a nested-set structure. Only the 5-most gene of each mRNA is functional for protein translation. Each mRNA has a leader sequence of 7090 nucleotides derived from the 5-end of the genome RNA. mRNA tr
10、anscription is carried out by a discontinuous transcription mecha- nism which fuses the leader RNA to the transcription start signal (intergenic sequence). The mRNA leader sequence is usually derived in trans from a different RNA molecule.3,4Therefore, the coronaviral polymer- ase must jump between
11、the leader sequence and intergenic sequences in different RNA molecules during positive- or negative-strand RNA synthesis. Recombination between viral genomes The fi rst coronavirus recombinant was isolated by coinfecting temperature-sensitive (ts) mutants of two mouse hepatitis virus (MHV) strains,
12、 A59 and JHM, and selecting progeny viruses which grew at the nonpermissive temperature.1The identity of this recombinant was established by genomic sequence analysis, which showed that it indeed had one cross- over site and contained sequences from both parents. Subsequently, additional recombinant
13、s were obtained using different pairs of ts mutants and other selection From the Howard Hughes Medical Institute, Department of Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, CA 90033-1054, USA seminars inVIROLOGY,Vol 7, 1996: pp 381388 1996
14、 Academic Press Ltd 1044-5773/96/060381 + 08 $25.00/0 381 markers, including monoclonal antibody neutral- ization epitopes and cellcell fusion ability.5-9 Although recombination frequency was not deter- mined in these early studies, the ease with which these recombinants were isolated suggested that
15、 the recom- bination frequency of MHV was very high. This was also suggested from the fi nding that many of these recombinants had multiple cross-overs, some of which were surprisingly located outside of the two selection markers used for the isolation of the recombinants. Therefore, MHV recombinati
16、on likely occurs at such a high frequency that recombinants are selected without specifi c selection pressure. The high fre- quency of recombination was also demonstrated in an experiment in which an A59 ts mutant and wild-type JHM were used for a mixed infection.8The recombi- nant viruses that grew at the nonpermissive tem- perature became the predominant virus population after only two tissue culture passages. This result was striking because one of the parental viruses (JHM) was Figure 1.
