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1、Recombination and Coronavirus Defective Interfering RNAs DavidA. Brian and Willy J. M. Spaan* Department of Microbiology, College of Veterinary Medicine, M409 Walters Life Sciences Building, University of Tennessee, Knoxville, Tennessee 37996-0845; and *Department of Virology, Institute of Medical M
2、icrobiology, Leiden University, 2300 RC Leiden, The Netherlands Naturally occurring defective interfering RNAs have been found in 4 of 14 coronavirus species. They range in size from 2.2 kb to approximately 25 kb, or 80% of the 30-kb parent virus genome. The large DI RNAs do not in all cases appear
3、to require helper virus for intracellular replication and it has been postulated that they may on their own function as agents of disease. Coronavirus DI RNAs appear to arise by internal deletions (through nonhomologous recombination events) on the virus genome or on DI RNAs of larger size by a poly
4、merase strand-switching (copy-choice) mechanism. In addition to their use in the study of virus RNA replication and virus assembly, coronavirus DI RNAs are being used in a major way to study the mechanism of a high-frequency, site-specifi c RNA recombination event that leads to leader acquisition du
5、ring virus replication (i.e., the leader fusion event that occurs during synthesis of subgenomic mRNAs, and the leader-switching event that can occur during DI RNA replication), a distinguishing feature of coronaviruses (and arteriviruses). Coronavirus DI RNAs are also being engineered as vehicles f
6、or the generation of targeted recombinants of the parent virus genome. r1997Academic Press KEYWORDS: RNArecombination; leader fusion; recombinant coronaviruses. DISCUSSION Recombination and the Origin of Coronavirus DI RNAs Naturally occurring DI RNAs have been reported for the mouse hepatitis virus
7、 (MHV) (13), bovine coronavirus (BCV) (4), porcine transmissible gastroen- teritis virus (TGEV) (5), and avian infectious bronchitis virus (IBV) (6). They range in size from 2.2 kb to approximately 25 kb, or close to the full length of the parental virus genome of nearly 30 kb. A survey of DI RNA st
8、ructures indicates they are in all but one case a mosaic of linear segments of the virus genome and contain the genomic 58 and 38 termini (Fig. 1). In the large (,24 kb) DI ssA RNA of MHV (7) a rearrange- ment of sequence was found in which much of the leader, encoded by the virus genome 58 terminus
9、, was also found internally on the DI RNAgenome. Similarly rearranged sequences (i.e., nonlinear with the parental virus genome) have been found in the genomes of virus recombinants (10, 11). In the absence of evidence supporting a splicing process in coronavirus RNA synthesis (1214), the most likel
10、y mechanism for the origin of DI RNA, the one presumed in this review, appears to be template strand switching by the RNA polymerase from a donor template to an acceptor template of the same polarity during replication of a virus genome or of a previously formed DI RNA(15). It is unclear whether thi
11、s happens during plus-strand or minus-strandsynthesis.Fromtheclassifi cationofcopy- choice recombination patterns described by Lai (16), it will be recognized that the generation of DI RNA molecules must, in general, happen by nonhomolo- gous recombination. That is, the switch between tem- plates wo
12、uld take place between nonhomologous re- gions of the genome that demonstrate little or no sequence similarity. Although the general pattern in Seminars in VIROLOGY8, 101111 (1997) Article No.VI970109 1044-5773/97 $25.00 Copyrightr1997 byAcademic Press All rights of reproduction in any form reserved
13、.101 FIG. 1. Structures of the naturally occurring defective interfering RNAs identifi ed in coronavirus-infected cells. Sequences found in DI RNAs are aligned with their place of origin on the parent virus genome (vgRNA). Boxes identify genes. The 58 and 38 untranslated regions are identifi ed as n
14、arrow lines. The leader is identifi ed by a box with heavy crosshatches. Regions not confi rmed by sequencing are boxed by a dashed line.Asynthetic DI RNAreplicon for MHV-A59, B36 (9), patterned after the naturally occurring BCV DI RNA(4), is marked by a dagger. Asterisks identify recombination site
15、s that occur within 27 nt of the UCUAAAC consensus intergenic sequence motif. 102Brian and Spaan Copyrightr1997 byAcademic Press coronavirus DI RNA formation would suggest nonho- mologous recombination, at some sites, short regions of sequence similarity may have directed polymerase reentry (for exa
16、mple, at intergenic consensus hepta- meric sequence motifs discussed below in the context of leader acquisition). Thus, in the microenvironment of the recombination site a short region of sequence identity might play a role in the mechanism of strand switching. Biological Signifi cance of Coronavirus DI RNAs Many of the coronavirus DI RNAs appear to have the same attributes ascribed to DI RNAs of other RNA viruses (1719), namely, the ability to competitively inhibit parental virus genome repli
