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1、Characterizations of Coronavirus cis-Acting RNA Elements and the Transcription Step Affecting Its Transcription Efficiency Sungwhan An* and Shinji Makino*,1 *Department of Microbiology, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712-1095 Rece
2、ived November 14, 1997; returned to author for revision December 19, 1997; accepted January 26, 1998 Seven to eight species of viral subgenomic mRNAs are produced in coronavirus-infected cells. These mRNAs are produced in different quantities, and their molar ratios remain constant during viral repl
3、ication. We studied RNA elements that affect coronavirus transcription efficiency by characterizing a series of cloned coronavirus mouse hepatitis virus (MHV) defective interfering (DI) RNAs containing an inserted intergenic sequence, from which subgenomic DI RNA is transcribed in MHV-infected cells
4、. Certain combinations of upstream and downstream flanking sequences of the intergenic sequence suppressed subgenomic DI RNA transcription, yet changing one of the flanking sequences to a different sequence eliminated transcription suppression. The suppressive effect of certain combinations of flank
5、ing sequences, but not all combinations, could be counteracted by altering the intergenic sequence. Thus, the combination of intergenic sequence and flanking sequence affected transcription efficiency. We also characterized another set of DI RNAs designed to clarify which transcription step determin
6、es the relative molar ratios of coronavirus mRNAs. Our study indicated that if subgenomic mRNAs were exclusively synthesized from negative-strand genomic RNA, then the relative molar ratios of coronavirus mRNAs were most likely determined after synthesis of the genomic-sized template RNA. If negativ
7、e-strand subgenomic RNAs were templates for subgenomic mRNAs, then the relative molar ratios of coronavirus mRNAs probably were determined after synthesis of the genomic-sized template RNA used for subgenomic-sized RNA transcription but prior to the completion of the synthesis of subgenomic-sized RN
8、As containing the leader sequence. The relative molar ratios of coronavirus mRNAs, therefore, seem to have been established prior to a putative replicon-type amplification of subgenomic mRNAs. 1998 Academic Press INTRODUCTION Many eukaryotic RNA viruses express their gene(s) by producing subgenomic
9、mRNA(s) in infected cells. Coro- navirus, an enveloped virus containing a large positive- sense single-strand RNA, belongs to this group. Cells infected with mouse hepatitis virus (MHV), a prototypic coronavirus, produce seven to eight species of virus- specific mRNAs that make up a 39 coterminal ne
10、sted set structure (Lai et al., 1981; Leibowitz et al., 1981). These mRNAs, which are named mRNAs 1 through 7 in de- creasing order of size (Lai et al., 1981; Leibowitz et al., 1981), are produced in different quantities, and their molar ratios remain constant during MHV replication. The 59-end of t
11、he MHV genomic RNA and the sub- genomic mRNAs start with a leader sequence that is approximately 72 to 77 nucleotides (nt) long (Lai et al., 1983, 1984; Spaan et al., 1983); the presence of the leader sequence in subgenomic mRNAs is a unique character- istic in coronavirus and arterivirus (de Vries
12、et al., 1990), which is closely related to coronavirus. Curiously, on the genome, the leader sequence is not found any place besides the 59-end, yet the subgenomic mRNAs have the leader sequences fused with the mRNA body se- quences. The mRNA body sequences begin from a UC- UAAAC transcription conse
13、nsus sequence or a very sim- ilar sequence of intergenic sequences, which is located upstream of each MHV gene. Coronavirus transcription undergoes a discontinuous transcription step, in which the leader sequence somehow joins to the body of the subgenomic RNA (Jeong and Makino, 1994; Zhang et al.,
14、1994). Genomic-sized and subgenomic-sized negative- strand RNAs, each of which corresponds to one of the subgenomic mRNA species, are also present in corona- virus-infected cells (Sethna et al., 1989). These negative- strand RNAs contain an antileader sequence at the 39- end and a poly U sequence at
15、 the 59-end (Sethna et al., 1991). Several models have been proposed to explain coro- navirus subgenomic RNA synthesis. One model pro- poses that negative-strand RNA synthesis starts on genomic RNA and terminates at the intergenic sequence (Sawicki and Sawicki, 1990). In this model, the leader seque
16、nce joins to the body of subgenomic RNA either by relocalization of negative-strand subgenomic RNA to the leader sequence of the genomic RNA (antileader se- quence joins to negative-strand subgenomic RNA), or during subgenomic mRNA synthesis on negative-strand subgenomic RNA template (Sawicki and Sawicki, 1990). 1To whom correspondence and reprint requests should be ad- dressed. Fax: (512) 471-7088. E-mail: makinomail.utexas.edu. VIROLOGY243, 198207 (1998) ARTICLE NO. VY989059 0042-6822/98 $25.
