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1、Virus Research, 13 (1989) 87-100 Elsevier 87 VRR 00499 Nucleotide sequence of coronavirus TGEV genomic RNA: evidence for 3 mRNA species between the peplomer and matrix protein genes Ronald D. Wesley, Andrew K. Cheung, David D. Michael and Roger D. Woods US?. 7859, 27744 and 9287, respectively. The p
2、otential translation product of ORF B (27744 Da) is considerably larger than previously reported and could be difficult to distinguish by size from the El-matrix protein. Coronavirus; TGEV; RNA sequencing Transmissible gastroenteritis virus (TGEV) is an economically important coronavirus of swine th
3、at produces an often fatal diarrhea especially in nursing pigs Correspondence ro: R.D. Wesley, National Animal Disease Center, P.O. Box 70, Ames, IA 50010, U.S.A. 016%1702/89/$03.50 0 1989 Elsevier Science Publishers B.V. (Biomedical Division) 88 less than 2 weeks of age (Saif and Bohl, 1986). Like
4、other members of the family Coronaviridae, TGE virions consist of 3 major structural proteins; the El-matrix and EZpeplomer surface glycoproteins and a phosphorylated nucleocapsid protein (N) that associates with the 23.6 kilobase (kb), non-segmented, positive-stranded RNA genome (Garwes and Pocock,
5、 1975; Brian et al., 1984; Hu et al., 1984; Jacobs et al., 1986; Wesley and Woods, 1986). The nucleotide and deduced amino acid sequences of these structural proteins have been determined for the avirulent Purdue 115 strain of TGEV (Kapke and Brian, 1986; Laude et al., 1987; Rasschaert and Laude, 19
6、87). In the replication of TGEV, as with other Coronadae, transcption proceeds via a discontinuous, nested-set mechanism in which several distinct mRNA species of subgenomic size are synthesized (Hu et al., 1984; Jacobs et al., 1986; Rasschaert et al., 1987). These mRNAs share the co-terminal 3 poly
7、adenylated end of the TGEV genome and extend for different lengths in the 5 direction. This transcrip- tion mechanism has been well documented in 2 other coronaviruses, murine hepatitis virus (MHV) and infectious bronchitis virus (IBV) (Lai et al., 1983; Spaan et al., 1983; Brown et al., 1984). In a
8、ddition, the 5 end of each subgenomic mRNA contains a short RNA leader sequence, derived from the 5 end of the genome, that primes transcription. This leader sequence is 72 bases long in the case of MHV and approximately 60 bases in length for IBV. The leader and the body sequences of each subgenomi
9、c mRNA are joined by a discontinuous transcption mechanism. The freely dissociated leader sequence binds to an intergenic recognition sequence and serves as a primer for the transcription of subgenomic mRNAs (Lai, 1986). Thus each subgenomic mRNA contains a homologous recognition sequence ap- proxim
10、ately 60-70 bases downstream from the actual 5 end. The core recognition sequence for MHV is SAATCZAAAC and for IBV it is 5CTEAACAA. Further- more, nucleotide sequences that flank the core homologous sequence are also important in leader RNA-primed transcription (Boursnell et al., 1987). In general,
11、 the translated region of each coronavirus subgenomic mRNA is the 5-most open reading frame (ORF) that is not present in the smaller subgenomic mRNAs. This is the case for mRNAs that encode the coronavirus structural genes. For TGEV, mRNAs 3, 6 and 7 have been shown by in vitro translation studies t
12、o code for the E2-peplomer, El-matrix and nucleocapsid proteins, respectively (Jacobs et al., 1986). In some instances for subgenomic mRNAs that do not appear to code for any of the major virion structural proteins, internal initiation of translation is thought to occur. These include mRNA D for IBV
13、 and mRNA 5 for MHV for which a 12.4 kDa IBV polypeptide and a 10.2 kDa MHV polypeptide have been shown to be translated in infected cells (Skinner et al., 1985; Smith et al., 1987). In the case of TGEV, 2 mRNA species have been identified by Northern hybridization to be present between the E2-peplo
14、mer and El-matrix structural genes (Hu et al., 1984; Jacobs et al., 1986; Rasschaert et al., 1987). The larger of these 2 mRNAs, designated mRNA 4 by Jacobs et al. (1986) and designated mRNA 3 by Rasschaert et al. (1987), coded for 2 non-overlapping ORFs that were separated by a non-cod- ing interve
15、ning region of 334 bases containing only a single ternation codon 267 bases upstream from the start of the second ORF. The next smaller TGEV mRNA 89 transcript contained only one unique 5 coding sequence for a single hydrophobic polypeptide. In this paper we present evidence for 3 mRNA species betwe
16、en the El and E2 structural genes of a virulent strain of TGEV (Miller strain). Nucleotide sequence analysis revealed 3 large ORFs each preceded by an appropriate octanucleotide recognition sequence which could direct transcription initiation. Materials and Methods Cells and virus Swine testicular (ST) cells (McClurkin and Norman, 1966) were grown in modified Eagles MEM (Gibco) supplemented with fetal bovine serum (lo%), sodium bicarbonate (0.22%), lactalbumin hydrolysate (0.2
