Transcription Regulatory Sequences and mRNA Expression Levels in the Coronavirus Transmissible Gastroenteritis Virus

Abstract

The transcription regulatory sequences (TRSs) of the coronavirus transmissible gastroenteritis virus (TGEV) have been characterized by using a helper virus-dependent expression system based on coronavirusderived minigenomes to study the synthesis of subgenomic mRNAs. The TRSs are located at the 5 end of TGEV genes and include a highly conserved core sequence (CS), 5-CUAAAC-3, that is essential for mediating a 100-to 1,000-fold increase in mRNA synthesis when it is located in the appropriate context. The relevant sequences contributing to TRS activity have been studied by extending the CS 5 upstream and 3 downstream. Sequences from virus genes flanking the CS influenced transcription levels from moderate (10-to 20-fold variation) to complete mRNA synthesis silencing, as shown for a canonical CS at nucleotide (nt) 120 from the initiation codon of the S gene that did not lead to the production of the corresponding mRNA. An optimized TRS has been designed comprising 88 nt from the N gene TRS, the CS, and 3 nt 3 to the M gene CS. Further extension of the 5-flanking nucleotides (i.e., by 176 nt) decreased subgenomic RNA levels. The expression of a reporter gene (␤-glucuronidase) by using the selected TRS led to the production of 2 to 8 g of protein per 10 6 cells. The presence of an appropriate Kozak context led to a higher level of protein expression. Virus protein levels were shown to be dependent on transcription and translation regulation.Transmissible gastroenteritis virus (TGEV) is a member of the family Coronaviridae, which, together with the family Arteriviridae, forms the order Nidovirales (6). The coronavirus RNA genome has a length ranging from 27.6 to 31.5 kb. About two-thirds of the entire RNA comprises open reading frames (ORFs) 1a and 1ab, encoding the replicase gene. The 3Ј onethird of the genome comprises the genes encoding the structural and nonstructural proteins. The organization of the non-ORF 1 nonstructural protein genes, which are interspersed between the known structural protein genes, varies significantly among different coronavirus strains (5).Coronavirus transcription is based on RNA-dependent RNA synthesis. Coronavirus mRNAs consist of six to eight types of various sizes, depending on the coronavirus strain. The largest mRNA is the genomic RNA, which also serves as the mRNA for ORFs 1a and 1ab; the others are subgenomic mRNAs (sgmRNAs), composed of noncontiguous sequences of the parental genome. The mRNAs and the genomic RNA form a nested set of RNAs of different lengths with common 5Ј and 3Ј ends. Except for the smallest mRNA, all of the mRNAs are structurally polycistronic; in general, however, only the 5Ј-most ORF (not present in the next smallest mRNA) is translated. However, there are exceptions: some mRNAs, e.g., mRNA 5 of mouse hepatitis virus (MHV), mRNA 3 of infectious bronchitis virus, and the bovine coronavirus (BCoV) nucleocapsid mRNA, are translated by internal initiation into two or three proteins (21,25). All the mRNAs possess a 5Ј sequence of 70 to 90 nucleotides (nt) that ...

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…TRSs include a conserved core sequence (CS) 6–7 nt in length and variable 5′ and 3′ flanking sequences (the 5′ TRS and 3′ TRS, respectively) (16). Because the CS is identical for the genome leader (CS-L) and all mRNA coding sequences (CS-B), the CS-L could base-pair with the nascent negative strand complementary to each CS-B (cCS-B), allowing for leader-body joining (Figure 1 c ).…”

Virus-based vectors for gene expression in mammalian cells: Coronavirus

“…Both genome-size and subgenomic negative-strand RNAs, which correspond in number of species and size to those of the virus-specific mRNAs have been detected [44,45]. The three that we [21] consider most relevant are: (i) potential base pairing between the leader 3 0 end and sequences complementary to the TRS located at the 5 0 end of each nidovirus gene (cTRS), that guide the fusion between the nascent negative strand and the leader TRS. At the start site of every transcription unit on the viral genomic RNA, there is a TRS that includes a highly conserved core sequence (CS) that is nearly homologous to the 3 0 end of the leader RNA.…”

“…The expression of GUS, PRSSV ORF5, or CAT using TGEV-or IBV-derived minigenomes in general increases until passages three or four, expression levels are maintained for about four additional passages, and steadily decrease during successive passages [20][21][22]29]. The highest expression levels (2-8 mg of GUS per 10 6 cells) have been obtained using a two-step amplification system based on TGEV derived minigenomes with optimized TRSs [20,21]. The highest expression levels (2-8 mg of GUS per 10 6 cells) have been obtained using a two-step amplification system based on TGEV derived minigenomes with optimized TRSs [20,21].…”

Rapid differentiation of vaccine strain and Chinese field strains of transmissible gastroenteritis virus by restriction fragment length polymorphism of the N gene

“…There was no deletion or insertion of nucleotides in the ORFs of the 11 strains. These strains had a transcription regulatory sequence (TRS) of 5 0 -CUAAAC-3 0 , as previously recognized in Purdue strain [33]. The nucleotide and deduced amino acid sequences of these TGEVs were determined and compared with the sequences of the published TGEVs, as well as with PRCV-ISU-1.…”