Transcription regulatory sequences and mRNA expression levels in the coronavirus transmissible gastroenteritis virus.
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 coronavirus-derived 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 (beta-glucuronidase) by using the selected TRS led to the production of 2 to 8 microg 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.
Alonso
Sara
S
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Izeta
Ander
A
Sola
Isabel
I
Enjuanes
Luis
L
D024509:3' Flanking Region / Q000502:physiology*; D024506:5' Flanking Region / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence / Q000502:physiology*; D000086862:Coronavirus M Proteins; D000086462:Coronavirus Nucleocapsid Proteins; D004279:DNA, Viral; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D016679:Genome, Viral; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019251:Nucleocapsid / Q000235:genetics; D019590:Nucleocapsid Proteins; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology*; D064370:Spike Glycoprotein, Coronavirus; D013552:Swine; D014158:Transcription, Genetic; D005760:Transmissible gastroenteritis virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics; D014763:Viral Matrix Proteins / Q000235:genetics
D000086862:Coronavirus M Proteins; D000086462:Coronavirus Nucleocapsid Proteins; D004279:DNA, Viral; C000706238:M protein, Transmissible gastroenteritis virus; D008562:Membrane Glycoproteins; D019590:Nucleocapsid Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins