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Journal of Virology, February 2002, p. 1293-1308, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1293-1308.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Sara Alonso, Ander Izeta, Isabel Sola, and Luis Enjuanes*

Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain

Received 27 April 2001/ Accepted 19 October 2001

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 (ß-glucuronidase) by using the selected TRS led to the production of 2 to 8 µg of protein per 106 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.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Cantoblanco, 28049 Madrid, Spain. Phone: 34-91-585 4555. Fax: 34-91-585 4915. E-mail: L.Enjuanes{at}cnb.uam.es.

Journal of Virology, February 2002, p. 1293-1308, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1293-1308.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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