| Transmissible gastroenteritis virus (TGEV) and severe acute respiratory associated coronavirus (SARS-CoV) are positive sense single-stranded RNA coronaviruses in the family Coronaviridae, order Nidovirales. Genetic analysis of the structure and function of RNA virus genomes has been profoundly advanced by the availability of genome-length cDNA clones. The large size of the coronavirus genome, the largest RNA genomes found in nature, and inability to clone portions of the polymerase gene in microbial vectors has hampered the ability to perform precise manipulations and genetic analyses of viruses in the Coronaviridae family. The goals of these studies were to construct infectious clones of the coronaviruses TGEV Purdue strain and SARS-CoV for vaccine development, genetic analysis and to further the current understanding of coronavirus replication and gene function.; Using a novel assembly strategy, full-length cDNA copies of the TGEV and SARS-CoV genomes were constructed from which infectious transcripts were generated in vitro. This system allowed for the development of recombinant TGEV replicons and viruses that expressed green fluorescent protein (GFP), demonstrating the feasibility of coronavirus derived expression vectors for vaccine development. Using the full-length and replicon RNA systems, we analyzed the necessity of 5 TGEV open reading frames (ORF) to viral replication and assembly (3a, 3b, E, M, and N). We report that 3a and 3b are unnecessary for viral replication in vitro, and, our research corroborates previous reports with a related coronavirus, mouse hepatitis virus (MHV), demonstrating the requirement of E to viral particle assembly.; Coronaviruses use a copy choice discontinuous transcription mechanism for the synthesis of a 3′ coterminal nested set of subgenomic mRNAs. A transcription regulatory sequence (TRS), containing a highly conserved sequence (CS), precedes each of the coronavirus ORFs and is involved in the regulation of subgenomic mRNA transcription. We report our findings in defining the SARS-CoV CS sequence, the mapping of the TGEV N gene TRS and the effect of two closely positioned TRS elements on TGEV mRNA transcription. Additionally, our research reveals important insights into the anatomy of a coronavirus TRS element and its role in regulating subgenomic mRNA transcription. |
