Transmissible gastroenteritis coronavirus packaging signal is located at the 5′ end of the virus genome

To locate the transmissible gastroenteritis coronavirus (TGEV) packaging signal, the incorporation of TGEV subgenomic mRNAs (sgmRNAs) into virions was first addressed. TGEV virions were purified by three different techniques, including an immunopurification using an M protein-specific monoclonal antibody. Detection of sgmRNAs in virions by specific reverse transcription-PCRs (RT-PCRs) was related to the purity of virus preparations. Interestingly, virus mRNAs were detected in partially purified virus but not in virus immuno-purified using stringent conditions. Analyses by quantitative RT-PCR confirmed that virus mRNAs were not present in highly purified preparations. Lack of sgmRNA encapsidation was probably due to the absence of a packaging signal (psi) within these mRNAs. This information plus that from the encapsidation of a collection of TGEV-derived minigenomes suggested that psi is located at the 5' end of the genome. To confirm that this was the case, a set of minigenomes was expressed that included an expression cassette for an mRNA including the beta-glucuronidase gene (GUS) plus variable sequence fragments from the 5' end of the virus genome potentially including psi. Insertion of the first 649 nucleotides (nt) of the TGEV genome led to the specific encapsidation of the mRNA, indicating that a psi was located within this region which was absent from all of the other virus mRNAs. The presence of this packaging signal was further confirmed by showing the expression and rescue of the mRNA including the first 649 nt of the TGEV genome under control of the cytomegalovirus promoter in TGEV-infected cells. This mRNA was successfully amplified and encapsidated, indicating that the first 649 nt of TGEV genome also contained the 5' cis-acting replication signals. The encapsidation efficiency of this mRNA was about 30-fold higher than the genome encapsidation efficiency, as estimated by quantitative RT-PCR. In contrast, viral mRNAs presented significantly lower encapsidation efficiencies (about 100-fold) than those of the virus genome, strongly suggesting that TGEV mRNAs in fact lacked an alternative TGEV psi .