RESEARCH SUMMARY

The main focus of our research is the study of the molecular basis of coronavirus (CoV) replication and virulence, and the identification of signalling pathways modified by the virus, to control disease. The information from these basic projects will be used to design protection strategies against CoV-induced diseases, particularly human severe pneumonia that can end in acute respiratory distress syndrome (ARDS).

The CoV are single-stranded positive-sense RNA viruses with genomes of around 30 kb, responsible for respiratory and enteric diseases with high impact on animal and human health. Our group is interested in the molecular basis of replication and transcription, assembly, and virus-host interaction using transmissible gastroenteritis coronavirus (TGEV) and the severe and acute respiratory syndrome virus (SARS-CoV) as models.

Virus replication and transcription, and virus-host interactions are mediated by binding of virus RNA motifs to viral and host cell proteins, and by protein-to-protein recognition. These interactions are analysed in all the processes we study, including the replication complex. We postulated that coronavirus transcription and replication involve 5' and 3' genome end interactions, mediated by proteins that are being identified using functional genomics and proteomics. The relevance of host cell factors involved in these processes is being evaluated by inhibiting their expression using siRNA.

Coronavirus transcription requires discontinuous RNA synthesis to link the leader to coding sequences in the subgenomic RNA, a process similar to high frequency copy-choice, similarity-assisted RNA recombination. Based on a large amount of data generated by reverse genetics, we described two mechanisms that regulate transcription in coronaviruses at different levels. We showed that basepairing between the nascent minus RNA chain and the genomic RNA leader regulates the amount of all subgenomic RNA produced. A transcription enhancer that regulates expression of specific mRNA was also identified recently. There are additional regulatory mechanisms that influence the amount of subgenomic RNA, such as RNA-protein interactions, one area in which our laboratory is highly active at present. The role of viral and cell proteins, such as RNA chaperones, that might be involved in CoV replication, transcription and packaging is being addressed; we showed that coronavirus N protein is essential for coronavirus RNA synthesis and acts as an RNA chaperone.

A main interest area in our laboratory is the study of the molecular basis of virus virulence, virus-host interaction, and signalling pathways affecting virus replication or, alternatively, cell pathways altered by coronavirus infection leading to diseases such as those associated with inflammation of respiratory tissues. Comparative genomic and proteomic information is essential in these studies. We described that specific virus structural proteins, such as the TGEV and SARS-CoV envelope (E) proteins, influence virus virulence and modulate signalling pathways. The deletion of E protein led to the generation of propagation-deficient TGEV, to attenuated phenotypes in the case of SARS-CoV, and to upregulation of the cell stress response, which affects the immune response. Deletion of non-essential virus components such as TGEV protein 7 significantly affects viral and cell translation and apoptosis. Viral and host cell factors involved in these signalling pathways are being studied.

The information derived from academic studies is being applied to the engineering of coronavirus vectors. Using reverse genetic approaches based on two infectious cDNA clones produced in our laboratory for TGEV and SARS-CoV, viral vectors have been engineered using virus-attenuated phenotypes. These vectors have led to promising recombinant vaccine candidates for animal and human health, including SARS.