Coronavirus-Host Interaction: Innate Immune Evasion and Coronavirus Replication.
Over the past years the research, we have been able to clone and propagate full length coronavirus (CoV) cDNAs from several CoVs using vaccinia virus as eukaryotic cloning vector. The reverse genetic system is based upon the in vitro transcription of infections RNA from a cloned full-length cDNA copy of a CoV genome, and the introduction of nucleotide changes, deletions or insertions is facilitated by vaccinia virus-mediated recombination. The reverse genetic system has proven to be rapid, robust and versatile and is available in the laboratory of Dr. Thiel for the generation of recombinant prototype viruses of all major CoV phylogenetic lineages, namely for HCoV 229E, type-I and type-II Feline CoVs (genus Alphacoronavirus), Mouse Hepatitis Virus strain A59, SARS-CoV (genus Betacoronavirus), Avian Infectious Bronchitis Virus (genus Gammacoronavirus).
One of our long-term goals is to study CoV replication in order to develop strategies to prevent and control CoV infections. Thus, we have applied the reverse genetic system to the analysis of mechanisms and enzymes involved in CoV genome expression on the molecular level. We could identify and characterize mechanisms of translational regulation of CoV gene expression, such as internal ribosomal entry on subgenomic mRNAs, and ribosomal frame-shifting on the CoV genomic RNA. Furthermore, we have studied enzymatic activities encoded by the CoV replicase gene and analyzed CoV replication, transcription and packaging using recombinant CoVs, replicon RNAs, defective minigenomes, and virus-like particles. We have extended these studies to the identification and evaluation of CoV replicase inhibitors, the analysis of cis-acting elements involved in RNA replication, the analysis of CoV target cell tropism, the generation of CoV-based vaccine vectors, the analysis of virus-host interactions in vivo using a murine model of CoV infection, and in depths analyses of virus-induced innate immune responses. In order to study virus-host interactions of respiratory viruses we have recently established primary airway epithelial cultures that grow under so-called “air-liquid-interface” conditions. This culture system is now available for several species (human, camelid, bats) and allows us to characterize particular virus-host interactions (e.g. innate immune evasion), and zoonotic transmission of CoVs in an environment that resembles the authentic primary target tissue of CoV infection.