Expression of cellular anti-viral antagonists from vaccinia virus to study the innate immune response and Z -DNA

The Vaccinia Virus (VACV) E3L protein is a major inhibitor of the cellular anti-viral response. Functional studies replacing the E3L amino-terminus with Z-DNA binding proteins confirm E3L as a member of the Z alpha class of Z-DNA binding proteins. Furthermore, mutations that interrupt Z-DNA binding correlate to reduction in VACV pathogenesis in mice. A murine cell line has been discovered that requires an intact E3L Z-DNA binding domain for VACV replication. Like the mouse pathogenesis studies, mutations that abrogate Z-DNA binding reduce VACV replication. In this cell line the E3L Z-DNA binding domain regulates interferon-beta. Mutations that reduce Z-DNA binding will result in interferon-beta production. The Z-DNA binding affinities for E3L and derivatives have also been resolved to complement the biological assays in understanding the biological function of Z-DNA.;VACV can be used to express foreign genes by inserting them into the VACV genome by homologous recombination. However, the process to make recombinant VACV is inefficient and time consuming. A novel way to generate recombinant VACV has been developed that is at least 10-fold more efficient than conventional techniques. This system relies on 3 components: psoralen-ultraviolet light inactivated VACV, purified pre-recombinant VACV genome, and the gene of interest as a PCR product with left and right recombination arms. This system has been demonstrated to generate recombinant VACV in one week compared to two months.;Viruses within the family Coronaviridae play a role in emerging infectious diseases. It has been discovered that the coronavirus mouse hepatitis virus (MHV) is resistant to the cellular type I interferon response. Infection with MHV did not lead to the activation of cellular double-stranded RNA (dsRNA) sensors such as dsRNA-dependent protein kinase or 2'-5' oligoadenylate synthetase. To resolve the specific MHV genetic component that confers MHV interferon resistance, MHV genes were inserted into the E3L locus of VACV by homologous recombination. This demonstrated that the MHV nucleocapsid protein can rescue an interferon-sensitive VACV. As a result, the MHV nucleocapsid protein is a cellular type I interferon antagonist.