The influenza A NS1 gene knockout virus delNS1: Replicatopm in dsRNA activated protein kinase (PKR) deficient systems and tumoor ablative potential

Research project P 14053 Influenza A NS1 Gene Knockout Virus delNS1 Thomas MUSTER 24.01.2000 We established a reverse genetics system for the NS gene segment of influenza A virus. This system permitted the rescue of transfectant viruses with large deletions in the NS I protein. Viable transfectant viruses only expressing the N-terminal 124, 80 or 3 8 amino acids of the NS 1 protein and a transfectant virus lacking the entire NS I gene (influenza deINS 1) were obtained. Whereas growth on Madin-Darby canine kidney cells decreased with increasing length of the deletions, all deletion mutants gre w to high titers in interferon (UN)-deficient systems such as Vero cells. This result demonstrates that the NS I protein of influenza A virus is dispensable for growth in IFN deficient systems. Moreover, it suggests that this protein counteracts the IFN-mediated antiviral response. The availability of the deINS 1 virus permits us to study the role of the NS 1 protein in the IFNpathway in more detail. Activation of the dsRNA-activated protein kinase (PKR) represents a major effector of the IFN antiviral response at the level of protein synthesis. It was shown that the influenza virus NS I protein inhibits activation of PKR in vitro. However, the biological significance of this interaction is not defined. The first objective of this proposal is to use the deINS I virus to study the effect of the interaction between NS I and PKR in the life cycle and pathogenicity of influenza virus. Since the deINS1 virus lacks the gene which counteracts the WN-mediated antiviral response of the host, infection by deINS I virus efficiently induces type I IFN in cells. The induction of IFN in turn is known for its diverse biological fimetions such as growth inhibition and immune cell stimulation. These findings suggest that the deINS I virus could induce anti-tumor activity. The second objective of this proposal is therefore to test the hypothesis that the deINS I virus possesses tumor-ablative potential.

Type 1 interferon (IFN)-induced cellular antiviral response is the first line of defense against viral infection within a mammalian host. To counteract the antiviral effects of IFN induction, many eukaryotic viruses have developed mechanisms to block the activity of IFN. In the case of influenza virus the NS1 protein counteracts the IFN- mediated response. We generated an influenza virus which lacks the NS1 protein (ONCOFLU). Due to the lack of its IFN-antagonist, ONCOFLU is a viral agent with two tumor-ablative properties: i) ONCOFLU fails to replicate in normal IFN-sensitive cells, but induces a lytic infection in IFN-resistant cells. Since human malignant tumor cells are frequently resistant to IFN, - as they contain defects in the IFN-signalling pathway - ONCOFLU is capable of selectively attacking such tumor cells. For example, it was demonstrated that melanoma cells frequently contain reduced levels of STAT1, a protein essential in the IFN-signaling pathway. ONCOFLU effectively destroys such melanoma cells. Another key protein in the IFN-signaling pathway is the double-stranded RNA-activated protein kinase (PKR). PKR is inhibited in cells expressing oncogenic (activated) ras. This oncogene-mediated blockage of PKR allows ONCOFLU to replicate in such cells. Moreover, it permits the exploitation of the activated ras pathway for anticancer therapy in the mouse model (Fig. 2). Activating mutations of the proto-oncogene ras occur in about 30% of all human tumors. ii) ONCOFLU is also capable of inducing a strong local and systemic IFN-response. Via this pathway ONCOFLU can induce IFN-mediated killing of IFN-sensitive melanomas. In summary, ONCOFLU is capable of destroying IFN-resistant tumor cells through lysis and IFN-sensitive tumor cells by IFN-mediated killing. The concept of ONCOFLU will now be further developed by the company Green Hills Biotechnology to treat melanoma patients.