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Brzozka, Krzysztof (2006): The rabies virus phosphoprotein P: a key regulator of innate immune responses. Dissertation, LMU München: Faculty of Biology
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Abstract

Interferons are the key cytokines of innate immunity and represent the first line of defense against invading viruses. By activating immediate antiviral mechanisms and stimulating the adaptive immune response, interferon signaling is decisive for the outcome of disease and virus clearance. The work presented in this thesis reveals a central role of the phosphoprotein P of rabies virus (Rhabdoviridae family), known as a polymerase cofactor, as an inhibitor of the host interferon system. Counter-mechanism to escape form recognition by the immune system were previously unknown for the neurotropic rabies virus, which is characterized by the highest case-fatality ratio. In my work I have shown that rabies virus (strain SAD L16) is able to prevent both the production IFN-alpha/beta and the effector functions of IFN-alpha/beta and IFN-gamma. The factor responsible is the viral phosphoprotein P. P interferes with transcriptional activation of IFN-alpha/beta by preventing phosphorylation of the essential transcription factors IRF3 and IRF7 by their kinases TBK1 and IKKepsilon. Unphosphorylated IRFs are unable to dimerize and fail to enter the nucleus. In addition, rabies virus P prevents IFN-mediated JAK/STAT signaling and the expression of IFN-stimulated genes which include a broad spectrum of antiviral and immune regulatory genes. The inhibition of JAK/STAT signaling by P involves a unique mechanism, namely, specific binding of the tyrosine-phosphorylated STAT1 and STAT2 isoforms and their retention in the cytoplasm. The inhibitory activities of RV P on IFN induction and signaling are independent functions, as shown by site-directed mutagenesis of P and identification of different short amino acid stretches required for either function. Importantly, the inhibitory activities of P were demonstrated in the context of recombinant viruses. Using reverse genetics, a rabies virus was constructed, in which P expression was “knocked down” by moving the P gene to a promoter-distal position of the genome (SAD deltaPLP). This virus caused efficient IFN-alpha/beta production in infected cells and upregulation of interferon stimulated genes. The IFN sensitivity of SAD deltaPLP was confirmed in cell culture and is now being studied in animal experiments including IFN receptor knock of mice, to verify the relevance of P functions in vivo. The described work contributes to the understanding of host responses to virus infections in general and of rabies virus pathogenicity in particular. In addition, viruses with modified IFN antagonists provide interesting opportunities for development of attenuated vaccines and vectors.