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Marozin, Sabrina (2006): Interferon Escape of Respiratory Syncytial Virus: Functional Analysis of Nonstructural Proteins NS1 and NS2. Dissertation, LMU München: Tierärztliche Fakultät



Respiratory syncytial virus (RSV) is recognised as the most frequent cause of severe lung infections in infants and cattle worldwide. Currently, no effective treatments are available and the development of a successful vaccine has been hampered by the fact that natural infection does not provide complete and durable protection. RSV nonstructural proteins, NS1 and NS2, are strong inhibitors of IFN α/β-production by specifically preventing interferon regulatory factor (IRF)-3 phosphorylation. However, the exact mechanisms leading to NS protein-mediated inhibition of IRF3 remain to be unravelled. One of the objectives of this study was to identify amino acid domains in the human respiratory syncytial virus (HRSV) nonstructural proteins (NS) responsible for their ability to ablate the IFN-β signalling pathway. Furthermore, I wanted to find out at which level of this signalling pathway the NS proteins exert their suppressive activity and which are their major cellular targets. HRSV strains A2 and Long differ in their ability to block interferon type I synthesis. Sequence analysis of their NS proteins revealed the presence of an amino acid residue in the NS2 protein with a potential role for RSV IFN-inhibitory functions. Two recombinant bovine respiratory syncytial (BRSV) viruses harbouring HRSV NS1 and NS2 genes were generated and tested in their ability to restrict IFN-β synthesis. These recombinant viruses differed only in the identity of the residue at position 26 of the HRSV NS2 protein: rBRSVh1/2 has a Threonine as in the Long strain, while in rBRS h1/2*T26I this amino acid was mutated into an Isoleucin similarly to A2 virus. Sets of in vitro tests revealed that IFN-β induction was impaired by rBRSVh1/2*T26I when compared to rBRSV h1/2. Analysis of the transcriptional factors (AP-1, NF-kB and IRF3) involved in the activation of IFN-β synthesis provided evidence that the inhibitory ability of rBRSVh1/2*T26I was correlated to a selective block of IRF3. The mutation (T26I) in the NS2 protein did neither effect the NF-kB activation pathway nor perturbed the IFN-resistance characteristics of the chimeric viruses. IRF3 is activated upon phosphorylation mediated by IKK-related kinases (TBK1 and IKK-ε). TBK1 was therefore cloned from a human lung cDNA library and its biological activities regarding the induction of IFN-β were compared in mock-infected and infected cells. rBRSVh1/2*T26I and HRSV A2 precluded virus-induced IRF3 activation by interfering with TBK1 functions. No direct interaction between TBK1 and NS2 protein was demonstrated indicating that the kinase TBK1 may not be the sole target involved in RSV mechanisms of evasion of the innate immune response. Recombinant IFN-inducible rabies viruses expressing HRSV Long-derived (rGFP-Ph2/1) or HRSV A2-like (rGFP-Ph2*T26I/1) NS proteins were also generated. The HRSV NS2 protein expressing an Isoleucin (NS2*T26I) at position 26 was not able to suppress IFN-β induction and to rescue the growth of the recombinant eGFP-Ph2*T26I/1 in interferon-competent cells. A low expression of the mutated NS2*T26I protein was probably the reason of this failure. In summary, these results show that the HRSV NS2 protein possesses an intrinsic IFN-β inhibitory activity, which is achieved throughout a selective inhibition of the IRF3 activation pathway. The block appears to be exerted at the level of IRF3-kinase TBK1. Interferonantagonist functions of the HRSV NS2 protein are linked to a particular amino acid motif in the N-terminus of the protein. Identification of this amino acid domain and of TBK1 as the cellular target provide a better insight of how the HRSV NS2 protein prevents the establishment of the antiviral innate immune response and therefore it might contribute to the development of an effective vaccine.