Reverse genetics of influenza C viruses

Influenza A, B and C viruses, three separate genera of the family Orthomyxoviridae, are enveloped viruses with a segmented negative single-strand RNA genome. Infections with influenza C viruses result mainly in upper- respiratory-tract disease. Influenza C viruses are distributed worldwide and can cause epidemics. Compared to influenza A and B viruses, the molecular mechanisms of infections by influenza C viruses are poorly understood. Techniques to study the functions of viral genes and their encoded proteins by reverse genetics are established for influenza A and B viruses. In this project, we want to develop a similar system to generate recombinant influenza C viruses entirely from plasmids. First, a suitable vector will be constructed, which will then be used to separately clone all seven RNA segments of the viral genome. Then, the seven different plasmids will be transfected into cells which support replication and assembly of influenza C viruses. Similar to methods described for the generation of recombinant influenza A viruses, RNAs will be transcribed from the plasmids and translated within the cells in a way that finally allows assembly and release of recombinant viruses. For identification of recombinant viruses, silent mutations will be introduced a cloned cDNA. RT-PCR and sequence analysis then allows identification of recombinant viruses. Once the procedure to generate viruses from plasmids is established, we want to study the function of the viral non-structural protein NS1 by reverse genetics. The NS1 protein of influenza A viruses inhibits the activation of the unspecific antiviral interferon response by different mechanisms. Available data indicate that the NS1 protein of influenza C viruses may have similar functions. Therefore, we want to generate recombinant viruses with deletions in the NS1 gene and study their replication properties. If the NS1 protein is essential for suppression of the interferon response, we anticipate that recombinant viruses with deletions in the NS1 gene are strongly attenuated and will replicate only in cells with a defect in the interferon response. In several instances cell lines derived from tumors exhibit defects in the interferon pathways. In the past, we have identified a number of human tumors, e.g. human malignant metastatic melanomas, which strongly overexpress 9-O-acetylated sialic acids, the receptor for influenza C viruses. Moreover, approximately 70% of human melanomas are defective in interferon pathways. Thus, recombinant influenza C viruses with deletions in the NS1 gene may in the future represent safe and efficient oncolytic viruses and/or vectors for target-specific gene delivery.

Influenza A, B and C viruses, three separate genera of the family Orthomyxoviridae, are enveloped viruses with a segmented negative single-strand RNA genome. Infections with influenza C viruses result mainly in upper- respiratory-tract disease. Influenza C viruses are distributed worldwide and can cause epidemics. Compared to influenza A and B viruses, the molecular mechanisms of infections by influenza C viruses are poorly understood. Techniques to study the functions of viral genes and their encoded proteins by reverse genetics are established for influenza A and B viruses. In this project, we developed a similar system to generate recombinant influenza C viruses entirely from plasmids. First, a suitable vector was constructed, which was then be used to separately clone all seven RNA segments of the viral genome. Then, the seven different plasmids were transfected into cells which support replication and assembly of influenza C viruses. Viral genes were transcribed from the plasmids and translated within the cells in a way that finally allowed assembly and release of recombinant viruses. Once the procedure to generate viruses from plasmids was established, we studied the function of the viral non-structural protein NS1. We could show that this protein counteracts the cellular antiviral interferon response. By expressing shorter forms of the NS1 protein we could determine which part of the viral protein is able to reduce the antiviral response. Furthermore, we found that the NS1 protein functions with at least two different mechanisms. In the future these results will be combined in order to prepare recombinant viruses which express a modified NS1 protein which can no longer suppress the interferon response. Such viruses can no longer replicate in cells of the respiratory tract, but should still be able to infect tumour tissues (e. g. melanoma) which express the receptor for influenza C viruses. Therefore, such recombinant viruses may represent a tool to specifically attack human malignant metastasizing melanoma. In order to proof this hypothesis, we recently started an international collaboration with a Canadian laboratory specialized in melanoma and breast cancer research. The final goal of this collaboration is to cure mice from metastasizing melanoma.