Classical swine fever virus NS5B protein suppresses the inhibitory effect of NS5A on viral translation by binding to NS5A

In order to investigate molecular mechanisms of internal ribosome entry site (IRES)-mediated translation in classical swine fever virus (CSFV), an important pathogen of pigs, the expression level of NS3 was evaluated in the context of genomic RNAs and reporter RNA fragments. All data showed that the NS5A protein has an inhibitory effect on IRES-mediated translation and that NS5B proteins suppress the inhibitory effect of NS5A on viral translation, but CSFV NS5B GDD mutants do not. Furthermore, glutathione S-transferase pull-down assay and immunoprecipitation analysis, associated with deletion and alanine-scanning mutations, were performed. Results showed that NS5B interacts with NS5A and that the region aa 390-414, located in the C-terminal half of NS5A, is important for binding of NS5B to NS5A. Furthermore, amino acids K399, T401, E406 and L413 in the region were found to be essential for NS5A-NS5B interact ion, virus rescue and infection. The above-mentioned region and four amino acids were observed to overlap with the site responsible for inhibition of IRES-mediated translation by the NS5A protein. We also found that aa 63-72, aa 637-653 and the GDD motif of NS5B were necessary for the interaction between NS5A and NS5B. These findings suggest that the repression activity of the NS5B protein toward the role of NS5A in translation might be achieved by NS5A-NS5B interaction, for which aa 390-414 of NS5A and aa 63-72, aa 637-653 and the GDD motif of NS5B are indispensable. This is important for understanding the role of NS5A-NS5B interaction in the virus life cycle.