Abstract
Influenza A virus can infect humans, birds, and several kinds of mammals. In the 20th century, 4 variants of the virus have arised as human transmissible pathogens. Moreover, the outbreak of H5N1 avian influenza, was recently reported in Europe and Asia. The virus has generated serious concern for its high death rates and the epidemics all over the world. The genome of influenza A virus is separated into 8 RNP segements which are coated with nucleoproteins, a common genetic property of Orthomyxoviridae family. The oligomerization and RNA-binding characteristics of nucleoproteins indicate that they can interact with vRNA-dependant RNA polymerase complex (PA, PB1, and PB2) and vRNA to form ribonucleoprotein complex, RNP. According to previous literatures, the leading factor of oligomerization in NP-NP contacts, salt-bridge between one nucleoprotein E339 and another one R416, can be broken by single point mutation (E339A or R416A), leading to the nucleoprotein oligomers switch to the monomers. Thus, we have created the same mutations on nucleocapsid protein (NP of H1N1 and H3N2), and then obtained soluble and native nucleoproteins to perform FPLC. The FPLC patterns show that the two mutations can indeed break the salt bridge of NP-NP contacts and disrupt the original oligomerization behavior of wild-type NP to form monomeric form. In addition, the nucleoprotein will bind to the DNA during purification process, which is monitored from the OD260/OD280 ratio. However, the DNA can not be dissociated or hydrolyzed completely by using high-salt buffer and DNase I. Additionally, the FPLC patterns show that the oligomerization state of the wild-type NP is retained and maintained. However, the two mutant NPs (NP-E339A and NP-R416A) are not, indicating that the mutant NP polymers will turn into NP monomers over time. Finally, we calculated the average rate of the monomer formation and characterized their possible conversion mode.