Interaction Between Classical Swine Fever Virus NS5A Protein With The Host Cellular Protein

Viruses are simple intracellular parasites with ecological characteristic. Viral replication depends on molecular ecological conditions of host cell. The moleculer environment conditions required by viral proliferation in host cell includ changes of cellular genome, structure, expression regulation and host factors involving the life cycle of viral infection and replication.Classical swine fever virus (CSFV) is a enveloped virus with a single stranded positive RNA genome, belonging to the genus pestivirus within the family Flaviviridae. CSFV genome encodes four structural proteins of Erns, E1, E2, C and eight non-structural proteins of Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B. CSFV is the causative agent of classical swine fever (CSF), a highly contagious disease with high morbidity and mortality, featuring symptoms of hemorrhagic fever and immune suppression. At present, CSF is still the most harmful contagious disease for pig industry in China.To identify the host protein which interacts with CSFV NS5A and participates in replication of CSFV, the recombinant expression vector pEGX-6p-NS5AC was induced to express the C-terminal fragment of NS5A protein (NS5AC). Recombinant protein GST-NS5AC was then incubated with PK-15 lysate to pull down cellular protein. The interacting cellular protein was identified by using mass spectrometry. In addition, the eukaryotic expression vector pCMV-myc-NS5AC was constructed and transfected into mammalian cell PK-15. The results of pull down and mass spectrometry was further validated by co-immunoprecipitation and western blot. The results of pull down and co-immunoprecipitation showes that heat shock protein 27 (HSP27) of host cell interacts with CSFV NS5AC. This result will provide new ideasfor further revealing the molecular mechanisms of host factors involving in viral replication and the development of new vaccines, targeting agents and innovative control strategies.