Classical swine fever virus (CSFV) is a fatal pig pestivirus and causes serious financial losses to the pig industry. CSFV NS4B
protein is one of the most important
viral replicase proteins. Rab5, a member of the small
Rab GTPase family, is involved in
infection and replication of numerous viruses including hepatitis C virus and dengue virus. Until now, the effects of Rab5 on the proliferation of CSFV are poorly defined. In the present study, we showed that Rab5 could enhance CSFV proliferation by utilizing lentivirus-mediated constitutive overexpression and eukaryotic plasmid transient overexpression approaches. On the other hand, lentivirus-mediated
short hairpin RNA knockdown of Rab5 dramatically inhibited virus production. Co-immunoprecipitation,
glutathione S-transferase pulldown and
laser confocal microscopy assays further confirmed the interaction between Rab5 and CSFV NS4B
protein. In addition, intracellular distribution of NS4B-Red presented many granular fluorescent signals (GFS) in CSFV infected PK-15 cells. Inhibition of basal Rab5 function with Rab5 dominant negative mutant Rab5S34N resulted in disruption of the GFS. These results indicate that Rab5 plays a critical role in facilitating the formation of the NS4B related complexes. Furthermore, it was observed that NS4B co-localized with viral NS3 and NS5A
proteins in the cytoplasm, suggesting that NS3 and NS5A might be components of the NS4B related complex. Taken together, these results demonstrate that Rab5 positively modulates CSFV propagation and interacts with NS4B
protein to facilitate the NS4B related complexes formation.