Rab5 Enhances Classical Swine Fever Virus Proliferation and Interacts with Viral NS4B Protein to Facilitate Formation of NS4B Related Complex

Abstract

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 lentivirusmediated 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.

Antiviral Role of IFITM Proteins in Classical Swine Fever Virus Infection

“…Accumulating evidence indicates that CSFV is transmitted from early endosomes (Rab5-dependent) to late endosomes (Rab7-dependent) and then into lysosomes [31,32]. Rab5 has been shown interact with CSFV NS4B to enhance CSFV replication by facilitating formation of NS4B complexes [43]. In this study, we found that IFITMs partly colocalized with Rab5, Rab7, and Lamp1.…”

FHC, an NS4B-interacting Protein, Enhances Classical Swine Fever Virus Propagation and Acts Positively in Viral Anti-apoptosis

“…Mitogen-activated protein kinase 2 interacts with the main CSFV antigen E2 to promote CSFV growth through attenuation of the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway 30 . The Rab5 protein interacts with CSFV NS4B to facilitate the formation of NS4B-related complex 10 , and in Huh7 hepatoma cells, hepatitis C virus (HCV) NS5A interacts with an immunosuppressant FK506-binging protein (FKBP38) to inhibit apoptosis 30 , 31 . We herein report interaction between CSFV NS4B and FHC.…”

“…However, a chimeric low-virulence GPE-derived virus carrying the complete Eystrup NS4B sequence showed enhanced pathogenicity and high replication efficiency 9 . In addition, the findings of our previous study indicated that an NS4B-related complex, the formation of which is facilitated by Rab5, is thought to be the CSFV replication complex 10 . Although many studies have focused on the roles of CSFV NS4B in viral virulence and genomic replication, few studies have investigated cellular NS4B-interacting proteins and their roles in CSFV replication.…”

MiR-140 inhibits classical swine fever virus replication by targeting Rab25 in swine umbilical vein endothelial cells

“…Rab1A takes part in particle assembly of CSFV [38] and Rab2 can promote CSFV replication [39]. In addition, Rab5 promotes NS4B induced complex formation and thereby increases CSFV replication [40]. However, the effects of Rab25 on CSFV replication have received little attention.…”