Intracellular Vimentin Regulates the Formation of Classical Swine Fever Virus Replication Complex through Interaction with NS5A Protein.

Vimentin (VIM), an indispensable protein, is responsible for the formation of intermediate filament structures within cells and plays a crucial role in viral infections. However, the precise role of VIM in classical swine fever virus (CSFV) infection remains unclear. Herein, we systematically investigated the function of VIM in CSFV replication. We demonstrated that both knockdown and overexpression of VIM affected CSFV replication. Furthermore, we observed by confocal microscopy the rearrangement of cellular VIM into a cage-like structure during CSFV infection. Three-dimensional (3D) imaging indicated that the cage-like structures were localized in the endoplasmic reticulum (ER) and ringed around the double-stranded RNA (dsRNA), thereby suggesting that VIM was associated with the formation of the viral replication complex (VRC). Mechanistically, phosphorylation of VIM at serine 72 (Ser72), regulated by the RhoA/ROCK signaling pathway, induced VIM rearrangement upon CSFV infection. Confocal microscopy and coimmunoprecipitation assays revealed that VIM colocalized and interacted with CSFV NS5A. Structurally, it was determined that amino acids 96 to 407 of VIM and amino acids 251 to 416 of NS5A were the respective important domains for this interaction. Importantly, both VIM knockdown and disruption of VIM rearrangement inhibited the localization of NS5A in the ER, implying that VIM rearrangement recruited NS5A to the ER for VRC formation. Collectively, our results suggest that VIM recruits NS5A to form a stable VRC that is protected by the cage-like structure formed by VIM rearrangement, ultimately leading to enhanced virus replication. These findings highlight the critical role of VIM in the formation and stabilization of VRC, which provides alternative strategies for the development of antiviral drugs. Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly infectious disease that poses a significant threat to the global pig industry. Therefore, gaining insights into the virus and its interaction with host cells is crucial for developing effective antiviral measures and controlling the spread of CSF. Previous studies have shown that CSFV infection induces rearrangement of the endoplasmic reticulum, leading to the formation of small vesicular organelles containing nonstructural protein and double-stranded RNA of CSFV, as well as some host factors. These organelles then assemble into viral replication complexes (VRCs). In this study, we have discovered that VIM recruited CSFV NS5A to form a stable VRC that was protected by a cage-like structure formed by rearranged VIM. This enhanced viral replication. Our findings not only shed light on the molecular mechanism of CSFV replication but also offer new insights into the development of antiviral strategies for controlling CSFV.