Microfilaments and microtubules alternately coordinate the multi-step endosomal trafficking of Classical Swine Fever Virus.

Cytoskeleton, as a ubiquitous structure in the cells, plays an important role in the process of virus entry, replication, and survival. However, the action mechanism of cytoskeleton in the invasion of Pestivirus into host cells remains unclear. In this study, we systematically dissected the key roles of the main cytoskeleton components, microfilaments and microtubules in the endocytosis of porcine Pestivirus, Classical swine fever virus (CSFV). We observed the dynamic changes of actin filaments in CSFV entry. Confocal microscopy showed that CSFV invasion induced the dissolution and aggregation of stress fibers, resulting in the formation of lamellipodia and filopodia. Chemical inhibitors and RNA interference were used to find that the dynamic changes of actin were caused by EGFR-PI3K/MAPK-RhoA/Rac1/Cdc42-cofilin signaling pathway, which regulates the microfilaments to help CSFV entry. Furthermore, co-localization of the microfilaments with clathrin and Rab5 (early endosome), as well as microtubules with Rab7 (late endosome) and Lamp1 (lysosome) revealed that microfilaments were activated and rearranged to help CSFV trafficking to early endosome after endocytosis. Subsequently, recruitment of microtubules by CSFV also assisted membrane fusion of the virions from late endosome to lysosome with the help of a molecular motor, dynein. Unexpectedly, vimentin, which is an intermediate filament, had no effect on CSFV entry. Taken together, our findings comprehensively revealed the molecular mechanisms of cytoskeletal components that regulated CSFV endocytosis and facilitated further understanding of Pestivirus entry, which would be conducive to explore antiviral molecules to control classical swine fever.