Microfilaments and Microtubules Alternately Coordinate the Multistep Endosomal Trafficking of Classical Swine Fever Virus

Abstract

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. IMPORTANCE Endocytosis, an essential biological process mediating cellular internalization events, is often exploited by pathogens for their entry into target cells. Previously, we have reported different mechanisms of CSFV endocytosis into the porcine epithelial cells (PK-15) and macrophages (3D4/21); however, the details of microfilaments/microtubules mediated virus migration within the host cells remained to be elucidated. In this study, we found that CSFV infection induced rearrangement of actin filaments regulated by cofilin through EGFR-PI3K/MAPK-RhoA/Rac1/Cdc42 pathway. Furthermore, we found that CSFV particles were trafficked along actin filaments in early and late endosomes, and through microtubules in lysosomes after entry. Here, we provide for the first time a comprehensive description of the cytoskeleton that facilitates entry and intracellular transport of highly pathogenic swine virus. Results from this study will greatly contribute to the understanding of virus-induced early and complex changes in host cells that are important in CSFV pathogenesis.

Cellular ESCRT components are recruited to regulate the endocytic trafficking and RNA replication compartment assembly during classical swine fever virus infection

“…transport from early endosomes (Rab5) to late endosomes (Rab7) and finally to the nucleus for replication [31,48]. Similarly, we also found that Tsg101 assists in transporting CSFV from early endosomes to late endosomes through microfilaments with the help of motor protein dynein [26,49]. Meanwhile, Tsg101 regulates virus replication through forming the replication complex together with CSFV NS4B/5B nonstructural proteins and dsRNA in the endoplasmic reticulum.…”

EGF Receptor Signaling Modulates YAP Activation and Promotes Experimental Proliferative Vitreoretinopathy

“…A recent study showed that RhoA played an important role in G protein-coupled receptor–regulated YAP activation, 23 whereas another report indicated that EGFR can promote RhoA activity. 24 Thus, we determined whether activated EGFR can bypass RhoA to modulate the expression of YAP in ARPE-19 cells. Western blot analysis showed that EGF upregulated the production of YAP and RhoA in ARPE-19 cells ( Fig.…”

25-hydroxycholesterol inhibits human papillomavirus infection in cervical epithelial cells by perturbing cytoskeletal remodeling

“…For example, coronavirus induces filopodia to facilitate internalization through macropinocytosis (26). Classical swine fever virus (CSFV) promotes microfilament reorganization by activating the EGFR-PI3K/MAPK-RhoA/Rac1/Cdc42 signaling axis and employs stress fibers to sustain CSFV trafficking in early and late endosomes (27). Cofilin-mediated F-actin dynamics is crucial for replication of intracellular herpes simplex virus 1 (HSV1) (20).…”