Entry of Classical Swine Fever Virus into PK-15 Cells via a pH-, Dynamin-, and Cholesterol-Dependent, Clathrin-Mediated Endocytic Pathway That Requires Rab5 and Rab7

Abstract

Classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae, is a small, enveloped, positive-strand RNA virus. Due to its economic importance to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. However, the mechanisms of CSFV entry into cells are not well characterized. In this study, we used systematic approaches to dissect CSFV cell entry. We first observed that CSFV infection was inhibited by chloroquine and NH 4 Cl, suggesting that viral entry required a low-pH environment. By using the specific inhibitor dynasore, or by expressing the dominant negative (DN) K44A mutant, we verified that dynamin is required for CSFV entry. CSFV particles were observed to colocalize with clathrin at 5 min postinternalization, and CSFV infection was significantly reduced by chlorpromazine treatment, overexpression of a dominant negative form of the EPS15 protein, or knockdown of the clathrin heavy chain by RNA interference. These results suggested that CSFV entry depends on clathrin. Additionally, we found that endocytosis of CSFV was dependent on membrane cholesterol, while neither the overexpression of a dominant negative caveolin mutant nor the knockdown of caveolin had an effect. These results further suggested that CSFV entry required cholesterol and not caveolae. Importantly, the effect of DN mutants of three Rab proteins that regulate endosomal traffic on CSFV infection was examined. Expression of DN Rab5 and Rab7 mutants, but not the DN Rab11 mutant, significantly inhibited CSFV replication. These results were confirmed by silencing of Rab5 and Rab7. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab7 during the early phase of infection within 45 min after virus entry. These results indicated that after internalization, CSFV moved to early and late endosomes before releasing its RNA. Taken together, our findings demonstrate for the first time that CSFV enters cells through the endocytic pathway, providing new insights into the life cycle of pestiviruses. IMPORTANCEBovine viral diarrhea virus (BVDV), a single-stranded, positive-sense pestivirus within the family Flaviviridae, is internalized by clathrin-dependent receptor-mediated endocytosis. However, the detailed mechanism of cell entry is unknown for other pestiviruses, such as classical swine fever (CSF) virus (CSFV). CSFV is the etiological agent of CSF, a highly contagious disease of swine that causes numerous deaths in pigs and enormous economic losses in China. Understanding the entry pathway of CSFV will not only advance our knowledge of CSFV infection and pathogenesis but also provide novel drug targets for antiviral intervention. Based on this objective, we used systematic approaches to dissect the pathway of entry of CSFV into PK-15 cells. This is the first report to show that the entry of CSFV into PK-15 cells requires a low-pH environment and involves dynamin-and cholesteroldependent, clathrin-mediated endocytosis that requires Rab5 and Rab7....

Porcine sapelovirus enters PK-15 cells via caveolae-dependent endocytosis and requires Rab7 and Rab11

“…3F). Additionally, the functionality of the Cav1 WT and Cav1 DN constructs was validated in PK-15 cells (Shi et al, 2016). As expected, a significantly decrease of VP1 protein expression level was observed for Cav1 DN construct-transfected cells compared to pEGFP-N1 vector or WT construct-transfected cells (Fig.…”

“…2E). The functionality of Eps15 WT and EPS15 DN constructs was confirmed in PK-15 cells by using labeled transferrin as a control (Shi et al, 2016). We then examined PSV infections of cells overexpressing the Eps15 WT and Eps15 DN constructs and no effect was observed on PSV VP1 production with any of the constructs (Fig.…”

“…The dynamics of the endocytic pathway are tightly regulated by Rab GTPases, which are involved in compartmentalization of the endocytic pathway into early, recycling, late, and lysosomal routes (Wandinger-Ness and Zerial, 2014). Rab proteins have been reported to be implicated in the life cycles of many virus, including Japanese encephalitis virus (Liu et al, 2017), classical swine fever virus (Shi et al, 2016) and porcine hemagglutinating encephalomyelitis virus . However, the mechanism by which Rab proteins are required for PSV infection still remains poorly understood.…”

PEDV enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway

“…The confocal results showed that the Vero and IPEC-J2 cells overexpressing wild-type dynamin II (GFP-Dyn-WT) were infected with PEDV while the cells overexpressing mutant dynamin II (GFP-Dyn-M) were barely infected ( Figure 2B). siRNA interference was also used to identify the importance of dynamin II on virus entry [23,35,42]. Sus scrofa and Chlorocebus sabaeus siRNAs of dynamin II (siDyn) were designed and synthesized.…”

Porcine Mx1 Protein Inhibits Classical Swine Fever Virus Replication by Targeting Nonstructural Protein NS5B

“…Rab proteins play major roles in endocytic vesicle trafficking (34). Of these, Rab5 is involved in the life cycles of multiple viruses in the family Flaviviridae and is an important factor in the process of CSFV endocytosis (35). Here, we tested whether overexpression of Mx protein affects CSFV endocytosis.…”