Echovirus 1 Entry into Polarized Caco-2 Cells Depends on Dynamin, Cholesterol, and Cellular Factors Associated with Macropinocytosis

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....

Section: Resultsmentioning

confidence: 99%

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

Shi

Liu

Zhou

et al. 2016

“…Although we had excluded clathrin-and caveolin-mediated endocytosis, we still tested the role of dynamin since it contributes to some forms of macropinocytosis, to fusion pore expansion, and to remodeling of the cytoskeleton (66,75,98). The dynamin inhibitor dynasore reduced HSV-1 gene expression in the four cell lines, again with the weakest effect on Vero cells.…”

Section: Discussionmentioning

confidence: 99%

Herpes Simplex Virus Internalization into Epithelial Cells Requires Na ⁺ /H ⁺ Exchangers and p21-Activated Kinases but neither Clathrin- nor Caveolin-Mediated Endocytosis

Devadas

Koithan

Diestel

et al. 2014

Herpes simplex virus 1 (HSV-1) is an alphaherpesvirus that has been reported to infect some epithelial cell types by fusion at the plasma membrane but others by endocytosis. To determine the molecular mechanisms of productive HSV-1 cell entry, we perturbed key endocytosis host factors using specific inhibitors, RNA interference (RNAi), or overexpression of dominant negative proteins and investigated their effects on HSV-1 infection in the permissive epithelial cell lines Vero, HeLa, HEp-2, and PtK 2 . HSV-1 internalization required neither endosomal acidification nor clathrin-or caveolin-mediated endocytosis. In contrast, HSV-1 gene expression and internalization were significantly reduced after treatment with 5-(N-ethyl-N-isopropyl)amiloride (EIPA). EIPA blocks the activity of Na ؉ /H ؉ exchangers, which are plasma membrane proteins implicated in all forms of macropinocytosis. HSV-1 internalization furthermore required the function of p21-activated kinases that contribute to macropinosome formation. However, in contrast to some forms of macropinocytosis, HSV-1 did not enlist the activities of protein kinase C (PKC), tyrosine kinases, C-terminal binding protein 1, or dynamin to activate its internalization. These data suggest that HSV-1 depends on Na ؉ /H ؉ exchangers and p21-activated kinases either for macropinocytosis or for local actin rearrangements required for fusion at the plasma membrane or subsequent passage through the actin cortex underneath the plasma membrane. IMPORTANCE After initial replication in epithelial cells, herpes simplex viruses (HSVs) establish latent infections in neurons innervating these regions.Upon primary infection and reactivation from latency, HSVs cause many human skin and neurological diseases, particularly in immunocompromised hosts, despite the availability of effective antiviral drugs. Many viruses use macropinocytosis for virus internalization, and many host factors mediating this entry route have been identified, although the specific perturbation profiles vary for different host and viral cargo. In addition to an established entry pathway via acidic endosomes, we show here that HSV-1 internalization depended on sodium-proton exchangers at the plasma membrane and p21-activated kinases. These results suggest that HSV-1 requires a reorganization of the cortical actin cytoskeleton, either for productive cell entry via pHindependent fusion from macropinosomes or for fusion at the plasma membrane, and subsequent cytosolic passage to microtubules that mediate capsid transport to the nucleus for genome uncoating and replication.

“…In most cells, macropinocytosis is not constitutively active but needs to be activated (53). A series of classical studies on other viruses, including the poxvirus vaccinia virus (VACV), respiratory syncytial virus, influenza A virus, echovirus 1 (58), and African swine fever virus, revealed that the pathogens are able to activate the pathway (42,(58)(59)(60)(61). As a consequence, virus attachment to the plasma membrane induces membrane "blebbing," triggers actin depolymerization, and increases bulk fluid uptake.…”

Section: Dystroglycan-mediated Lasv Entry Involves An Unusual Pathwaymentioning

confidence: 99%

Lassa Virus Cell Entry Reveals New Aspects of Virus-Host Cell Interaction

Torriani

Galan-Navarro

Kunz

2017

Viral entry represents the first step of every viral infection and is a determinant for the host range and disease potential of a virus. Here, we review the latest developments on cell entry of the highly pathogenic Old World arenavirus Lassa virus, providing novel insights into the complex virus-host cell interaction of this important human pathogen. We will cover new discoveries on the molecular mechanisms of receptor recognition, endocytosis, and the use of late endosomal entry factors.