Porcine sapovirus Cowden strain enters LLC-PK cells via clathrin- and cholesterol-dependent endocytosis with the requirement of dynamin II

Abstract

Caliciviruses in the genus Sapovirus are a significant cause of viral gastroenteritis in humans and animals. However, the mechanism of their entry into cells is not well characterized. Here, we determined the entry mechanism of porcine sapovirus (PSaV) strain Cowden into permissive LLC-PK cells. The inhibition of clathrin-mediated endocytosis using chlorpromazine, siRNAs, and a dominant negative (DN) mutant blocked entry and infection of PSaV Cowden strain, confirming a role for clathrin-mediated internalization. Entry and infection were also inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin and was restored by the addition of soluble cholesterol, indicating that cholesterol also contributes to entry and infection of this strain. Furthermore, the inhibition of dynamin GTPase activity by dynasore, siRNA depletion of dynamin II, or overexpression of a DN mutant of dynamin II reduced the entry and infection, suggesting that dynamin mediates the fission and detachment of clathrin- and cholesterol-pits for entry of this strain. In contrast, the inhibition of caveolae-mediated endocytosis using nystatin, siRNAs, or a DN mutant had no inhibitory effect on entry and infection of this strain. It was further determined that cell entry of PSaV Cowden strain required actin rearrangements for vesicle internalization, endosomal trafficking from early to late endosomes through microtubules, and late endosomal acidification for uncoating. We conclude that PSaV strain Cowden is internalized into LLC-PK cells by clathrin- and cholesterol-mediated endocytosis that requires dynamin II and actin rearrangement, and that the uncoating occurs in the acidified late endosomes after trafficking from the early endosomes through microtubules.Electronic supplementary materialThe online version of this article (10.1186/s13567-018-0584-0) contains supplementary material, which is available to authorized users.

Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

“…7A ). Confirming the results of our recent report ( 33 ), these data indicated that uncoating of PSaV particles was completed at 90 mpi.…”

“…The above-described results imply that PSaV-induced early activation of PI3K/Akt and MEK/ERK signaling pathways might be involved in the virus entry process. In our previous report, we demonstrated that PSaV enters cells through clathrin- and cholesterol-mediated endocytosis and travels from early to late endosomes ( 33 ). Since these membrane lipid rafts compartmentalize cellular processes by acting as a signaling platform ( 44 ), we tested whether cholesterol-perturbing drugs could affect these signaling pathways in response to PSaV infection.…”

“…However, inhibition of both signaling cascades by pretreatment with wortmannin and U0126, which are specific inhibitors of PI3K and MEK, respectively, sequestered PSaV particles within the early endosomes, as indicated by colocalization with the early endosome marker EEA1. This prevented PSaV trafficking to late endosomes even after 3 h postinfection, by which time PSaV particles should have been uncoated and their genomes released into the cytoplasm ( 33 ). These results suggested that PSaV-induced early activation of PI3K/Akt and MEK/ERK facilitates PSaV trafficking from early to late endosomes.…”

“…Recently, we demonstrated that PSaV is internalized by clathrin- and cholesterol-mediated endocytosis, with the requirement of dynamin II and actin rearrangement, and that its uncoating occurs in the acidified late endosomes, after it has moved from early endosomes via microtubules ( 33 ). Although the PI3K/Akt and MEK/ERK signaling pathways are known to be involved in the entry of some viruses, their roles during PSaV entry have remained elusive.…”

Sialoglycovirology of Lectins: Sialyl Glycan Binding of Enveloped and Non-enveloped Viruses

“…Each VP1 monomer has 2 domains: a shell (S) domain and a protrusion (P) domain. Binding of the P domains to host receptors triggers receptor-mediated endocytosis that depends on clathrin, dynamin II, and/or cholesterol [108][109][110] as shown in Table 2, and the endocytosis is followed by penetration of the viral genome into the host cytosol for multiplication. The use of neoglycoproteins for binding studies has revealed that virus-like particles (VLPs) of Chron1 (GII.3), Dijon (GII.4), and Norwalk (GI.1) strains bind to Le b and H type 1 chain glycoconjugates.…”