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The receptor(s) for porcine sapelovirus (PSV), which causes diarrhea, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs, remains largely unknown. Given the precedent for other picornaviruses which use terminal sialic acids (SAs) as receptors, we examined the role of SAs in PSV binding and infection. Using a variety of approaches, including treating cells with a carbohydrate-destroying chemical (NaIO4), mono- or oligosaccharides (N-acetylneuraminic acid, galactose, and 6'-sialyllactose), linkage-specific sialidases (neuraminidase and sialidase S), lectins (Maakia amurensis lectin and Sambucus nigra lectin), proteases (trypsin and chymotrypsin), and glucosylceramide synthase inhibitors (DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and phospholipase C), we demonstrated that PSV could recognize α2,3-linked SA on glycolipids as a receptor. On the other hand, PSVs had no binding affinity for synthetic histo-blood group antigens (HBGAs), suggesting that PSVs could not use HBGAs as receptors. Depletion of cell surface glycolipids followed by reconstitution studies indicated that GD1a ganglioside, but not other gangliosides, could restore PSV binding and infection, further confirming α2,3-linked SA on GD1a as a PSV receptor. Our results could provide significant information on the understanding of the life cycle of sapelovirus and other picornaviruses. For the broader community in the area of pathogens and pathogenesis, these findings and insights could contribute to the development of affordable, useful, and efficient drugs for anti-sapelovirus therapy. [ABSTRACT FROM AUTHOR]

The genus Sapovirus belongs to the family Caliciviridae, and its members are common causative agents of severe acute gastroenteritis in both humans and animals. Some caliciviruses are known to use either terminal sialic acids or histoblood group antigens as attachment factors and/or cell surface proteins, such as CD300lf, CD300ld, and junctional adhesion molecule 1 of tight junctions (TJs), as receptors. However, the roles of TJs and their proteins in sapovirus entry have not been examined. In this study, we found that porcine sapovirus (PSaV) significantly decreased transepithelial electrical resistance and increased paracellular permeability early in infection of LLC-PK cells, suggesting that PSaV dissociates TJs of cells. This led to the interaction between PSaV particles and occludin, which traveled in a complex into late endosomes via Rab5- and Rab7-dependent trafficking. Inhibition of occludin using small interfering RNA (siRNA), a specific antibody, or a dominantnegative mutant significantly blocked the entry of PSaV. Transient expression of occludin in nonpermissive Chinese hamster ovary (CHO) cells conferred susceptibility to PSaV, but only for a limited time. Although claudin-1, another TJ protein, neither directly interacted nor was internalized with PSaV particles, it facilitated PSaV entry and replication in the LLC-PK cells. We conclude that PSaV particles enter LLC-PK cells by binding to occludin as a coreceptor in PSaV-dissociated TJs. PSaV and occludin then form a complex that moves to late endosomes via Rab5- and Rab7- dependent trafficking. In addition, claudin-1 in the TJs opened by PSaV infection facilitates PSaV entry and infection as an entry factor. [ABSTRACT FROM AUTHOR]

Enteric caliciviruses in the genera Norovirus and Sapovirus are important pathogens that cause severe acute gastroenteritis in both humans and animals. Cyclooxygenases (COXs) and their final product, prostaglandin E2 (PGE2), are known to play important roles in the modulation of both the host response to infection and the replicative cycles of several viruses. However, the precise mechanism(s) by which the COX/PGE2 pathway regulates sapovirus replication remains largely unknown. In this study, infection with porcine sapovirus (PSaV) strain Cowden, the only cultivable virus within the genus Sapovirus, markedly increased COX-2 mRNA and protein levels at 24 and 36 h postinfection (hpi), with only a transient increase in COX-1 levels seen at 24 hpi. The treatment of cells with pharmacological inhibitors, such as nonsteroidal anti-inflammatory drugs or small interfering RNAs (siRNAs) against COX-1 and COX-2, significantly reduced PGE2 production, as well as PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE2 pathway. We observed that pharmacological inhibition of COX-2 dramatically increased NO production, causing a reduction in PSaV replication that could be restored by inhibition of nitric oxide synthase via the inhibitor N-nitro-L-methylarginine ester. This study identified a pivotal role for the COX/PGE2 pathway in the regulation of NO production during the sapovirus life cycle, providing new insights into the life cycle of this poorly characterized family of viruses. Our findings also reveal potential new targets for treatment of sapovirus infection. [ABSTRACT FROM AUTHOR]

Sapoviruses of the Caliciviridae family of small RNA viruses are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture, making it a suitable model for understanding the infectious cycle of sapoviruses and the related enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus genome-encoded viral protein genome (VPg) protein, which is covalently linked to the 5′ end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg, and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the 4F subunit of the eukaryotic translation initiation factor (eIF4F) complex in infected cells and bound directly to the eIF4E protein. As has been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall, our study provides new insights into the novel mechanism of sapovirus translation, suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction. [ABSTRACT FROM AUTHOR]

Some viruses within the Caliciviridae family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs), and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Ley HBGAs expressed in the bovine digestive tract and are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to a large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of α1,2-linked fucose and α1,3/4-linked fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of α1,2- linked fucose and α1,3/4-linked fucose epitopes of targeted HBGAs. However, removal of terminal α2,3- and α2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored. IMPORTANCE Enteric caliciviruses such as noroviruses, sapoviruses, and recoviruses are the most important etiological agents of severe acute gastroenteritis in humans and many other mammalian host species. They initiate infection by attachment to cell surface carbohydrate moieties, HBGAs, and/or terminal SAs. However, the attachment factor(s) for BNeV, a recently classified enteric calicivirus genus/type species, remains unexplored. Here, we demonstrate that BNeV VLPs have a wide spectrum of binding to synthetic HBGAs, bovine and human saliva samples and bovine duodenal sections. We further discovered that α1,2-linked fucose and α1,3/4-linked fucose epitopes are essential for binding of BNeV VLPs. However, BNeV VLPs do not bind to terminal SAs on cell carbohydrates. Continued investigation regarding the proteinaceous receptor(s) will be necessary for better understanding of the tropism, pathogenesis, and host range of this important viral genus. [ABSTRACT FROM AUTHOR]