Very little is known about the mechanism of cell entry of avian reovirus (ARV). The aim of this study was to explore the mechanism of ARV entry and subsequent infection. Cholesterol mainly affected the early steps of the ARV life cycle, because the presence of cholesterol before and during viral adsorption greatly blocked ARV infectivity. Although we have demonstrated that ARV facilitating p38 MAPK is beneficial for virus replication, its mechanism remains unknown. Here, we show that ARV-induced phosphorylation of caveolin-1 (Tyr 14 ), dynamin-2 expression, and Rac1 activation through activation of p38 MAPK and Src in the early stage of the virus life cycle is beneficial for virus entry and productive infection. The strong inhibition by dynasore, a specific inhibitor of dynamin-2, and depletion of endogenous caveolin-1 or dynamin-2 by siRNAs as well as the caveolin-1 colocalization study implicate caveolin-1-mediated and dynamin-2-dependent endocytosis as a significant avenue of ARV entry. By means of pharmacological inhibitors, dominant negative mutants, and siRNA of various cellular proteins and signaling molecules, phosphorylation of caveolin-1, dynamin-2 expression, and Rac1 activation were suppressed, suggesting that by orchestrating p38 MAPK, Src, and Rac1 signaling cascade in the target cells, ARV creates an appropriate intracellular environment facilitating virus entry and productive infection. Furthermore, disruption of microtubules, Rab5, or endosome acidification all inhibited ARV infection, suggesting that microtubules and small GTPase Rab5, which regulate transport to early endosome, are crucial for survival of ARV and that exposure of the virus to acidic pH is required for productive infection.
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Avian reovirus (ARV) is a non-enveloped virus with a segmented double-stranded RNA genome surrounded by a double icosahedral capsid shell. ARVs are associated with viral arthritis, immunosuppression, and enteric diseases in poultry. The sC protein was involved in induction of apoptosis and neutralization antibody. In the present study, sC-His protein was expressed in Sf9 insect cells and purified by immobilized metal affinity chromatography. Eight monoclonal antibodies (mAbs) against sC-His and three mAbs against His were screened from hybridoma cells produced by fusion of splenocytes from immunized mice with NS1 myeloma cells. Among the eight mAbs against sC protein, all belonged to the IgG isotype except three for IgM. It was discovered that all anti-His mAbs were mixtures of IgG and IgM isotypes. mAbs reacted with sC-His protein in a conformation-independent manner based on dot blot and western blotting assays. The competitive binding assay indicated that all mAbs recognized the same epitope on sC protein that was conserved in different isolates. Compared with the commercial anti-ARV S1133 polyclonal antibody, mAb (D15) had universal reactivity to all serotypes or genotypes of ARVs tested. This monoclonal antibody may therefore be useful for the development of an antigen-capture enzyme-linked immunosorbent assay for rapid detection of field isolates.
Hyperglycemia and advanced glycation end products (AGEs) are associated with an elevated risk of developing several cancers in diabetic patients. However, the detailed mechanisms remain to be elucidated. The mechanism of AGE-bovine serum albumin (BSA) on gap junction intercellular communication in human hepatoma cell line, SKHep 1, was investigated. Both Cx32 and Cx43 are major gap junction forming proteins in the liver, the loss of which has been shown to facilitate tumorigenesis. Although the MTT assay results showed that AGE-BSA significantly increased cell growth by 31%, AGE-BSA down-regulated Cx32 and Cx43 expression in a dose- and time-dependent manner. The present study also demonstrated that ERK1/2 and JNK/SAPK were significantly activated by AGE-BSA and that Src, ERK1/2, and JNK/SAPK inhibitors significantly reversed the reduction of Cx32 and Cx43 proteins by AGE-BSA. Taken together, these results strongly support the hypothesis that Src-dependent ERK1/2 and JNK/SAPK/AP1 signaling pathways play a key role in AGE-BSA-mediated down-regulation of Cx32 and Cx43 protein expression in SKHep 1 cells.
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