| Previous studies reported that zinc exhibited antiviral activity in vitro and in vivo.Zinc iones have been widely used as a therapeutic or supplemental ingredient for various disease indications, including viral infections, and recently added into anti-HIV microbicide formulation as preventive medicines. However, up to now, the mechanism of the antiviral activity of zinc ions has yet to be illustrated. In this study,we studied the antiviral properties of two zinc ionophores (Pyrrolidine dithiocarbamate, PDTC; pyrithione, PT), and the results showed that these two zinc ionophores could inhibit HSV replication through blocking ubiquitin-proteasome pathway. Various studies have demonstrated that many viruses can hijack the ubiquitin-proteasome system (UPS) of the host cells to fight the host defense and help themselves complete infection cycle. We further explored the effect of UPS on viral replication and the roles of other components in UPS pathway, such as ubiquitin-like proteins. Finally, we focused on USP14, a 26S proteasome-associated deubiquitinating enzyme, and its effect on HSV infection and the mechanism. To summarize, the major observations of this thesis are:1. PDTC and PT could inhibt HSV-1 and HSV-2 replication, and these two compounds blocked both HSV immediately-early gene (ICP4) and late gene product(gD) to prevent the formation of intact virions. As is known that PDTC and PT are zinc ionophores, we found that the antiviral properties of PDTC and PT were dependent on zinc ions in the medium. And these two compounds could inhibit HSV-induced NF-κB activation by blocking virus-mediated IκB-α degradation and p65 nuclear transloacation. Further studies found that PDTC and PT mediated zinc influx, and increased intracellular zinc ions inhibited 26S proteasome activity. This finding implied that the antiviral property of zinc ions might be associated with cellular UPS pathway. Meanwhile, zinc ionophores could inhibit virus-mediated PML degradation, which was related to cellular proteasome activity.2. The effect of ubiquitin on HSV infection was investigated by using dominant negative mutants of ubiquitin on the viral replication efficiency, and we found that the expression of ubiquitin mutant could effectively inhibit the expression of viral IE and L gene products. We also studied whether HSV infection could influence the expression of the main components of 26S proteasome, and observed that it did not have any effect on them. Immunofluoresence assay showed that ICPO could co-localize with Ub and proteasome. Furthermore, at the late stage of of HSV infection, proteasome would focus together in cytoplasma. The relationship between REGy and viral replication was also investigated through RNAi or overexpression of dominant negative mutant. It was found that REGγ might not participate in HSV replication. In addition, we also knocked down SUMO and NEDD8 to study the effect on HSV infection and found that the knockdown inhibited HSV infection and viral protein expression, implying that these two ubiquitin-like tag proteins are associated with viral replication. Finally, we employed PYR-41 and WP1130 in HSV replication studies and found that these two specific inhibitors on El ubiquitin-activating enzyme and DUBs, respectively, could inhibit HSV infection. We conclude that the intact USP pathway is a prerequisite for HSV replication.3. The function of USP14 on HSV replication was also studied. Knocking down USP 14 could inhibit HSV replication and viral protein expression. Subsequently, we also studied other 26S proteasome-associated DUBs, UCH37 and Rpnll on HSV replication. And we found that USP14-mediated HSV replication was independent on its deubiquitinating activity. In a further study, USP14 inhibited IRE1, and IRE1 activity will block HSV replication. It was found that the overexpression of XBP1 and downstream ERAD would also inhibit viral replication. Therefore, we concluded that USP14 links the relationship between ubiquitin-proteasome pathway and endoplasmic reticulum (ER) stress/unfolded protein response. And the activation of IRE1/XBP1 pathway will influence HSV replication negatively. |
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