Endoplasmic Reticulum-Associated Degradation Controls Virus Protein Homeostasis, Which Is Required for Flavivirus Propagation

Abstract

Many positive-stranded RNA viruses encode polyproteins and viral proteins are generated by processing the polyproteins. This system produces an equal amount of each viral protein, though their required amounts are different. In this study, we found that the extra membrane-anchored non-structural (NS) proteins of Japanese encephalitis virus and dengue virus are rapidly and selectively degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. Our gene targeting study revealed that ERAD involving Derlin2 and SEL1L, but not Derlin1, is required for the viral genome replication. Derlin2 predominantly localized in the convoluted membrane (CM) of viral replication organelle, and viral NS proteins degraded in the CM. Hence, these results suggest that viral protein homeostasis is regulated by Derlin2-mediated ERAD in the CM, and this process is critical for the propagation of these viruses. Importance The results of this study reveal that the cellular ERAD system controls the amount of each viral protein in virus-infected cells; this “viral protein homeostasis” is critical for viral propagation. Furthermore, we clarified that the “convoluted membrane (CM),” which was previously considered a structure with unknown function, serves as a kind of waste dump where viral protein degradation occurs. We also found that the Derlin2/Sel1L/HRD1-specific pathway is involved in this process, whereas the Derlin1-mediated pathway is not. This novel ERAD-mediated fine-tuning system for the stoichiometries of polyprotein-derived viral proteins may represent a common feature among polyprotein-encoding viruses.

Flavivirus recruits the valosin-containing protein–NPL4 complex to induce stress granule disassembly for efficient viral genome replication

“…Hence, the cells infected with the virus for 2 h were treated with specific small-molecule VCP ATPase inhibitors, such as DBeQ (26), for 4 h. Treatment with the VCP ATPase inhibitors impaired the production of infectious JEV in the supernatant (Fig. 3C), which was consistent with the results of a previous study (13). A 4-h pulse treatment with DBeQ just after virus inoculation was sufficient to reduce the viral titers.…”

“…Previously, proteome analysis revealed that both NPL4 and VCP co-precipitated with JEV NS4B and DENV NS4B (13). Hence, these interactions were confirmed using the reciprocal pull-down assay.…”

“…A recent study has reported that the ATPase activity of VCP is required for flavivirus propagation (13). Hence, the cells infected with the virus for 2 h were treated with specific small-molecule VCP ATPase inhibitors, such as DBeQ (26), for 4 h. Treatment with the VCP ATPase inhibitors impaired the production of infectious JEV in the supernatant (Fig.…”

“…Furthermore, various co-factors, including NPL4, UFD1, p47, p37, and UBXD1, bind to the N-terminal domain of VCP through specific interaction domains or motifs and consequently modulate the VCP-mediated processes (11). Previously, we had reported that siRNA-mediated knockdown of VCP markedly downregulated the production of JEV or DENV particles (>10 6 -fold titer reductions) without exerting cytotoxic effects (13). This indicates that VCP is a potential therapeutic target for flaviviral infections.…”

“…During capsid uncoating, the VCP complex promotes the transportation of ubiquitinated viral capsids to the proteasome, which leads to the release of the RNA genome into the cytoplasm (15). In viral genome RNA replication, the VCP-mediated ERAD pathway is involved in the degradation of ubiquitinated NS proteins, which is important to maintain the homeostatic levels of viral proteins for efficient flaviviral genome replication (13). However, the mechanisms underlying the antiviral effect of VCP depletion have not been elucidated.…”