Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4

Abstract

Viperin (virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an interferon-inducible protein that mediates antiviral activity. Generally, rabies virus (RABV) multiplies extremely well in susceptible cells, leading to high virus titres. In this study, we found that viperin was significantly up-regulated in macrophage RAW264.7 cells but not in NA, BHK-21 or BSR cells. Transient viperin overexpression in BSR cells and stable expression in BHK-21 cells could inhibit RABV replication, including both attenuated and street RABV. Furthermore, the inhibitory function of viperin was related to reduce cholesterol/sphingomyelin on the membranes of RAW264.7 cells. We explored the up-stream regulation pathway of viperin in macrophage RAW264.7 cells in the context of RABV infection. An experiment confirmed that a specific Toll-like receptor 4 (TLR4) inhibitor, TAK-242, could inhibit viperin expression in RABV-infected RAW264.7 cells. These results support a regulatory role for TLR4. Geldanamycin, a specific inhibitor of interferon regulatory factor 3 (IRF3) (by inhibiting heat-shock protein 90 (Hsp90) of the IRF3 phosphorylation chaperone), significantly delayed and reduced viperin expression, indicating that IRF3 is involved in viperin induction in RAW264.7 cells. Taken together, our data support the therapeutic potential for viperin to inhibit RABV replication, which appears to involve upstream regulation by TLR4.

The radical‐SAM enzyme Viperin catalyzes reductive addition of a 5′‐deoxyadenosyl radical to UDP‐glucose in vitro

“…Uridine diphosphate glucose is a metabolite essential for the N‐linked glycosylation machinery and many other cellular processes. In vitro identification of UDP‐glucose as a substrate of Viperin is consistent with the in vivo effect of Viperin on different processes directly linked to UDP‐glucose, e.g., expression of viral glycoproteins , formation of lipid rafts , cellular ATP level and glucose import , and cholesterol level . We speculate that the product of Viperin activity is an inhibitor of a glycosyltransferase of the N‐linked glycosylation machinery.…”

Monophosphoryl-Lipid A (MPLA) is an Efficacious Adjuvant for Inactivated Rabies Vaccines

“…Virus titer of RABV was measured by using direct immunofluorescence assays in BSR cells, as described previously [26,27]. Briefly, viruses were 10-fold serially diluted and added into 96-well plates, then BSR cells (2 × 10 4 cells/well) were incubated with serially diluted RABV at 37 • C for 48 h. After incubation, the culture medium was discarded, and the adherent cells were fixed with 80% ice-cold acetone at −20 • C for 1 h. After 3 washes with PBS, the cells were stained with FITC-conjugated anti-RABV N protein antibodies for 1 h at 37 • C. After 3 washes with PBS, antigen-positive fluorescent foci on the cells were counted under an Olympus IX51 fluorescence microscope (Olympus, Tokyo, JPN), and virus titers were calculated as fluorescent focus units per ml.…”

Interferon Control of the Sterol Metabolic Network: Bidirectional Molecular Circuitry-Mediating Host Protection

“…This has not, however, been confirmed (160). Very recently, a TLR4/IRF3-dependent Viperin-induced reduction in membrane cholesterol and sphingomyelin was found to be key to the inhibition of Rabies virus replication in RAW264.7 cells (161). Taken together, the above studies highlight the importance of Viperin as a very early IFN-induced antiviral protein.…”