Study On The Mechanism Of Viperin Inhibits Rabies Virus

Viperin (virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an interferon-inducible protein that mediates antiviral activity against several viruses by apparently diverse mechanisms. The viperin inhibits rabies virus (RABV) replication in the previous study has not been reported. Generally, RABV multiplies well in its susceptible cells and with high virus titers in the cells cultures. In the present study, we found that RABV replicates slowly in a kind of macrophage cell RAW264.7, and its virus titers in the cells cultures is significantly lower than that of in the susceptible cells (e.g. neuroblastoma cells, NA). The further study found that the viperin expressed weakly or not expressed in susceptible NA cells infected with RABV, but viperin was significantly up-regulated in the macrophage RAW264.7 cells.In the present study, to explore whether the antiviral protein viperin inhibits RABV replication, we constructed viperin eukaryotic expression vector, and the vector was transiently transfected into BSR-T7 cells for overexpression of viperin, confirmed that overexpression of viperin could inhibit rRC-HL replication, the expression of N, P and M proteins in BSR-T7 cells was significantly inhibited, and virus titer was significantly lower compare with the control cells in a certain extent. We further screened a BHK-21 cell-line with steadily viperin-expressing and then infected with RABV, the results showed that the steadily viperin-expressing cell-line could effectively inhibit RABV replication rather than those control susceptible cells. Viperin significantly inhibits synthesis of RABV proteins. And the steadily viperin-expressing cell-line showed the identical significant inhibitory effect on virulent RABV strains GX01 and GXN119, representative street RABV isolates of group I and group III derived from Guangxi province of China respectively.Based on above research, to further explore the major functional domain of viperin inhibits RABV, we constructed a viperin mutant with deletion of N-terminal amphiphilic a helix region and a viperin mutant with 3 amino acids mutation in the radical SAM (S-adenosyl-L-methionine) enzymes motif using site-mutagenesis, then analysis of the viperin mutants’functions were carried out. The results showed that the functional domain of viperin inhibiting RABV replication was mainly located in its N-terminus, the deletion of viperin N-terminus could significantly decrease the anti-RABV ability. Three key amino acid (C83A/C87A/C90A) mutated in the radical SAM enzymes motif of viperin also obviously decrease the ability of viperin inhibiting RABV replication.Previous studies found that the N-terminus of viperin can bind to intracellular endoplasmic reticulum and lipid droplet, inhibit budding and releasing of virus by disrupting lipid raft microdomains on the plasma membrane. In this experiment, to understand the mechanism of viperin inhibiting RABV replication, we explored the biological effects of viperin to cholesterol and sphingomyelin, two composition components, of lipid raft. The viperin was overexpressed in BSR-T7 cells and the change of cholesterol and sphingomyelin in the BSR-T7 cells was detected. The results showed that overexpression of viperin can reduce the content of cholesterol and sphingomyelin in the BSR-T7 cells. It is confirned that viperin might disrupt cholesterol and sphingomyelin by inhibiting the release of RABV. Then, a cholesterol inhibitor MβCD and a sphingomyelin inhibitor myroicin were used to inhibit the synthesis of cholesterol and sphingomyelin in BSR-T7 cells, respectively. The results indicated that the cellular cholesterol and sphingomyelin were damaged. However, the damage of cholesterol and sphingomyelin in BSR-T7 cells could not influence adsorption and entry of RABV, but could affect budding and releasing of RABV, confirming that the destruction of cholesterol and sphingomyelin in cells menbrance is one of the important ways for viperin inhibiting RABV replication.Viperin, an interferon-inducible protein, is induced and regulated by interferon. In this experiment, to further explore the up-stream signaling transduction pathways of viperin in macrophage-line RAW264.7 cells infected with RABV, the microarray was used to analyze the transcriptional profiles of signal transduction pathway, including 84 immune response gene and 5 housekeeping genes in RAW264.7 cells infected RABV. Of the 84 genes,26 genes were up-regulated (=2.0 folds) at 12,24 and 36 hour post-inoculation (hpi),2 genes were up-regulated (=2.0 folds) at 12 hpi, but not changed at 24 and 36 hpi, one gene was up-regulated (=2.0 folds) at 12 and 24 hpi, but not changed at 36 hpi,15 genes no changed at 12 hpi, but up-regulated (=2.0 folds) at 24 and 36 hpi,17 genes was not changed at 12 and 24 hpi, but up-regulated (=2.0 folds) at 36 hpi,25 genes were not changed at 12,24 and 36 hpi. One gene was down-regulated (=2.0 folds) at 12,24 and 36 hpi. The cxc110 gene raised 377.85,333.14 and 183.12 folds; IFNβ gene raised 370.07,68.12 and 13.09 folds in 12,24 and 36 hpi, respectively. Also, the TLRs were up-regulated in different level of RAW264.7 cells infected with RABV, such as, TLR3 gene was up-regulated 156.68,380.03 and 164.66 folds at 12,24, and 36 hpi respetively; TLR2 and TLR8 genes were upregulated in certain extent; TLR1, TLR4, TLR5, TLR6, TLR7 and TLR9 also have a significant change.These experiments verified that TLRs involved in immune signaling transduction pathway in RAW264.7 cells infected with RABV. Several studies reported that TLR4 can recognize viral envelope protein and transfer immune signals, activate innate immune responses, TLR3 can recognize the double-stranded RNA (dsRNA) and intermediate compounds produced in the process of viral replication. In this study, the TLR4 was detected in RAW264.7 cells infected with RABV by flow cytometry, and the TLR4 was significantly up-regulated, indicating the TLR4 was activated by RABV. The inactivated RABV was used to incubate with RAW264.7 cells, and the results showed that RABV G protein which retained the basic natural characteristics can induce viperin. When the TLR4-specific inhibitor TAK-242 was used to block TLR4 signal transduction, the expression of viperin was significantly reduced compared with controls. These results suggested that TLR4 was involved in recognition of RABV and induction of viperin expression. To determine whether TLR3 signaling pathways involves in the interaction between RABV and host cells, and induces the innate immune response of viperin expression, the TLR3 gene knocking out RAW264.7 cell line TLR3-/- RAW264.7 was generated, then the TLR3-/- RAW264.7 cell line was infected with RABV, then detected change of viperin expression. The results showed that the viperin expression was significantly delayed and reduced in the TLR3-/- RAW264.7 cell line. These findings suggested that TLR3 was involved in recognization of RABV dsRNA and induction of viperin expression.NF-κB and IRF3 are two important immune factors in innate immune signaling pathway, the specific inhibitors of NF-κB was used to inhibit its signal transduction, then the RAW264.7 cells were infected with RABV, the results showed that inhibition of NF-κB signal transduction pathway did not affect the viperin expression, indicating that up-regulation of viperin by RABV did not related with NF-κB signal pathway directly in RAW264.7 cells. After the RAW264.7 cells were treated with Geldanamycin, the specific inhibitor of IRF3, which can inhibit Hsp90 of IRF3 chaperone. The viperin expression was significantly delayed and reduced, indicating that the IRF3 involved in induction of viperin expression in RAW264.7 cells infected with RABV.In summary, the study concluded that RABV infected RAW264.7 cells to induce viperin expression and the viperin can inhibit attenuated and virulent RABV replication. The inhibition of viperin to RABV replication was mainly caused by hindering bud and release of RABV via disrupting cholesterol and sphingomyelin on RAW264.7 cell membrane. Further research using microarray technology found that most TLRs were up-regulated in RAW264.7 cells infected with RABV. The TLR3 gene in RAW264.7 cells was knocked out to generate a TLR3-locking TLR3-/- RAW264.7 cell-line. When the TLR3-/- RAW264.7 cell-line was infected with RABV, the expression of viperin was significantly reduced and delayed, indicating that TLR3 involves in recognition of RABV. Further experiment confirmed that the specific TLR4 inhibitor TAK-242 can inhibit induction of viperin by RABV infection, not only activating RABV but also inactivating RABV. Taken together, the viperin which is an IFN-inducible protein induced in RAW264.7 cells by RABV is regulated by TLR3/TLR4 signal transduction pathways. In this study we clarified the functions of viperin and its signal transduction pathway on up-stream in the RAW264.7 cells infected with RABV, these systematic research advances imply important theoretical and practical significance to explore for pathogenic mechanism of RABV.