The antiviral action of resveratrol against Zika virus through the regulation of interferon responses by HMGB1

doi:10.21203/rs.3.rs-1495329/v1

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The antiviral action of resveratrol against Zika virus through the regulation of interferon responses by HMGB1

https://doi.org/10.21203/rs.3.rs-1495329/v1

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Zika virus (ZIKV) infection is a public health concern and currently there is no specific therapeutic or approved vaccine. Resveratrol (RESV), a natural antiviral compound, has been shown to possess antiviral properties, but the mechanisms of action against ZIKV remain unknown. This study aimed to investigate the role of the high mobility group box 1 protein (HMGB1) in the underlying anti-ZIKV mechanisms of RESV. HMGB1 protein expression and ZIKV replication in both RESV-treated wild-type (WT) and HMGB1-knockdown (shHMGB1) Huh7 cells were analyzed using ELISA, immunofluorescence assay, immunoblot assay, focus-forming assay and qRT-PCR. HMGB1’s role was explored by evaluating the changes in the type-1 interferon (IFN) response genes using qRT-PCR and immunoblot assays. The treatment of ZIKV-infected WT Huh7 cells with RESV significantly reduced ZIKV titers by >90% (P < 0.001) and inhibited ZIKV-induced HMGB1 translocation (P < 0.001), resulting in nuclear HMGB1 accumulation. Compared to WT Huh7 cells, shHMGB1 Huh7 cells without RESV treatment showed a significant increase in the infectious virus titers and RNA with a maximum rise of 74% (P < 0.001) and 65% (P < 0.01), respectively. RESV treatment of ZIKV-infected WT Huh7 cells significantly increased the MxA (one of the classical interferon-stimulated genes, ISGs) and IFN-β levels (p < 0.05). The treatment of infected shHMGB1 Huh7 cells with RESV showed a less effective antiviral response (P > 0.05) and did not cause changes in the expressions of MxA and IFN-β. Findings presented here suggest that RESV possesses therapeutic activity against ZIKV infection and the mechanism of action is mainly attributed to HMGB1 nuclear retention, which could upregulate the type-1 IFN and ISGs.

Mosquito-borne Zika virus (ZIKV) was a neglected tropical pathogen until its explosive spread in the Pacific and Americas ^1,2. Zika fever is generally mild and self-limiting. However, it has been associated with more severe, life-threatening neurological disorders and congenital nervous system malformations ^3,4. In the infected host, pathogen recognition activates the innate immune responses, including the production of type-1 interferon (IFN), which triggers the JAK-STAT signaling pathway and the transcriptional induction of hundreds of interferon-stimulated genes (ISGs) ⁵. Immunosuppressive mouse models lacking the IFN signaling pathway are highly susceptible to both African and Asian ZIKV strains, developing severe ZIKV neurological symptoms such as hind-limb weakness, paralysis and death ^6,7. Strategies for effectively modulating interferon expression may drive the development of more effective antiviral agents. To date, there is no specific antiviral drug or licensed vaccine against ZIKV infection. Numerous potential anti-ZIKV natural compounds, including quercetin ⁸, baicalein and baicalin ⁹, pinocembrin ¹⁰, silymarin ¹¹ and resveratrol ^12,13 have been studied.

Resveratrol (3, 5, 4’–trihydroxy-trans-stilbene, RESV), a natural non-flavonoid polyphenol compound found in plants such as grapes, berries and peanuts, as well as processed foods like red wine ^14,15, possess potential antiviral properties ¹⁶. Previous studies reported the ability of RESV to suppress viral gene expression and viral protein synthesis, leading to inhibition of enterovirus 71 ¹⁷, influenza A virus ¹⁸, Epstein-Barr virus ^19,20, human immunodeficiency virus (HIV) ^21,22, herpes simplex virus ^23,24, respiratory syncytial virus ²⁵, cytomegalovirus ²⁶, severe acute respiratory syndrome coronavirus‐2 ²⁷, as well as flaviviruses including dengue virus (DENV) ²⁸ and ZIKV ^12,13 replication. However, the antiviral mechanisms of RESV against ZIKV are yet to be determined.

A previous study reported that RESV retains its antiviral properties against DENV via regulating the high mortality group box 1 (HMGB1) ²⁸, an abundant non-histone nucleosomal protein that regulates DNA transcription and nucleosome stability. Upon infection, acetylated HMGB1 translocates from the nucleus to the extracellular milieu, where it acts as an alarmin to trigger inflammatory responses ^29-31. The intracellular and extracellular HMGB1 have been suggested to play various roles in virus replication, including enhancing and inhibiting virus replication ^28,32-35. In addition, HMGB1 intracellular translocation is crucial for ZIKV replication in Huh7 cells, where inhibition of the HMGB1 translocation by dexamethasone resulted in a reduction of ZIKV replication ³⁶. The present study is intended to determine the role of HMGB1 in RESV’s antiviral mechanisms against ZIKV infection.

Resveratrol treatment inhibited ZIKV replication in a dose-dependent manner.

The human hepatoma cell line, Huh7, was used because of its high susceptibility to ZIKV infection ³⁷, as well as good basal expression and functional RIG- and Toll-like receptors ^38,39. The potential toxicity of RESV on Huh7 cells was first determined using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay after 72 hours of treatment. The data was further analyzed using GraphPad Prism 7 by plotting a dose-response curve to obtain the cytotoxicity concentration of 50% (CC50) and maximum non-toxic dose (MNTD) values (Fig. 1A). The calculated CC50 and MNTD of RESV on Huh7 cells were at 1841.05 μM and 343.72 μM, respectively (Fig. 1A). Additionally, RESV was found to have low cytotoxicity for uninfected and ZIKV-infected Huh7 cells, with ≥ 90% cell viability following treatments with 20 to 200 μM of RESV (Fig. 1B).

To examine the antiviral effects of RESV against ZIKV, Huh7 cells were treated with various concentrations of RESV (0 μM to 80 μM) following ZIKV infection at an MOI of 1 and virus titers were determined by the focus-forming assay (FFA) as previously reported ⁴⁰ at 24, 48 and 72 hr pi (hour post-infection) (Fig. 2). Significant decrease in virus titers (p < 0.001) were detected in all RESV-treated cells compared to the mock-treated cells (ZIKV infected-Huh7 cells without RESV treatment) at 48 and 72 hr pi and the virus inhibitory effect was in a dose-dependent manner (Fig. 2B and C). Treatment of ZIKV-infected cells with 10 μM, 30 μM, 50 μM and 80 μM of RESV significantly reduced the infectious virus titers by 22% (P > 0.05), 47 % (P < 0.001), 61% (P < 0.001) and 54% (P < 0.001) at 24 hr pi; by 42% (P < 0.001), 91% (P < 0.001), 91% (P < 0.001) and 94% (P < 0.001) at 48 hr pi; and by 50% (P < 0.001), 90% (P < 0.001), 93% (P < 0.001) and 95% (P < 0.001) at 72 hr pi, respectively, in comparison to the mock-treated infected cells (Fig. 2D). Because of its non-toxic effects on Huh7 cells and ability to lower > 90% of ZIKV titers (p < 0.001) at 48 and 72 hr pi, 80 μM of RESV was chosen for subsequent studies.

Resveratrol treatment prevented ZIKV-induced nuclear HMGB1 translocation and extracellular release.

In our previous study, it was demonstrated that ZIKV infection in Huh7 cells induces the nuclear-to-cytoplasmic translocation and extracellular release of HMGB1 ³⁶. In the present study, the effects of RESV treatment (80 μM) on the intracellular HMGB1 protein expression in the ZIKV-infected Huh7 cells were investigated using the immunofluorescence assay (IFA) and immunoblot assays at 48 hr pi, whereas, extracellular HMGB1 production was determined by ELISA and immunoblot assays at 72 hr pi. The uninfected Huh7 cells without RESV treatment (cell control) and uninfected Huh7 cells with RESV treatment (RESV control) were used as controls. IFA results showed that RESV-treated cells had a higher intensity of HMGB1, with most HMGB1 concentrated in the nucleus region (Fig. 3A, RESV control and RESV-treated ZIKV-infected Huh7 cells compared to cell control and mock-treated infected cells). On the contrary, ZIKV infection in Huh7 cells caused decreased HMGB1 intensity in the nucleus but increased HMGB1 intensity in the cytoplasm (Fig. 3A, mock-treated infected cells compared to cell control), similar to our prior findings ³⁶. RESV treatment after ZIKV-infection resulted in HMGB1 retention in the nuclei of ZIKV-infected cells and reduced ZIKV replication (Fig. 3A, RESV-treated ZIKV-infected Huh7 cells as compared to mock-treated infected cells). The immunoblotting analysis of nuclear and cytosolic HMGB1 protein expression was performed to verify the retention of nuclear HMGB1 by RESV treatment during ZIKV infection. In comparison to the cell control (51%) and mock-treated infected cells (16%), RESV treatment significantly increased nuclear HMGB1 levels in both the mock-infected (100%, P < 0.001) and ZIKV-infected (39 %, P < 0.01) cell groups, respectively (Fig. 3B). In contrast, RESV treatment resulted in lower cytosolic HMGB1 levels in mock-infected (8%, P > 0.05) and ZIKV-infected (18%, P < 0.001) cells than the cell control (13%) and mock-treated infected cells (100%), respectively (Fig. 3C), consistent with the results obtained from the IFA (Fig. 3A).

Apart from inhibiting ZIKV replication, RESV treatment of infected cells decreased extracellular HMGB1 levels from 1562 47.50 pg/ml in mock-treated infected cells to 922 ± 97.50 pg/ml (p < 0.01), 764.50 ± 30 pg/ml (p < 0.01), 709.50 ± 75.00 pg/ml (P < 0.001) and 502 ± 38.89 pg/ml (P < 0.001) by RESV treatment at 10, 30, 50 and 80 μM, respectively at 72 hr pi (Fig. 3D), consistent with the immunoblotting results (Fig. 3E). Collectively, these findings suggested that the treatment with RESV prevented HMGB1 nucleocytoplasmic translocation and extracellular release of HMGB1 induced by ZIKV infection.

HMGB1-knockdown enhanced ZIKV replication and compromised the antiviral actions of resveratrol.

The role of HMGB1 in RESV’ anti-ZIKV mechanisms was investigated using the HMGB1-knockdown cells, shHMGB1 cells (Supplementary Fig. S1). Both wild-type (WT) and shHMGB1 Huh7 cells were infected with ZIKV at an MOI of 1 and the infected cell culture supernatants were harvested at various time points (e.g., 24, 48 and 72 hr pi) for virus titration using FFA and qRT-PCR (Fig. 4). The infectious virus titers of shHMGB1 Huh7 cells significantly increased by 74% (P < 0.001) at 48 hr pi and 53% (P < 0.001) at 72 hr pi (Fig. 4A), as well as ZIKV RNA increased by 65% (P < 0.01) at 24 hr pi; 31% (P < 0.001) at 48 hr pi and 16% (P < 0.001) at 72 hr pi, as compared to WT Huh7 cells, respectively (Fig. 4B). These results suggested that HMGB1 depletion led to increased ZIKV replication.

To study the effects of HMGB1-knockdown in the antiviral mechanisms mediated by RESV, virus in both cell culture supernatants and cell lysates were harvested at 48 hr pi to assess the ZIKV replication in WT and shHMGB1 Huh7 cells using FFA and immunoblot assays, respectively (Fig. 5). The FFA results showed that both mock-treated (6.65 ± 0.034 log10 FFU/mL, p < 0.001) and RESV-treated (6.49 ± 0.016 log10 FFU/mL, p < 0.001) shHMGB1 Huh7 cells had significantly higher ZIKV titers than the mock-treated (5.07 ± 0.028 log10 FFU/mL) and RESV-treated (2.21 ± 0.176 log10 FFU/mL) WT Huh7 cells (Fig. 5A). Interestingly, a significant reduction of approximately 99% (P < 0.01) in ZIKV titers was observed in RESV-treated WT Huh7 cells, but only a slight reduction of 30% (P > 0.05) in virus titers was observed in RESV-treated shHMGB1 cells (Fig. 5A), consistent with the immunoblotting results showing that RESV treatment resulted in a reduction of ZIKV NS2B protein expression only in WT Huh7 cells (Fig. 5B). These findings suggested that the depletion of HMGB1 in the cells diminishes RESV’s antiviral activity against ZIKV replication, suggesting the important role of HMGB1 in the antiviral mechanisms of RESV.

HMGB1 was involved in the antiviral mechanisms of RESV against ZIKV infection by inducing ISG and IFN-β.

The impact of RESV treatment on type-1 interferon (IFN) responses was examined to understand better the mechanisms behind the RESV's antiviral activity against ZIKV. The gene and protein expression levels of two well-known established type-1 IFN response genes, including human myxovirus resistance protein 1 (MxA) and interferon-beta (IFN-β) in both WT and shHMGB1 Huh7 cells were assessed by qRT-PCR and immunoblot assays at 48 hr pi (Fig. 6). Both MxA and IFN-β mRNA and protein expression levels were significantly higher in ZIKV-infected WT Huh7 cells than those without ZIKV infection (Fig. 6A, 6C and 6E). Treatment of ZIKV-infected WT Huh7 cells with RESV led to significantly higher MxA (p < 0.05) and IFN-β (p < 0.05) induction levels compared to the mock-treated infected cells (Fig. 6A, 6C and 6E), suggesting that RESV's antiviral action against ZIKV could be attributable to IFN induction and ISG activation.

The less effective RESV-mediated antiviral response in shHMGB1 cells prompted us to study the effects of HMGB1 on type-1 IFN response. Overall, MxA and IFN-β mRNA and protein expression levels in the ZIKV-infected shHMGB1 cells were lower than in the infected WT cells, in the mock-treated and RESV-treated groups (Fig. 6B and 6D). As shown in Fig. 6E, MxA and IFN-β protein expression levels in shHMGB1 cells were too low to be detected by the immunoblot assay. Treatment of infected shHMGB1 Huh7 cells with RESV had no influence on MxA or IFN-β expressions (Fig. 6B, 6D and 6E), suggesting that the antiviral action of RESV via type-1 IFN induction may depend on HMGB1.

RESV downregulated the expression of pro-inflammatory cytokines in ZIKV-infected cells.

The effects of RESV treatment on the expression of pro-inflammatory cytokines such as nuclear factor-kappa B (NF-κB) and interleukin-1β (IL-1β) were investigated at 48 hr pi using qRT-PCR and immunoblot assays (Fig. 7). In comparison to the cell control, ZIKV infection of WT Huh7 cells significantly elevated both NF-kB (p < 0.001) and IL-1β (p < 0.001) mRNA and protein expression levels (Fig. 7A, 7C and 7E). RESV treatment in WT Huh7 cells after ZIKV infection caused a significant downregulation of NF-kB (p < 0.05) and IL-1β (p < 0.001) expressions as compared to the mock-treated infected cells (Fig. 7A, 7C and 7E), suggesting that RESV treatment had anti-inflammatory effects on ZIKV-infected cells.

HMGB1's role in RESV's anti-inflammatory action was further investigated using shHMGB1 Huh7 cells. NF-κB and IL-1β mRNA expressions were lower in the ZIKV-infected shHMGB1 cells than in the infected WT cells in the mock-treated and RESV-treated groups (Fig. 7B and 7D). However, as illustrated in Fig. 7E, NF-κB and IL-1β protein expressions in shHMGB1 cells were almost undetectable. The RESV treatment, on the other hand, reduced the mRNA expression of NF-κB (p < 0.05) (Fig. 7B) in infected shHMGB1 cells but did not affect the expression of IL-1 (Fig. 7D).

Although RESV has recently been identified as an in vitro anti-ZIKV agent ^12,13, its antiviral mechanisms against ZIKV is still unclear. The present study’s findings reveal that the treatment of ZIKV-infected Huh7 cells with RESV significantly inhibits virus replication, HMGB1 nucleocytoplasmic translocation and the induction of inflammatory responses. The antiviral mechanisms of RESV against ZIKV was found from the study likely to be mediated through induction of IFN and activation of the ISG, both of which are regulated by HMGB1 nuclear retention (Fig. 8).

In addition to its anticancer, antioxidant, cardioprotective, and neuroprotective effects, RESV is also proposed as a potential antiviral agent against both human and animal viruses ^16,41,42. Results from the present study showed that the treatment with RESV significantly reduced the extracellular ZIKV particles in a dose-dependent manner at 48 and 72 hr pi but not at 24 hr pi. Similarly, immunoblotting results also showed a reduction of intracellular ZIKV NS2B protein synthesis after RESV treatment at 48 hr pi. These findings corroborate recent findings on the antiviral activities of RESV against ZIKV at 48 hr pi in Huh7 and human retinal pigment epithelial cells, which showed that post-infection treatments of 50 µM and 80 µM RESV reduced ZIKV titers by 75% and 93%, respectively ^12,13. RESV exerted antiviral effects after ZIKV infection, which was not attributable to cytotoxicity, suggesting that its antiviral actions are most likely by regulating host cell signaling pathways. Previous study suggested that RESV displayed potent antiviral activity against DENV, a flavivirus closely related to ZIKV, through regulating the localization of HMGB1 ²⁸. Hence, RESV’s inhibitory mechanism of action against ZIKV infection could be similar to that reported for DENV infection.

HMGB1 is one of the damage-associated molecular patterns (DAMPs) with distinct intracellular and extracellular functions ⁴³. Instead of accumulating in the nucleus to regulate gene transcription during homeostasis, infection may trigger HMGB1 to undergo post-translational modifications including acetylation, phosphorylation and methylation, causing it to translocate from the nucleus to the extracellular milieu ^44,45, where it acts as an initiator of inflammatory immune responses ²⁹. Our earlier study revealed that ZIKV infection can induce nuclear-to-cytoplasmic translocation and extracellular release of HMGB1 ³⁶, consistent with the findings in various virus infections ^30,46-48. In addition, extracellular HMGB1 binds to RAGE, TLR2 or TLR4 receptors, activating the MyD88 and NF-kB signaling pathways, which in turn triggers a ‘cytokine storm’ that aids in viral clearance ⁴⁹. Hence, extracellular HMGB1 could serve to protect neighboring cells from ZIKV infection, albeit more research is needed. Excessive extracellular HMGB1 during virus infection, on the other hand, has been linked to the pathogenesis of diseases such as dengue hemorrhagic fever or dengue shock syndrome (DHF/DSS), neuroinvasion and hepatocellular carcinoma metastasis ^50-53. Thus, preventing extracellular HMGB1 overproduction may be a viable option for developing therapeutic alternatives for viral diseases.

In addition to suppressing ZIKV replication, the present study discovered that treatment with RESV blocked ZIKV-induced HMGB1 translocation and extracellular release by retaining HMGB1 within the nucleus of infected cells. According to previous studies, RESV treatment promotes sirtuin1 (Sirt1), an NAD⁺-dependent histone deacetylase, which leads to HMGB1 deacetylation and prevents HMGB1 translocation out of the nucleus ^54,55. Moreover, the effects of RESV treatment on nuclear HMGB1 retention have also been shown to have a protective role against DENV infection by inducing innate immune responses ²⁸. RESV treatment may thereby restrict ZIKV replication through the impact of retaining HMGB1 in the nucleus and inhibiting its extracellular release.

Results from the present study showed that ZIKV replication was higher in HMGB1-knockdown (shHMGB1) Huh7 cells than in WT Huh7 cells, suggesting that HMGB1 depletion in the cells increased ZIKV replication. Furthermore, less effective antiviral effects were observed in RESV-treated shHMGB1 Huh7 cells, suggesting that RESV's antiviral activity depends on the presence of intracellular HMGB1. These findings are consistent with prior studies revealing the antiviral role of intracellular HMGB1 against DENV ²⁸, duck Tembusu, duck reovirus, duck plague virus ³⁵ and HIV ⁵⁶. For example, it was shown that overexpression of nuclear HMGB1 activates the IFN-I/IRF7 signaling pathway, increasing the transcriptional levels of PRRs, pro-inflammatory cytokines, IFNs, and ISGs to eliminate viruses ^28,35. Intracellular HMGB1 has also been found to interact directly or indirectly with viral and/or cellular proteins, such as restricting HIV replication via suppressing the viral long terminal repeat (LTR)-mediated transcription ⁵⁶, highlighting the important role of HMGB1. In contrast to hepatitis C and influenza viruses, intracellular HMGB1 promotes virus replication by directly binding to viral RNA or nucleoprotein ^33,34.

In the present study, ZIKV infection of WT Huh7 cells was found to elevate MxA and IFN-β expressions, consistent with the findings in other cell lines ^57-59. Upregulation of ISG and IFN by ZIKV infection alone, however, is likely insufficient to completely inhibit virus replication. Treatment with RESV significantly increased MxA and IFN-β induction levels in ZIKV-infected WT Huh7 cells, and then suggested that RESV’s antiviral effects against ZIKV could be mediated through IFN induction and ISG activation. Vice versa, both MxA and IFN-β expression levels were significantly lowered in infected shHMGB1 Huh7 cells with or without RESV treatment, suggesting that RESV's antiviral effects is dependent on HMGB1. These findings correlate with an earlier study demonstrating that nuclear HMGB1 accumulation induced by RESV treatment reduced DENV replication by binding to the MxA gene promoter, which increases MxA expression ²⁸. It has been established that type-1 IFN and ISGs such as MxA, viperin and OAS2 can effectively suppress ZIKV replication ^60-63. ISGs have been shown to directly inhibit multiple steps in the virus replication cycle and trigger a positive feedback regulation of the type-1 IFN ^64,65. Thus, the anti-ZIKV mechanisms of RESV is most likely mediated by HMGB1 nuclear retention, which enhances the production of type-1 IFN and ISGs.

Multiple organ inflammations have been linked to ZIKV infection, including hepatitis, enteritis, acute tubulitis and acute pancreatitis ^66-69. Our results showed that ZIKV infection increased the expression of pro-inflammatory cytokines such as NF-κB and IL-1β, similar to other published findings indicating that patients with ZIKV infections have higher levels of interferon and pro-inflammatory cytokines in their sera ^70-72. Besides its remarkable antiviral properties, RESV is a potent anti-inflammatory agent by suppressing the NF-κB and JAK/STAT signaling pathways ^73-75. Similarly, the treatment with RESV was found to suppress the ZIKV- induced inflammatory effects by markedly downregulating NF-κB and IL-1β expression. On the other hand, infected shHMGB1 Huh7 cells had significantly lower NF-κB and IL-1β expression than infected WT Huh7 cells in the mock- and RESV-treated groups. This is because HMGB1 is a well-characterized DAMP, as previously described ⁴³. Hence, RESV appears to exert anti-inflammatory effects against ZIKV infection, most likely by preventing the extracellular release of HMGB1 and decreasing the production of pro-inflammatory cytokines. However, the observed decrease in cytokine levels could also be due to RESV's inhibitory effects against ZIKV replication itself.

One drawback of RESV is its low oral bioavailability, caused by the rapid metabolization to glucuronides and sulfates in the liver system ^76-78. Numerous approaches have been proposed to increase this compound’s bioavailability, including using RESV-loaded nanostructured lipid carriers, zein-based nanoparticles and solid lipid nanoparticles ^79,80. Moreover, the present study was limited in that it only examined a single ZIKV strain in a single cell line. It is thus necessary to conduct both in vivo and human clinical investigations to determine RESV's antiviral and anti-inflammatory properties against ZIKV infection and assess the compound's safety, especially in pregnant women.

In conclusion, the present study demonstrates that treatment with RESV significantly inhibits ZIKV replication, HMGB1 nucleocytoplasmic translocation and inflammatory responses in infected Huh7 cells. RESV’s antiviral action against ZIKV is probably mediated by the HMGB1 nuclear retention, resulting in the induction of IFN and activation of ISG. This study sheds light on the possibility of RESV as an antiviral agent against ZIKV and a suitable candidate of therapeutic treatment of Zika through mechanisms mediated by HMGB1. Additional in vivo studies are required to determine the role of RESV and HMGB1 in the treatment of ZIKV infection.

Cell lines and virus. Human hepatocellular carcinoma cells (Huh7, JCRB 0403) were maintained in Roswell Park Memorial Institute 1640 medium (RPMI; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Bovogen, Australia) and L-glutamine (Gibco, NY, USA). The cells were incubated at 37°C with 5% CO2 and sub-cultured when they achieved 80% confluency. P6-740 strain of Asian Zika virus (ZIKV), archived at the Research & Education Tropical Infectious Diseases Centre (TIDREC) of the University of Kuala Lumpur in Malaysia (University of Malaysia), was used in this study. The virus was propagated and titrated using African Green monkey kidney cells (Vero, ATCC- CCL81) in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, NY, USA). Stable knockdown shHMGB1 Huh7 cell line generation. The short hairpin RNA (shRNA) method was employed to establish a stable knockdown shHMGB1 Huh7 cell line 81. The recombinant lentiviruses carrying shRNA plasmids targeting the human HMGB1 gene were procured from OriGene Technologies (TL316576V, Rockville, MD, USA). In the presence of 8 µg/ml of polybrene, a 50% confluent Huh7 cell monolayer was infected with the recombinant lentiviruses at an MOI of 20 and incubated at 37°C overnight. The culture media was then replaced with a selective puromycin-containing media after 24 hours with a final concentration of 1 μg/ml (Sigma-Aldrich, St. Louis, MO). Antiviral activity assay of RESV. Huh7 cells were seeded at 1 x 〖10〗^6 cells per well in a 6-well plate and incubated overnight at 37°C. The cells were infected with ZIKV at an MOI of 1 and incubated at 37°C with 5% CO2 for 1 hour after gently rocking for 20 minutes at room temperature to enhance virus adsorption 82. Following the infection, the cells were washed twice with serum-free RPMI to remove the unabsorbed virus. Subsequently, the cells were mock-treated or treated with resveratrol (RESV, R5010, St. Louis, MO, USA) at various concentrations (0 µM, 10 µM, 30 µM, 50 µM and 80 µM) in RPMI supplemented with 2% fetal bovine serum. Focus-forming assay (FFA). 200 µl of the infected cell culture supernatants were harvested at various time points (e.g., 24 hr pi, 48 hr pi and 72 hr pi) for virus titration using a focus-forming assay and expressed as Focus-Forming-Unit per ml (FFU/ml). The assay was performed as previously reported 40. Enzyme-linked immunosorbent assay (ELISA). The concentration of HMGB1 in cell culture supernatants was measured with a commercially available human HMGB1 ELISA kit (Cloud-Clone Corp, USA; cat. SEA399Hu 96 tests) according to the manufacturer’s protocol. Immunofluorescence assay (IFA). Huh7 cells were fixed by 4% paraformaldehyde for 20 min and then permeabilized with 0.1% Triton X-100 for 15 min. Fixed cells were rinsed with PBS and incubated overnight with primary antibodies, including rabbit anti-HMGB1 antibody (ab79823; 1: 1:500 dilution; Abcam, Cambridge, UK) and mouse anti-flavivirus group E antibody 4G2 (1: 1:500 dilution; Merck Millipore, Bedford, USA) at 4°C in PBS. Cells were then probed with secondary antibodies, including donkey anti-rabbit-IgG Alexa Fluor 594 (ab150076; 1: 1:500 dilution; Abcam, Cambridge, UK) and goat anti-mouse IgG Alexa Fluor 488 (ab150113; 1: 1:500 dilution; Abcam, Cambridge, UK) for 1 h at RT. 4′ 6′-diamino-2-phenylindole dihydrochloride (DAPI) at a dilution of 1:600 was used to stain the cell nuclei. Cells were visualized using a Nikon TE 2000 epifluorescence microscope. Immunoblot assay. Nuclear and cytosolic proteins were isolated from Huh7 cells using the NE-PER cytoplasmic and nuclear protein extraction kit (Thermo Scientific Pierce, Rockford, IL, USA) as indicated by the manufacturer. For total protein extraction, Huh7 cells were rinsed with cold PBS and lysed for 30 minutes at 4°C in RIPA lysis buffer (Merck Millipore, Bedford, USA) containing a protease inhibitor cocktail (Thermo Scientific Pierce, Rockford, IL, USA). Cellular debris was separated by centrifugation at 12,000 g for 20 min. Micro BCA Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL, USA) was utilized to quantify the protein concentration of each sample. Equal amounts of proteins were denatured with sodium dodecyl sulphate–sample loading buffer, separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride membrane for 14 minutes by the Bio-Rad semi-dry transfer system (Hercules, CA). Afterward, the membranes were blocked with 3% skimmed milk in PBS for 1 h at room temperature before being incubated with primary antibodies overnight at 4°C. The primary antibodies that were used in this study as follows: rabbit anti-HMGB1 antibody (ab79823; 1: 1:10000 dilution; Abcam, Cambridge, UK), rabbit anti-Zika virus NS2B antibody (GTX133308; 1:10000 dilution; GeneTex, San Antonio, TX), rabbit anti-MX1 antibody (ab95926; 1:500 dilution; Abcam, Cambridge, UK), rabbit anti-interferon-β antibody (ab85803, 1 µg/ml; Abcam, Cambridge, UK), rabbit anti-NF-Kb p65 antibody (ab16502; 0.5 µg/ml; Abcam, Cambridge, UK) and rabbit anti-IL-1β antibody (12703S; 1:1000 dilution; Cell Signaling Technology, Danvers, MA). Rabbit anti-GADPH antibody (ab9485, 1:2500; Abcam, Cambridge, UK) and rabbit anti-PARP1 antibody (PLA0184, 1:5000 dilution, Sigma-Aldrich, St. Louis, MO, USA) were used as the loading controls for all total cell lysates and nuclear protein extracts samples, respectively. Blots were then incubated for 1 h at room temperature with a secondary goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) (65-6120, 1:3,000-1:10,000; Invitrogen, Carlsbad, CA, USA). The chemiluminescence Western blotting kit (BioRad, Hercules, CA) was used to detect the proteins. Quantitative real-time analysis (qRT-PCR) Total RNA was extracted from Huh7 cells using RNeasy Kit (Qiagen, USA) following the manufacturer’s protocol. qRT-PCR was performed using the SensiFAST SYBR Hi-Rox One-Step PCR kit (Bioline, UK). The primers used in this study are listed in Supplementary Table S1. Each 15 µl reaction mixture contained 400 nM forward primer, 400 nM reverse primer, 1x the SensiFAST SYBR Hi-Rox One-Step master mix, reverse transcriptase, RiboSafe RNase inhibitor and nuclease-free water. Thermal cycling conditions were as follows: 10 min of reverse transcription at 45°C, 2 min of polymerase activation at 95 °C, followed by 40 cycles of 5 sec of denaturation at 95°C and 10 sec of annealing at 60 °C and 5 sec of elongation at 72°C. The mRNA expressions were shown in fold induction and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used as a housekeeping gene. All samples were analyzed in duplicate. Statistical analysis. The statistically significant differences between the cell groups were accessed by employing one-way or two-way analysis of variance of variance (ANOVA) followed Bonferroni's multiple-comparison test with a 95% confidence interval (C.I.) to determine the significance of the variance. Graph-Pad Prism 8 was used to perform all statistical tests. The statistical significances of the groups were as follows: *P<0.05, **P<0.01, ***P<0.001.

Acknowledgements

This study was supported in parts by the Ministry of Education Malaysia Higher Institution Centre of Excellence (HICoE) Program (Project MO002-2019), the Long Term Research Grant Scheme (LRGS MRUN Phase 1: LRGS MRUN/F1/01/2018), and the Fundamental Research Grant Scheme (FRGS FP015-2019A).

Author Contributions

K.L.C., N.Z., S.A.B. and S.S.S. involved in the study and experimental design. K.L.C and N.Z. conducted the experiments and analyzed the results. K.L.C., N.Z., S.A.B. and S.S.S. wrote, reviewed and edited the manuscript. All the authors have reviewed the manuscript.

Additional Information

Competing interests: The authors declare no competing interests.

Data Availability

All data generated or analysed during this study are included in this published article [and its supplementary information files]

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No competing interests reported.

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The antiviral action of resveratrol against Zika virus through the regulation of interferon responses by HMGB1

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