Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase

Abstract

Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection.IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used to treat HEV cases, there are known side effects and limitations of such therapy. Our discovery of the ability of zinc salts to block HEV replication by virtue of their ability to inhibit the activity of viral RdRp is important because these findings pave the way to test the efficacy of zinc supplementation therapy in HEV-infected patients. Since zinc supplementation therapy is known to be safe in healthy individuals and since high-dose zinc is used in the treatment of Wilson's disease, it may be possible to control HEV-associated health problems following a similar treatment regimen.

Keywords: hepatitis E virus.

FIG 1

Zinc salts inhibit the activity of g-1 HEV-EGFP replicon. (A) qRT-PCR detection of the HEV sense-strand RNA level in Huh7 cells expressing in vitro-synthesized capped RNA of a g-1 HEV-EGFP replicon treated with different salts, as indicated. Values were normalized to the level of GAPDH and are represented as means ± SEM of triplicate samples. (B) qRT-PCR detection of the HEV antisense-strand RNA level in the samples described in panel A. Values were normalized to the level of GAPDH and are represented as means ± SEM of triplicate samples. (C) MTT assay-mediated cell viability estimation of aliquots of the samples described in panel A. The value for an untreated (no salt) sample was considered to be 100%, and all other values were calculated with reference to that. Values are means ± SEM of triplicate samples. (D) Silver staining of total protein of whole-cell extract prepared from Huh7 cells treated with different salts for 48 h, as indicated. (E) MTT assay-mediated cell viability estimation of Huh7 cells treated for 24 h with increasing concentrations of Zn sulfate and Zn acetate, as indicated. The value for the untreated (no salt) sample was considered to be 100%, and all other values were calculated with reference to that. Values are means ± SEM of triplicate samples. (F) Detection of EGFP (green) and 4′,6′-diamidino-2-phenylindole (nucleus, blue) in Huh7 cells expressing in vitro-synthesized capped genomic RNA of g-1 HEV-EGFP replicon, treated with different salts, as indicated. Untransfected and no-salt cells refer to Huh7 cells lacking g-1 HEV-EGFP and g-1 HEV-EGFP-containing Huh7 cells without any additional salt treatment, respectively.

FIG 2

Inhibition of a g-3 HEV replicon and infectious g-1 HEV replication by zinc salts. (A) Measurement of Renilla luciferase activity in Huh7 cells expressing in vitro-synthesized capped RNA of a P6 HEV-Luc replicon, treated for 48 h with different salts, as indicated. Renilla luciferase values were normalized to the value of the cell viability assay and are represented as means ± SEM of triplicate samples. Mock treatment represents cells transfected with the transfection reagent only (without RNA). (B) qRT-PCR detection of HEV sense and antisense RNA levels in Huh7 cells expressing in vitro-synthesized capped genomic RNA of g-1 HEV or its replication-defective mutant (GAA HEV) and treated with different salts, as indicated. Values were normalized to the value of GAPDH and are represented as means ± SEM of triplicate samples. Mock treatment represents cells transfected with the transfection reagent only (without RNA). (C) Immunofluorescence detection of ORF2 (green) and nucleus (blue) in Huh7 cells expressing in vitro-synthesized capped genomic RNA of g-1 HEV treated with different salts, as indicated. Untransfected and no-salt cells represent Huh7 cells lacking the g-1 HEV genome and g-1 HEV genome-containing Huh7 cells without any additional salt treatment, respectively. (D) Quantitation of ORF2-positive cells in 12 random fields of the immunofluorescence slides represented in panel C. The percentage of green fluorescent cells with reference to blue fluorescent cells (total nuclear staining) is shown. Values are means ± SEM of two experiments.

FIG 3

Repeat treatment with zinc salts at 24-h intervals more effectively inhibits HEV replication. (A) Huh7 cells were transfected with in vitro-synthesized capped genomic RNA of g-1 HEV. At day 6 posttransfection, cells were treated once (1×) or twice (2×; treatment repeated at a 24-h interval) with the indicated concentrations of different salts. At 24 and 48 h posttreatment, total RNA was isolated from the cells, followed by qRT-PCR measurement of viral sense, antisense, and host GAPDH RNA levels. GAPDH-normalized sense (s) and antisense (as) RNA levels are represented as means ± SEM of triplicate samples. (B) Simultaneously processed samples (described for panel A) were used to measure cell viability by MTT assay. The value for the untreated (no salt) sample was considered to be 100%, and all other values were calculated with reference to that. Values are means ± SEM of triplicate samples.

FIG 4

Comparison of the replication efficiency of a g-1 HEV clinical isolate in Huh7 and ORF4-Huh7 cells. qRT-PCR detection of the HEV sense-strand RNA level in Huh7 or ORF4-Huh7 cells infected with a g-1 HEV clinical isolate. Total RNA was isolated at different days postinfection, followed by estimation of HEV sense and GAPDH RNA levels. The ratios of HEV sense/GAPDH values are represented as means ± SEM.

FIG 5

Zinc salts inhibit the replication of a g-1 HEV clinical isolate. (A) MTT assay-mediated estimation of cell viability in Huh7 cells infected with a g-1 HEV clinical isolate and treated with different salts, as indicated. The value for the untreated (no salt) sample was considered to be 100%, and all other values were calculated with reference to that. Values are means ± SEM of the triplicate samples. (B) MTT assay-mediated estimation of cell viability in ORF4-Huh7 cells infected with a g-1 HEV clinical isolate treated with different salts, as indicated. The value for the untreated (no salt) sample was considered to be 100%, and all other values were calculated with reference to that. Values are means ± SEM of the triplicate samples. (C) qRT-PCR detection of the HEV sense-strand RNA level in ORF4-Huh7 cells infected with a g-1 HEV clinical isolate treated with different salts, as indicated. On days 8 and 9 postinfection, 48- and 24-h treatments, respectively, were started, and all samples were processed for RNA isolation on the 10th day. HEV sense RNA values were normalized to the value for GAPDH and are represented as means ± SEM of triplicate samples. (D) qRT-PCR detection of the HEV antisense-strand RNA level in ORF4-Huh7 cells infected with a g-1 HEV clinical isolate treated with different salts, as indicated. Treatment was done as described for panel B, and values were normalized to the value of GAPDH and are represented as means ± SEM of triplicate samples. (E) qRT-PCR detection of the HEV sense-strand RNA level in HEV particles recovered from the medium of ORF4-Huh7 cells infected with a g-1 HEV clinical isolate, followed by treatment with different salts, as indicated. Treatment was done as described for panel B, and 10th-day medium from each plate was used for PEG precipitation. No amplification was observed in samples treated with 100 μM Zn acetate (denoted by *).

FIG 6

Zinc salts act by inhibiting the activity of HEV RNA-dependent RNA polymerase. (A) qRT-PCR detection of the HEV sense RNA level in ORF4-Huh7 cells infected for 1 h with a g-1 HEV clinical isolate. Cells were treated with the indicated salts at 15 min prior to the infection and maintained throughout the HEV infection period. HEV sense RNA values were normalized to the value of GAPDH and are represented as means ± SEM of triplicate samples. (B) Immunofluorescence detection of ORF2 (green) and nucleus (blue) in ORF4-Huh7 cells infected and treated with different zinc salts as described for panel A. (C) ORF4-Huh7 cells were pretreated with the Mg acetate or Zn acetate for 10 h, followed by infection with a g-1 HEV clinical isolate for 1 h. Infected cells were maintained in the presence of Mg acetate or Zn acetate for 3 days. On day 4, total RNA was isolated from the cells, followed by qRT-PCR detection of HEV sense and host GAPDH RNA levels. HEV sense RNA values were normalized to the level of GAPDH and are represented as means ± SEM of triplicate samples. (D) MTT assay-mediated cell viability estimation of aliquots of the samples described in panel C. The value for the untreated (no salt) sample was considered to be 100%, and all other values were calculated with reference to that. Values are means ± SEM of triplicate samples. (E) HEV RdRp assay in the presence of different zinc and magnesium salts, as indicated. Different salts were added to the reaction mixture containing RdRp protein and template RNA and maintained throughout the incubation period. Mock and RdRp (−NTPs) denote reaction mixtures devoid of RdRp protein and nucleoside triphosphates (ATP, CTP, GTP, and UTP), respectively. (F) HEV RdRp assay in the presence of increasing amounts of zinc salts, as indicated. Salt treatment was done as described for panel E.

FIG 7

Cotreatment with Zn sulfate and ribavirin marginally increases the inhibitory effect of either of them on HEV replication. Renilla luciferase activity was measured in Huh7 cells expressing in vitro-synthesized capped RNA of the P6 HEV-Luc replicon, treated every day for 3 days with 100 μM Zn sulfate and/or 10, 50 μM ribavirin, as indicated. Luciferase values were normalized to the value of the cell viability assay (detected by MTT assay) and are represented as means ± SEM of triplicate samples.