Mutational Analysis of the Hypervariable Region of Hepatitis E Virus Reveals Its Involvement in the Efficiency of Viral RNA Replication

ABSTRACT Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3′-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3′-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3′-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3′ termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3′ terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3′-terminal mutations in vivo. Incorporation of the 3′-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.

Download Full-text

Targeted cleavage of hepatitis E virus 3′ end RNA mediated by hammerhead ribozymes inhibits viral RNA replication

Virology ◽

10.1016/s0042-6822(03)00259-9 ◽

2003 ◽

Vol 312 (2) ◽

pp. 350-358 ◽

Cited By ~ 11

Author(s):

Bandi Sriram ◽

Deepshi Thakral ◽

Subrat Kumar Panda

Keyword(s):

Hepatitis E Virus ◽

Rna Replication ◽

Hepatitis E ◽

Viral Rna ◽

Hammerhead Ribozymes

Download Full-text

Hepatitis E Virus RNA‐Dependent RNA Polymerase is Involved in RNA Replication and Infectious Particle Production

Hepatology ◽

10.1002/hep.32100 ◽

2021 ◽

Author(s):

Noémie Oechslin ◽

Nathalie Da Silva ◽

Dagmara Szkolnicka ◽

François‐Xavier Cantrelle ◽

Xavier Hanoulle ◽

...

Keyword(s):

Rna Polymerase ◽

Hepatitis E Virus ◽

Particle Production ◽

Rna Replication ◽

Hepatitis E ◽

Rna Dependent Rna Polymerase ◽

Infectious Particle ◽

Virus Rna

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.00754-17 ◽

2017 ◽

Vol 91 (21) ◽

Cited By ~ 36

Author(s):

Nidhi Kaushik ◽

Chandru Subramani ◽

Saumya Anang ◽

Rajagopalan Muthumohan ◽

Shalimar ◽

...

Keyword(s):

Rna Polymerase ◽

Hepatitis E Virus ◽

Zinc Supplementation ◽

Hepatitis E ◽

Viral Rna ◽

Health Concern ◽

Healthy Individuals ◽

Genomic Rna ◽

Rna Dependent Rna Polymerase ◽

Zinc Salts

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.

Download Full-text

Hepatitis E Virus (HEV) Open Reading Frame 2 Antigen Kinetics in Human-Liver Chimeric Mice and Its Impact on HEV Diagnosis

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz171 ◽

2019 ◽

Vol 220 (5) ◽

pp. 811-819 ◽

Cited By ~ 8

Author(s):

Ibrahim M Sayed ◽

Lieven Verhoye ◽

Claire Montpellier ◽

Florence Abravanel ◽

Jacques Izopet ◽

...

Keyword(s):

Density Gradient ◽

Hepatitis E Virus ◽

Gradient Analysis ◽

Hepatitis E ◽

Open Reading Frame ◽

Viral Rna ◽

Humanized Mice ◽

Molecular Diagnostic ◽

Reading Frame ◽

Open Reading Frame 2

Abstract Background Hepatitis E virus infection (HEV) is an emerging problem in developed countries. Diagnosis of HEV infection is based on the detection of HEV-specific antibodies, viral RNA, and/or antigen (Ag). Humanized mice were previously reported as a model for the study of HEV infection, but published data were focused on the quantification of viral RNA. However, the kinetics of HEV Ag expression during infection remains poorly understood. Methods Plasma specimens and suspensions of fecal specimens from HEV-infected and ribavirin-treated humanized mice were analyzed using HEV antigen–specific enzyme-linked immunosorbent assay, reverse transcription–quantitative polymerase chain reaction analysis, density gradient analysis, and Western blotting. Result Open reading frame 2 (ORF2) Ag was detected in both plasma and stool from HEV-infected mice, and levels increased over time. Contrary to HEV RNA, ORF2 Ag levels were higher in mouse plasma than in stool. Interestingly, ORF2 was detected in plasma from mice that tested negative for HEV RNA in plasma but positive for HEV RNA in stool and was detected after viral clearance in mice that were treated with ribavirin. Plasma density gradient analysis revealed the presence of the noninfectious glycosylated form of ORF2. Conclusion ORF2 Ag can be used as a marker of active HEV infection and for assessment of the effect of antiviral therapy, especially when fecal samples are not available or molecular diagnostic tests are not accessible.

Download Full-text

5′ cis Elements Direct Nodavirus RNA1 Recruitment to Mitochondrial Sites of Replication Complex Formation

Journal of Virology ◽

10.1128/jvi.02040-08 ◽

2009 ◽

Vol 83 (7) ◽

pp. 2976-2988 ◽

Cited By ~ 24

Author(s):

Priscilla M. Van Wynsberghe ◽

Paul Ahlquist

Keyword(s):

Complex Formation ◽

Mutational Analysis ◽

Protein A ◽

Rna Replication ◽

Viral Rna ◽

Yeast Cells ◽

Positive Strand ◽

Replication Complex ◽

Negative Strand ◽

Positive Strand Rna

ABSTRACT Positive-strand RNA viruses replicate their genomes on intracellular membranes, usually in conjunction with virus-induced membrane rearrangements. For the nodavirus flock house virus (FHV), we recently showed that multifunctional FHV replicase protein A induces viral RNA template recruitment to a membrane-associated state, but the site(s) and function of this recruitment were not determined. By tagging viral RNA with green fluorescent protein, we show here in Drosophila cells that protein A recruits FHV RNA specifically to the outer mitochondrial membrane sites of RNA replication complex formation. Using Drosophila cells and yeast cells, which also support FHV replication, we also defined the cis-acting regions that direct replication and template recruitment for FHV genomic RNA1. RNA1 nucleotides 68 to 205 were required for RNA replication and directed efficient protein A-mediated RNA recruitment in both cell types. RNA secondary structure prediction, structure probing, and phylogenetic comparisons in this region identified two stable, conserved stem-loops with nearly identical loop sequences. Further mutational analysis showed that both stem-loops and certain flanking sequences were required for RNA1 recruitment, negative-strand synthesis, and subsequent positive-strand amplification in yeast and Drosophila cells. Thus, we have shown that protein A recruits RNA1 templates to mitochondria, as expected for RNA replication, and identified a new RNA1 cis element that is necessary and sufficient for RNA1 template recognition and recruitment to these mitochondrial membranes for negative-strand RNA1 synthesis. These results establish RNA recruitment to the sites of replication complex formation as an essential, distinct, and selective early step in nodavirus replication.

Download Full-text

A naturally occurring human/hepatitis E recombinant virus predominates in serum but not in faeces of a chronic hepatitis E patient and has a growth advantage in cell culture

Journal of General Virology ◽

10.1099/vir.0.037259-0 ◽

2012 ◽

Vol 93 (3) ◽

pp. 526-530 ◽

Cited By ~ 56

Author(s):

H. T. Nguyen ◽

U. Torian ◽

K. Faulk ◽

K. Mather ◽

R. E. Engle ◽

...

Keyword(s):

Hepatitis E Virus ◽

Cultured Cells ◽

Hypervariable Region ◽

Hepatitis E ◽

Structural Protein ◽

Chronic Infections ◽

Virus Growth ◽

Acute Hepatitis E ◽

A Minor ◽

Virus Genomes

Hepatitis E virus is the aetiological agent of acute hepatitis E, a self-limiting disease prevalent in developing countries. Molecular analysis of viral genomic RNA from a chronically infected patient confirmed the recent discovery that chronic infection correlated with extensive diversification of the virus quasispecies: the hypervariable region of some virus genomes in this USA patient contained large continuous deletions and a minor proportion of genomes in faeces and serum had acquired a mammalian sequence that encoded 39 aa of S19 ribosomal protein fused to the virus non-structural protein. Genomes with this insert were selected during virus passage in cultured cells to become the predominant species, suggesting that the inserted sequence promoted virus growth. The results demonstrated that hepatitis E virus can mutate dramatically during a prolonged infection and suggests it may be important to prevent or cure chronic infections before new variants with unpredictable properties arise.

Download Full-text