Viral Replication: Learning more about hepatitis E virus
A domain in the ORF1 polyprotein of the hepatitis E virus that was previously thought to be a protease is actually a zinc-binding domain.
- Structural Virology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States
Hepatitis E virus (HEV) is a single-stranded, positive-sense RNA virus that is spread by fecal-oral transmission. Although infection is usually self-limiting, it can result in death via acute liver failure. The World Health Organization estimates that HEV causes 20 million infections and 44,000 deaths per year, particularly among expectant mothers (WHO, 2022). The genome of the HEV contains three open reading frames that produce: (i) an enzyme that helps the virus to replicate itself; (ii) a capsid protein for the protein shell that surrounds the newly replicated viruses; (iii) a viroporin that helps the new viruses to escape from cells that have already been infected so that they can infect other cells.
In HEV, translation of the first open reading frame (ORF1) produces a polyprotein that contains seven domains. Multi-domain polyproteins are also made by other viruses, including HIV, hepatitis C virus, Chikungunya, Dengue, SARS coronavirus, rubella, influenza, and polio. In most other viral families, this polyprotein is then cleaved into individual proteins by enzymes called proteases that derive from the virus or its host (Yost and Marcotrigiano, 2013). Although the domain organization of the HEV ORF1 polyprotein is similar to other viruses (Figure 1), it is not clear if ORF1 undergoes cleavage. Previous studies have suggested that ORF1 contains a domain that acts as a protease, with a cysteine residue (Cys483) and a histidine residue (His590) acting as the catalytic sites. However, while Cys483 is highly conserved, His590 is not, and there is little evidence that this domain (which is called a putative papain-like cysteine protease, or pPCP for short) operates as a protease.
Figure 1
Comparing four RNA viruses.
The seven domains of the ORF1 polyprotein for the hepatitis E virus (HEV; top) are shown schematically and compared to polyproteins from rubella, Chikungunya (CHIKV), and hepatitis C virus (HCV). All four viruses contain a helicase enzyme (Hel) and an RNA polymerase enzyme (RdRp). Rubella, CHIKV and HCV contain proteases, but LeDesma et al. have shown that the PCP domain in HEV that was previously thought to be a protease is a zinc-binding domain. The locations of the zinc-binding motifs are represented by coloured spheres: green for 6Cys (HEV); orange for HisGluHis (HEV); blue for 3Cys1His (Rubella and HCV), yellow for 4Cys (CHIKV and HCV). MeT: methyltransferase; Y: Y-domain; PCP: papain-like cysteine protease; HVR: hypervariable region; X: macro-domain; AUD: alphavirus unique domain; NS/nsP: non-structural protein.
Now, in eLife, Alexander Ploss and colleagues at Princeton University – including Robert LeDesma as first author – report the results of experiments which shed light on the role of the pPCP domain (LeDesma et al., 2023). Their results indicate that this domain – while necessary for replication of the virus – is not a protease, but rather a structural organization and localization domain. Moreover, they also show that Cys483 facilitates zinc binding rather than being a catalytic site for a protease.
If the pPCP domain were a protease, LeDesma et al. hypothesized that it would be possible to rescue protease-defective mutants by expressing pPCP in trans, so they generated cell lines that expressed either the wild-type ORF1 polyprotein, two mutant ORF1 polyproteins (called C483A and Pol(–)), or the wild-type pPCP domain alone. The next step was to transfect each of these cell lines with a reporter RNA in which ORF1 was either wild type or one of the mutants. Their results suggest that the pPCP domain is either not a protease or not proteolytically active in isolation.
The researchers then turned their attention to the residue Cys483. If this residue were part of a protease catalytic site then it, and no other cysteines in the pPCP domain, would support replicase activity. However, alanine and triple-alanine mutation indicated that six of the eight cysteines in the PCP domain are critical for replicase activity.
Since there is no protease, they investigated what the pPCP domain and the residue Cys483 might do. LeDesma et al. noticed that a six-cysteine motif within the domain was similar to other proteins that may bind bivalent metal cations. Using inductively coupled plasma mass spectrometry and confocal microscopy, the researchers observed that the mutation C483A reduced the ability of the domain to bind zinc ions, and also resulted in ORF1 being unable to localize in the nucleus.
Like all the best science, this work raises more questions than it answers. Zinc-binding domains with unique folds have been identified in a number of positive-sense RNA viruses (Shin et al., 2012; Tellinghuisen et al., 2004; Tellinghuisen et al., 2005; see coloured circles in Figure 1), and if the six cysteines of the pPCP domain bind zinc, the structure will be novel. A transcriptional activator in yeast called Gal4 is the foundational example of a six-cysteine, zinc-binding motif (Hong et al., 2008), but the six-cysteine pattern of the pPCP domain does not align well with the sequence or structure of Gal4, which again suggests a novel structure.
In Chikungunya, a viral protease digests the polyprotein to generate a functional replication complex (Tan et al., 2022). In the absence of a protease, how is this achieved in HEV? Zinc-binding domains often function as dimers or as repeat domains. Does pPCP structurally organize the other domains within a single copy of the ORF1 polyprotein, or does it organize multiple ORF1s? Many zinc-binding domains bind double-stranded nucleic acids, and the six-cysteine region in pPCP has several basic residues that could facilitate this.
Given the significant effect that HEV infection has on human health, more information about ORF1 domain organization and function can assist in the development of drugs to combat disease.
References
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Molecular architecture of the Chikungunya virus replication complexScience Advances 8:eadd2536.https://doi.org/10.1126/sciadv.add2536
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The NS5A protein of hepatitis C virus is a zinc metalloproteinThe Journal of Biological Chemistry 279:48576–48587.https://doi.org/10.1074/jbc.M407787200
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Viral precursor polyproteins: keys of regulation from replication to maturationCurrent Opinion in Virology 3:137–142.https://doi.org/10.1016/j.coviro.2013.03.009
Article and author information
Author details
Altaira D Dearborn
Joseph Marcotrigiano
Publication history
- Version of Record published: March 22, 2023 (version 1)
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This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
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Viral Replication: Learning more about hepatitis E virus
eLife 12:e87047.
https://doi.org/10.7554/eLife.87047
Further reading
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- Medicine
- Microbiology and Infectious Disease
Background:
End-stage renal disease (ESRD) patients experience immune compromise characterized by complex alterations of both innate and adaptive immunity, and results in higher susceptibility to infection and lower response to vaccination. This immune compromise, coupled with greater risk of exposure to infectious disease at hemodialysis (HD) centers, underscores the need for examination of the immune response to the COVID-19 mRNA-based vaccines.
Methods:
The immune response to the COVID-19 BNT162b2 mRNA vaccine was assessed in 20 HD patients and cohort-matched controls. RNA sequencing of peripheral blood mononuclear cells was performed longitudinally before and after each vaccination dose for a total of six time points per subject. Anti-spike antibody levels were quantified prior to the first vaccination dose (V1D0) and 7 d after the second dose (V2D7) using anti-spike IgG titers and antibody neutralization assays. Anti-spike IgG titers were additionally quantified 6 mo after initial vaccination. Clinical history and lab values in HD patients were obtained to identify predictors of vaccination response.
Results:
Transcriptomic analyses demonstrated differing time courses of immune responses, with prolonged myeloid cell activity in HD at 1 wk after the first vaccination dose. HD also demonstrated decreased metabolic activity and decreased antigen presentation compared to controls after the second vaccination dose. Anti-spike IgG titers and neutralizing function were substantially elevated in both controls and HD at V2D7, with a small but significant reduction in titers in HD groups (p<0.05). Anti-spike IgG remained elevated above baseline at 6 mo in both subject groups. Anti-spike IgG titers at V2D7 were highly predictive of 6-month titer levels. Transcriptomic biomarkers after the second vaccination dose and clinical biomarkers including ferritin levels were found to be predictive of antibody development.
Conclusions:
Overall, we demonstrate differing time courses of immune responses to the BTN162b2 mRNA COVID-19 vaccination in maintenance HD subjects comparable to healthy controls and identify transcriptomic and clinical predictors of anti-spike IgG titers in HD. Analyzing vaccination as an in vivo perturbation, our results warrant further characterization of the immune dysregulation of ESRD.
Funding:
F30HD102093, F30HL151182, T32HL144909, R01HL138628. This research has been funded by the University of Illinois at Chicago Center for Clinical and Translational Science (CCTS) award UL1TR002003.
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- Microbiology and Infectious Disease
- Structural Biology and Molecular Biophysics
African trypanosomes replicate within infected mammals where they are exposed to the complement system. This system centres around complement C3, which is present in a soluble form in serum but becomes covalently deposited onto the surfaces of pathogens after proteolytic cleavage to C3b. Membrane-associated C3b triggers different complement-mediated effectors which promote pathogen clearance. To counter complement-mediated clearance, African trypanosomes have a cell surface receptor, ISG65, which binds to C3b and which decreases the rate of trypanosome clearance in an infection model. However, the mechanism by which ISG65 reduces C3b function has not been determined. We reveal through cryogenic electron microscopy that ISG65 has two distinct binding sites for C3b, only one of which is available in C3 and C3d. We show that ISG65 does not block the formation of C3b or the function of the C3 convertase which catalyses the surface deposition of C3b. However, we show that ISG65 forms a specific conjugate with C3b, perhaps acting as a decoy. ISG65 also occludes the binding sites for complement receptors 2 and 3, which may disrupt recruitment of immune cells, including B cells, phagocytes, and granulocytes. This suggests that ISG65 protects trypanosomes by combining multiple approaches to dampen the complement cascade.
