Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication
- Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, United States
- Department of Geosciences, Princeton University, United States
Figures
Figure 1 with 1 supplement
Mutations within the hepatitis E virus (HEV) putative protease domain render the virus replication incompetent.
(a) Genome organization of HEV and Kernow strain genotype 3 reporter replicon. ORF2 and ORF3 were replaced by a Gaussia Luciferase reporter in frame with the subgenomic promoter and translation …
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Figure 1—source data 1
Mutations within the hepatitis E virus (HEV) putative protease domain render the virus replication incompetent.
Spreadsheet of raw Gaussia luciferase (GLuc) data kinetics for (b–c) point and double mutant bearing replicons, (d) Kc1/p6 genotype 3 HEV replicons, (e) SHEV3 genotype 3 replicons, (f) SAR55 genotype 1 HEV replicons, (g) Tw6196E genotype 4 replicons, and (h) alternate codons of Kc1/p6 genotype 3 HEV replicons.
- https://cdn.elifesciences.org/articles/80529/elife-80529-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Alignment of one or more representative strain(s) from each known hepatitis E virus (HEV) genotype putative papain-like cysteine protease (PCP) domain with rubella virus (RUBV) protease reveals highly conserved octa-cysteine motif in HEV.
Black – HEV putative PCP. Brown – upstream flanking 32 AAs – downstream flanking 8 AAs. (*) Catalytic dyad of RUBV protease at putative catalytic dyad positions within HEV putative PCP domain. Bold …
Figure 2
Hepatitis E virus (HEV) ORF1 putative papain-like cysteine protease (pPCP) cannot function outside of the context of the full-length protein, and C483A replication deficiency can be rescued in trans.
(a) Schematic of the transcomplementation assay. HepG2C3A human hepatoma cells were transduced with lentivirus expressing HEV ORF1 wt, Pol (-), C483A, or pPCP only (ORF1 AAs 433–592 in Kc1/p6) and …
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Figure 2—source data 1
Hepatitis E virus (HEV) ORF1 putative papain-like cysteine protease (pPCP) cannot function outside of the context of the full-length protein, and C483A replication deficiency can be rescued in trans.
Spreadsheet of raw Gaussia luciferase (GLuc) data kinetics for transduced HepG2C3A cells (mock, ORF1 WT, ORF1 Pol [-], ORF1 C483A, and HEV pPCP domain) transfected with HEV Kc1/p6 replicons (WT, Pol [-], and C483A). Each sheet corresponds to different transduction condition.
- https://cdn.elifesciences.org/articles/80529/elife-80529-fig2-data1-v1.xlsx
Figure 3 with 1 supplement
Point mutations of highly conserved cysteines and alanine scanning mutagenesis within putative papain-like cysteine protease (pPCP) identifies residues and regions indispensable for viral replication.
(a) Partial sequence of Kc1/p6 hepatitis E virus (HEV) ORF1 including pPCP that shows eight cysteines that are highly conserved across all eight known HEV genotypes. Orange – upstream 40 amino acids …
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Figure 3—source data 1
Point mutations of highly conserved cysteines and alanine scanning mutagenesis within putative papain-like cysteine protease (pPCP) identifies residues and regions indispensable for viral replication.
Spreadsheet of raw Gaussia luciferase (GLuc) data kinetics for Kc1/p6 conserved cysteine point mutant replicons and alanine scanning mutagenized triplets.
- https://cdn.elifesciences.org/articles/80529/elife-80529-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Bioinformatic analysis (PROSITE) predicts CxC[X11]CC[X8]CxC motif (CxC motif) to be necessary for divalent metal ion coordination.
(a) Bioinformatic pipeline depicting hexa-cysteine motif search across all deposited proteins in UniProt and TrEMBLE databases. Exact hepatitis E virus (HEV) motif search yielded 25 protein hits, …
Figure 4 with 2 supplements
Structural prediction models of hepatitis E virus (HEV) putative papain-like cysteine protease (pPCP) demonstrates low-confidence scores, suggesting lack of highly ordered secondary structure.
(a) HEV ORF1-WT AlphaFold structure prediction. (b) HEV ORF1-WT AlphaFold structure prediction pseudo-colored by pLDDT score gradation (darker blue – higher pLDDT Score, darker red – lower pLDDT …
Figure 4—figure supplement 1
AlphaFold predicts structured domains of hepatitis E virus (HEV) ORF1 and viral protease of hepatitis A virus (HAV) with high confidence.
(a–c) Superimposition of AlphaFold (A.F.) prediction of corresponding Kernow C1/p6 ORF1 regions (Bronze) plus distance plot in (Å) and pLDDT score of A.F. prediction with: (a) the crystal structure …
Figure 4—figure supplement 2
Sequence alignment of Kc1/p6 macrodomain with macrodomains of Sindbis virus (SINV) and Chikungunya virus (CHIKV).
Alignment of ORF1 macrodomain (AAs 857–1000) with SINV macrodomain (AAs 1342–1,509 of PDB: 4GUA; Shin et al., 2012) and CHIKV macrodomain (PDB: 3DPG; Malet et al., 2009). Dash – gap in alignment. …
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Figure 4—figure supplement 2—source data 1
Similarity and identity percentages of Kc1/p6 macrodomain with macrodomains of Sindbis virus (SINV) and Chikungunya virus (CHIKV).
Left: percent similarity and identity with SINV macrodomain (AAs 1342–1,509 of PDB: 4GUA; Shin et al., 2012). Right: percent similarity and identity with CHIKV macrodomain (PDB: 3DPG; Malet et al., 2009).
- https://cdn.elifesciences.org/articles/80529/elife-80529-fig4-figsupp2-data1-v1.xlsx
Figure 5 with 1 supplement
Structural prediction models suggest conserved cysteines within CxC[x11]CC[x8]CxC motif form divalent ion coordination pockets and novel domain-domain interaction with upstream Y-domain.
(a) Alphafold structural predictions of domains within hepatitis E virus (HEV) ORF1. Left: pseudo zinc-finger (amino acids 451–493 within putative papain-like cysteine protease [pPCP]). Magenta: …
Figure 5—figure supplement 1
Replication end point analysis of WT-HA, D248A, and H249A Gaussia luciferase (Gluc) constructs.
HepG2C3A cells were transfected with in vitro transcribed RNA of WT, polymerase deficient, WT-HA-tagged, or point mutants in one of the upstream interacting Y-domain (D248A and H249A). Cell culture …
Figure 6 with 1 supplement
Inductively coupled plasma mass spectrometry (ICP-MS) shows divalent cation coordination by hepatitis E virus (HEV) ORF1.
(a) HA-epitope tag flanked by linker sequence inserted into hypervariable region (HVR) of HEV ORF1 lentiviral construct. (b) Western blot analysis of HUH7 human hepatoma cell produced epitope tagged …
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Figure 6—source data 1
Inductively coupled plasma mass spectrometry (ICP-MS) shows bivalent cation coordination by hepatitis E virus (HEV) ORF1.
- https://cdn.elifesciences.org/articles/80529/elife-80529-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Inductively coupled plasma mass spectrometry (ICP-MS) shows divalent cation coordination by hepatitis E virus (HEV) ORF1 disrutped when mutated at residue C483.
Figure 7
Immunofluorescence of epitope tagged ORF1 demonstrates loss of membrane association when divalent ion coordination residues are mutated.
Confocal microscopy images of HUH7 cells stably expressing ORF1 bicistronic for zsGreen. ORF1-HA tagged proteins were imaged using a rabbit-anti-HA antibody and AlexaFluor647 (goat anti-rabbit IgG …
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Figure 7—source data 1
Immunofluorescence of epitope tagged ORF1 demonstrates loss of membrane association when divalent ion coordination residues are mutated.
TIFF files of confocal microscopy used in subcellular localization analysis.
- https://cdn.elifesciences.org/articles/80529/elife-80529-fig7-data1-v1.xlsx
Author response image 1
Videos
Video 1
AlphaFold Prediction of ORF1 WT - putative papain-like cysteine protease (pPCP) Alanine Scan Gradation.
Best ranked model of ORF1 WT AlphaFold prediction algorithm that transitions to a visual representation of the alanine scanning mutagenesis data across the pPCP. Color gradation corresponds fold …
Video 2
AlphaFold Prediction of ORF1 WT.
Best ranked model of AlphaFold prediction algorithm of ORF1 WT, exhibiting the conserved hexa-cysteine motif (magenta), interacting Y-domain residues D248 and H249 (cornflower blue), …
Video 3
AlphaFold Prediction of ORF1 C483A.
Best ranked model of AlphaFold prediction algorithm of ORF1 C483A, exhibiting the conserved hexa-cysteine motif (magenta), interacting Y-domain residues D248 and H249 (cornflower blue), …
Video 4
AlphaFold Prediction of ORF1 C563A.
Best ranked model of AlphaFold prediction algorithm of ORF1 C563A, exhibiting the conserved hexa-cysteine motif (magenta), interacting Y-domain residues D248 and H249 (cornflower blue), …
Video 5
AlphaFold Prediction of ORF1 D248A.
Best ranked model of AlphaFold prediction algorithm of ORF1 D248A, exhibiting the conserved hexa-cysteine motif (magenta), interacting Y-domain residues D248 and H249 (cornflower blue), …
Video 6
AlphaFold Prediction of ORF1 H249A.
Best ranked model of AlphaFold prediction algorithm of ORF1 H249A, exhibiting the conserved hexa-cysteine motif (magenta), interacting Y-domain residues D248 and H249 (cornflower blue), …
Additional files
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Supplementary file 1
Source data for inductively coupled plasma mass spectrometry (ICP-MS) experimental runs and primer sequences used in plasmid generation.
Spreadsheet tabs 1–3: raw ion reads for inductively coupled plasma mass spectrometry data for each of the independent experimental runs, organized by date of run. Spreadsheet tab 4: primer sequences for each construct generated for use int his manuscript.
- https://cdn.elifesciences.org/articles/80529/elife-80529-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/80529/elife-80529-mdarchecklist1-v1.docx
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Source data 1
AlphaFold Predictions Figures 4 and 5.
These files are the best ranked (ranked 0) predictions generated by AlphaFold of HEV ORF1, its associated point mutants, and the hepatitis A virus (HAV) 3 C protease.
- https://cdn.elifesciences.org/articles/80529/elife-80529-data1-v1.zip
