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. 2018 Sep 26;92(20):e00684-18.
doi: 10.1128/JVI.00684-18. Print 2018 Oct 15.

Potent Inhibition of Hepatitis E Virus Release by a Cyclic Peptide Inhibitor of the Interaction Between Viral Open Reading Frame 3 Protein and Host Tumor Susceptibility Gene 101

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Potent Inhibition of Hepatitis E Virus Release by a Cyclic Peptide Inhibitor of the Interaction Between Viral Open Reading Frame 3 Protein and Host Tumor Susceptibility Gene 101

Saumya Anang et al. J Virol. .
Free PMC article

Abstract

Hepatitis E virus (HEV) generally causes self-limiting acute viral hepatitis in normal individuals. It causes a more severe disease in immunocompromised persons and pregnant women. Due to the lack of an efficient cell culture system or animal model, the life cycle of the virus is understudied, few antiviral targets are known, and very few antiviral candidates against HEV infection have been identified. Inhibition of virus release is one possible antiviral development strategy, which limits the spread of the virus. Previous studies have demonstrated the essential role of the interaction between the PSAP motif of the viral open reading frame 3 protein (ORF3-PSAP) and the UEV domain of the host tumor susceptibility gene 101 (TSG101) protein (UEV-TSG101) in mediating the release of genotype 3 HEV. Cyclic peptide (CP) inhibitors of the interaction between the human immunodeficiency virus (HIV) gag-PTAP motif and UEV-TSG101 are known to block the release of HIV. Using a molecular dynamic simulation, we observed that both gag-PTAP and ORF3-PSAP motifs bind to the same site in UEV-TSG101 by hydrogen bonding. HIV-released inhibitory CPs also displayed binding to the same site in UEV-TSG101, indicating that they may compete with ORF3-PSAP or gag-PTAP for binding to UEV-TSG101. Two independent assays confirmed the ability of a cyclic peptide (CP11) to inhibit the ORF3-TSG101 interaction. CP11 treatment also reduced the release of both genotype 1 and genotype 3 HEV by approximately 90%, with a 50% inhibitory concentration (IC50) of 2 μM. Thus, CP11 appears to be an attractive candidate for further validation of its anti-HEV properties.IMPORTANCE There is no specific therapy against hepatitis E virus (HEV)-induced hepatic and nonhepatic health problems. Prevention of the release of the progeny viruses from infected cells is an attractive strategy to limit the spread of the virus. Interactions between the viral open reading frame 3 and the host tumor susceptibility gene 101 proteins have been shown to be essential for the release of genotype 3 HEV from infected cells. In this study, we have identified a cyclic peptide inhibitor of the above-mentioned interaction and demonstrate the efficiency of the inhibitor in preventing virus release from infected cells. Thus, our findings uncover the possibility of developing a specific antiviral agent against HEV by blocking its release from infected cells.

Keywords: HEV release; cyclic peptide inhibitor; hepatitis E virus.

Figures

FIG 1
FIG 1
Computational analysis of the interaction between UEV-TSG101 and P(T/S)AP motif-containing peptides and cyclic peptides. (A) Docking of different peptides to the crystal structure of UEV-TSG101. Yellow, HIV gag-PTAP; green, HEV ORF3-PSAP (conformer 1); purple, HEV ORF3-PSAP (conformer 2). (B) Docking of the extended portion of HEV-ORF3 (30-aa region encompassing the PSAP motif [indicated in green]) and HIV gag-PTAP (yellow) to UEV-TSG101. A magnified view of the interaction region is represented in the inset. (C) Docking energy estimation of 500 conformers each for the interaction between UEV-TSG101 and HIV gag-PTAP, HEV ORF3-PSAP, CP11, CP6, and CP16. Brown circles denote the conformer selected for MM-GBSA/PBSA analysis. (D) Analysis of docking of different peptides to the crystal structure of UEV-TSG101. Yellow, PTAP-HIV-gag; green, HEV-ORF3 (30 aa); light blue, CP11; orange, CP6; purple, CP16. (E) Plot showing free energy of binding between TSG101 and the indicated peptides, estimated by MM-PBSA analysis.
FIG 2
FIG 2
Optimization of the yeast three-hybrid assay using cyclic peptide inhibitors of the HIV gag-TSG101 interaction. (A) Schematic of the binding domain vector for coexpression of the GAL4-BD (binding domain)-fused bait protein and the cyclic peptides. IC, C-terminal intein; IN, N-terminal intein; CBD, chitin binding domain; HA, hemagglutinin epitope tag; NLS, nuclear localization signal; TRP1, tryptophan selection marker; Ampr, ampicillin resistance cassette; PADH1, ADH1 promoter; TADH, ADH terminator; PMet25, MET25 promoter; TPGK, PGK terminator. The PacI site-containing SICLOPPS cassette from the pARCBD plasmid was subcloned into multiple cloning site 2 (MCS2) of the pBRIDGE vector to generate pBRIDGE SIC. (B) Western blotting of Y2H gold whole-cell extracts transformed with pBRIDGE (lane 1) (BD) and pBRIDGE SIC (lane 2) (BD-Sic) plasmids to check the expression of SICLOPPS in the Y2H gold strain, using anti-CBD (top) and anti-HA (bottom) antibodies. (C) Western blotting of Y2H gold whole-cell extracts transformed with the indicated plasmids and grown on LTM medium. Aliquots of the lysate were probed with following antibodies: gag (first panel), myc (second panel), and HA (third and fourth panels). “*” indicates a nonspecific band. Samples in the fourth panel were resolved by 20% SDS-PAGE to reveal the 6-kDa band, representing C-terminal intein. (D) Analysis of the HIV gag-TSG101 interaction in the presence and absence of CP11 and CP6. The Y2H gold strain was transformed in the indicated combinations and plated onto LT medium supplemented with 1 mM methionine. Eight random colonies from each plate were replica plated onto SD medium containing various selection markers, as indicated, and their growth was monitored over a period of 4 days. Two colonies are represented. AD, activation domain; L, leucine; T, tryptophan; M, methionine; H, histidine; A, adenine hemisulfate; Ar, aureobasidin A; 3AT, 3-amino-1,2,4-triazole. “−” indicates deficiency in the medium, and “+” indicates supplemented medium. (E) MTT assay-mediated cell viability estimation for Y2H gold cells with different cotransformants, as indicated. Values are means ± SEM of data from triplicate samples; values for cells only were considered 100%, and others were estimated with reference to that value.
FIG 3
FIG 3
Yeast three-hybrid analysis of the HEV ORF3-TSG101 interaction in the presence and absence of CP11 and CP6. (A) Analysis of the p6 HEV ORF3 and TSG101 interaction in the presence and absence of CP11 and CP6. The Y2H gold strain was transformed in the indicated combinations and plated onto LT medium supplemented with 1 mM methionine. Eight random colonies from each transformant were replica plated onto SD medium containing the indicated selection markers, and their growth was monitored over a period of 4 days. Two colonies each are represented. AD, activation domain; BD, binding domain; L, leucine; T, tryptophan; M, methionine; H, histidine; A, adenine hemisulfate; Ar, aureobasidin; 3AT, 3-amino-1,2,4-triazole. “−” indicates a deficiency in the medium, and “+” indicates supplemented medium. (B) MTT assay-mediated cell viability estimation for Y2H gold cells shown in panel A. Values are means ± SEM of data from triplicate samples; values for cells only were considered 100%, and others were estimated with reference to that value. (C) Analysis of the g1-HEV ORF3 and TSG101 interaction in the presence and absence of CP11 and CP6. The Y2H gold strain was transformed in the indicated combinations and plated onto LT medium supplemented with 1 mM methionine. Eight random colonies from each transformant were replica plated onto SD medium containing various selection markers, as indicated, and their growth was monitored over a period of 4 days. Two colonies are represented.
FIG 4
FIG 4
His pulldown assay reveals the inhibition of the HEV ORF3 and host TSG101 interaction by CP11. (A, left) Coomassie blue-stained image of purified ORF3-His protein. (Right) Western blot image of aliquots of the sample from the left panel, probed using anti-His antibody. (B) Western blotting of TNT-expressed proteins using anti-myc antibody. The mock lysate represents TNT of the empty pGBKT7 vector. (C) His pulldown assay. (Top) Anti-myc Western blotting of ORF3-His-bound proteins. (Bottom) Anti-His Western blotting of aliquots of the sample represented in top panel. *, nonspecific band. Note that a shorter-exposure image is represented for the ORF4-myc panel. (D) Quantitation of the TSG101 and ORF4 band intensities represented in panel C. Values were normalized to the respective ORF3-His band intensity and plotted in the graph.
FIG 5
FIG 5
CP11 inhibits release of p6 HEV from Huh7 cells. (A) Viability measurement of p6 HEV-expressing Huh7 cells, treated with 10 μM CP11, as indicated. Values are means ± SEM of data for triplicate samples. (B) QRT-PCR measurement of the intracellular level of p6 HEV sense-strand RNA in the samples shown in panel A. p6 HEV sense RNA values were normalized to that of GAPDH and are represented as means ± SEM of data from triplicate samples. (C) QRT-PCR estimation of p6 HEV genome copy numbers in the culture medium of Huh7 cells shown in panel A. Values are means ± SEM of data for triplicate samples. (D) ELISA of ORF2 VLPs using anti-ORF2 antibody. Values are represented as means ± SEM of the corrected absorbances (A450–650 [absorbance at 450 nm minus the absorbance at 650 nm]) for triplicate samples. (E) ELISA to quantitate ORF2 levels in the culture medium of Huh7 cells expressing p6 HEV and treated with 10 μM CP11, as indicated. Values are means ± SEM of the corrected absorbances (A450–650) for triplicate samples. (F) Measurement of secreted Gaussia luciferase in the culture medium of Huh7 cells expressing p6 HEV-Luc and treated with 10 μM CP11, as indicated. Luc values were normalized to cell viability values and are represented as means ± SEM. (G) IC50 estimation for CP11 by QRT-PCR-mediated quantitation of p6 HEV sense RNA levels in the culture medium of Huh7 cells expressing p6 HEV and treated for 24 h with the indicated quantities of CP11 (final concentration). Values are means ± SEM of data from triplicate samples. (H) QRT-PCR measurement of p6 HEV sense-strand RNA in S10-3 cells infected with the PEG-precipitated virus obtained from the culture medium of p6 HEV-electroporated cells treated with CP11 or mock treated. Note that equal amounts of virus (106 genome copies) from mock- and CP11-treated culture media were used for infection. As a positive control, 107 genome copies of the virus were used for infection. p6 HEV sense RNA values were normalized to that of GAPDH and are represented as means ± SEM of data from triplicate samples. (I) QRT-PCR estimation of the p6 HEV genome copy numbers in the culture medium of S10-3 cells shown in panel H. Media from all three time points were pooled, PEG precipitated, and processed for RNA isolation. Values are means ± SEM. Bars in the graph are the same as for panel H.
FIG 6
FIG 6
CP11 inhibits g1-HEV release. (A) QRT-PCR-mediated quantitation of HEV sense-strand RNA levels from PEG-precipitated virus obtained from ORF4-Huh7 cells infected with a g1-HEV clinical isolate and treated with 10 μM CP11, as indicated. (B) Viability measurement of ORF4-Huh7 cells used for panel A. Values are means ± SEM of data for triplicate samples.

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