Free Chlorine and Peroxynitrite Alter the Capsid Structure of Human Norovirus GII.4 and Its Capacity to Bind Histo-Blood Group Antigens

Abstract

Human noroviruses (HuNoVs) are one of the leading causes of acute gastroenteritis worldwide. HuNoVs are frequently detected in water and foodstuffs. Free chlorine and peroxynitrite (ONOO^-) are two oxidants commonly encountered by HuNoVs in humans or in the environment during their natural life cycle. In this study, we defined the effects of these two oxidants on GII.4 HuNoVs and GII.4 virus-like particles (VLPs). The impact on the capsid structure, the major capsid protein VP1 and the ability of the viral capsid to bind to histo-blood group antigens (HBGAs) following oxidative treatments were analyzed. HBGAs are attachment factors that promote HuNoV infection in human hosts. Overall, our results indicate that free chlorine acts on regions involved in the stabilization of VP1 dimers in VLPs and affects their ability to bind to HBGAs. These effects were confirmed in purified HuNoVs. Some VP1 cross-links also take place after free chlorine treatment, albeit to a lesser extent. Not only ONOO^- mainly produced VP1 cross-links but can also dissociate VLPs depending on the concentration applied. Nevertheless, ONOO^- has less effect on HuNoV particles.

Keywords: free chlorine; histo-blood group antigens; norovirus; peroxynitrite; viral protein; virus-like particles.

Figure 1

Morphology of GII.4 VLPs after oxidative treatments. Images of GII.4 VLPs, both untreated and treated with free chlorine (A) and ONOO⁻ (B) captured using TEM at X 15,000 (white scale bar at 0.2 μm) and X 27,500 (white scale bar at 100 nm). Representative numbers of GII.4 VLPs per field observed using TEM at X 15,000 are indicated for native and oxidized particles at different concentrations of free chlorine (C) and ONOO⁻ (D). The number of GII.4 VLPs was determined from eight representative fields, which were obtained from two independent experiments, conducted on different days. Error bars indicate standard deviations. *Unpaired Student’s t-test and Mann-Whitney U test were used to compare groups with p < 0.01.

Figure 2

Free chlorine and ONOO⁻ modify the capsid proteins of GII.4 VLPs. GII.4 VLPs were analyzed using SDS-PAGE following treatment with free chlorine (A) or ONOO⁻ (B) without heat treatment. The variation in intensity of protein bands obtained using SDS-PAGE gel was measured for both oxidative treatments (see the histograms placed below the gels of the respective conditions). The intensity of protein bands (I/I₀ ratio) was determined by dividing the mean intensity value of each band obtained at a specific molecular weight following oxidative treatment (I) by the mean intensity value of the same band obtained in the untreated control (I₀). Each data point is an average of three independent experiments conducted on different days (except for VLPs treated with free chlorine at final concentrations of 14,297 and 19,062 μM, for which one replication was performed). The intensity of protein bands was separated into groups, as indicated by the color code on the right-hand side of the gels (50–60 kDa, 75–100 kDa, and 250 kDa, and the wells are in yellow, orange, gray, and violet, respectively). Error bars indicate standard deviations. Black rectangles on SDS-PAGE gels correspond to the protein bands that were analyzed using MS.

Figure 3

Free chlorine and ONOO⁻ prevent HBGA-binding to GII.4 VLPs. Assays of HBGA-binding to GII.4 VLPs using ALe⁺ saliva following free chlorine (A) and ONOO⁻ (B) treatments were presented. The OD/OD₀ ratio values were determined by dividing the mean OD₄₅₀ values obtained by HBGA-binding ELISA at each oxidant concentration (OD) by the mean OD₄₅₀ values obtained in the untreated control (OD₀). Two concentrations of GII.4 VLPs were tested: 1 μg.ml⁻¹ (black circles and line) and 0.5 μg.ml⁻¹ (gray circles and line). Each data point corresponds to the mean OD₄₅₀ values emanating from three independent experiments with duplicate measures carried out on different days (except for VLPs treated with free chlorine at final concentrations of 14,297 and 19,062 μM, for which one replication was performed). Error bars indicate standard deviations. *Unpaired Student’s t-test was used to compare groups with p < 0.01.

Figure 4

Free chlorine and ONOO⁻ modify the capsid proteins of GII.4 HuNoVs. GII.4 HuNoVs were analyzed using SDS-PAGE following free chlorine (A) or ONOO⁻ (B) treatments without heat treatment. The variations in intensity of protein bands obtained using SDS-PAGE gel were measured for both oxidative treatments (see histograms below the gels of the respective conditions). The intensity of protein bands (I/I₀ ratio) was determined by dividing the mean intensity value of each band obtained at a specific molecular weight after oxidative treatment (I) by the mean intensity value of the same bands obtained in the control without oxidant (I₀). Each data point is an average of three independent experiments conducted on different days. The intensity of the protein bands was separated into groups, as indicated by the color code on the right-hand side of the gels (60 kDa, 100 kDa, 150 kDa, and 250 kDa are shown in yellow, brown, green, and gray colors, respectively). Error bars indicate standard deviations.

Figure 5

Free chlorine and ONOO⁻ decrease HBGA-binding to complete GII.4 HuNoVs. Assays of HBGA-binding to encapsidated GII.4 HuNoVs in ALe⁺ saliva following free chlorine (A) and ONOO⁻ (B) treatments were presented. Complete HuNoVs correspond to viral particles treated with RNase at a final concentration of 100 μg.ml⁻¹ for 1 h at 37°C. The black line and gray line represent complete HuNoVs quantified by RT-qPCR and complete HuNoVs after HBGA-binding followed by RNA amplification using RT-qPCR, respectively. The log (C/C₀) values were calculated by dividing the mean GII.4 HuNoV gc values following oxidative treatment (C) by the mean GII.4 HuNoV gc values in the untreated control (C₀). Each data point corresponds to the mean data value of duplicates of three independent experiments carried out on different days. Error bars indicate standard deviations. Arrows (↓) indicate data below the quantification limit. *Unpaired Student’s t-test and Mann-Whitney U tests were used to compare groups with p < 0.01.