Reactivity of Viral Proteins with Free Chlorine: Structural Insights and Implications for Virus Inactivation

Viruses exhibit diverse susceptibility to free chlorine inactivation, but the underlying mechanisms that drive these differences are poorly understood. To investigate this, we applied a multiplex and cost-effective tandem mass tag-based quantitative proteomics to characterize peptide decay kinetics and reactions in three model viruses, namely, MS2, PhiX174, and T4, when they were treated with free chlorine. Strong correlations were observed between peptide decay rate constants and both solvent-accessible surface areas (SASAs) of methionine residues (Pearson's r = 0.78, p < 0.0001) and SASAs per residue in methionine-lacking peptides (Pearson's r = 0.81, p < 0.0001). A multiple linear regression model fitted using these two independent structural variables accurately predicted peptide decay rate constants in the three viruses (R²= 0.91 for MS2 peptides, R²= 0.72 for PhiX174 peptides, and R²= 0.86 for T4 peptides). Notably, the peptide decay kinetics in viral proteins responsible for host attachment and genome injection better predicted virus resistance than that in other viral proteins. Furthermore, peptides containing methionine sulfoxide, 3-chlorotyrosine, and nonspecific peptide bond cleavages were detected in chlorine-treated viruses. This study combines proteomics and structural analysis, providing new insights into protein reactivity and virus inactivation by free chlorine.