Proteolytic Inactivation of Viruses in Lakewater

Enteric viruses are important contaminants of surface waters and are a significant burden on public health. Because these viruses cannot replicate outside their natural hosts, their ability to spread depends largely on their environmental stability. In aquatic ecosystems, enteric viruses are exposed to a range of abiotic and biotic stressors which can exert either inactivating or protective effects. These factors differentially impact virus types, resulting in variability in the stability of viral pathogens. Aquatic microbial communities have demonstrated antiviral activity through the action of secreted proteases. These enzymes hydrolyze peptide bonds and mediate virus inactivation through degradation of the virus capsid. In this thesis, I investigated the mechanisms of microbial inactivation of enteric viruses in lakewater, specifically the antiviral activity of microbial extracellular proteases. We first characterized the substrate specificity of the extracellular proteases present in Lake Geneva, Switzerland. Using Multiplex Substrate Profiling by Mass Spectrometry, we identified preferred proteolytic cleavage next to positively charged and certain, primarily aromatic, non-polar amino acids, while cleavage next to proline, negatively charged, and certain polar residues was disfavored. The proteolytic fingerprint of Lake Geneva was conserved throughout time and water depths and was shared with two other Swiss lakes. In contrast, we observed variability in the types of less prevalent cleavage patterns, suggesting that the degree of heterogeneity in lakewater proteolytic substrate specificity varies spatially and temporally. The proteolytic fingerprint of Lake Geneva was then used to investigate the structural features determining the stability of the enterovirus echovirus 11 (E11) in lakewater. By linking the lakewater proteolytic fingerprint to E11 structural features, we identified four surface-exposed amino acids on E11 proposed to be susceptible to proteolytic cleavage. The relevance of these amino acids for viral inactivation was tested by producing E11 mutants using reverse genetics and comparing their stability in lakewater relative to that of the E11 wild-type. This revealed that residue VP2.Y97 renders E11 sensitive to proteolytic decay. Strikingly, this residue is absent from several enteroviruses that display greater environmental stability than E11, indicating that substitution at a single capsid position is sufficient to shift virus fate in lakewater. Finally, we investigated the fate of two enteroviruses, E11 and coxsackievirus B5 (CVB5), and one adenovirus, human adenovirus 2 (HAdV2), in lakewater and explored the mechanisms underlying their microbial inactivation. Combining infectivity assays, genome quantification, and capsid integrity analysis, we identified distinct inactivation rates among these viruses and linked virus decay to differences in capsid structural integrity. We observed rapid, intermediate, and negligible decay