Impact of viral genetic determinants in modulating viral pathogenicity: focus on SARS-Cov-2 and chronic hepatitis viruses

Viruses are known to undergo a process of genetic diversification during their spreading across humans, and the knowledge about viral mutations and genetic variations may improve our understanding of viral pathogenicity, immunity, and drug design. The overall aim of this PhD thesis was to study the impact of viral genetic determinants in modulating viral pathogenicity, antigenicity and drug binding affinity. In particular, the attention was focused on i) evaluating the extent of genetic conservation across the druggable proteins: the main protease, RNA dependent RNA polymerase (RdRp), and Spike protein of SARS-CoV-2 and other human pathogenic coronaviruses, ii) defining key genetic-elements, single or in clusters, underlying SARS-CoV-2 evolutionary diversification across Continents, and their impact on drug binding-affinity and viral antigenicity, iii) investigating the existence of genetic determinants within the NS5A first domain associated with hepatocellular carcinoma (HCC) in patients with HCV chronic infection, sustained by HCV genotype 1b, iv) evaluating the correlation of HBx-mutations, particularly in the N-terminal domain, with HBVinduced HCC in vivo, and their in vitro impact on pathogenicity. First, the analysis of the main protease, RdRp, and Spike protein sequences from SARS-CoV2 and other pathogenic coronaviruses revealed highly conserved protein regions that could represent novel pharmacological targets for compounds with anti-pan-coronavirus activity, to be thoroughly investigated in future studies. Second, the analysis of SARS-CoV-2 amino acids sequences identified 35 key mutations (prevalence: >0.5%), residing in different viral proteins. 16/35 form tight clusters involving multiple SARS-CoV-2 proteins, highlighting inter-genic co-evolution. Some clusters (including D614GSpike+P323LRdRp+R203KN+G204RN) occur in all Continents, while others show a geographically restricted circulation. Structure-based virtual screening identified 20 best RdRp inhibitors and 21 best 3CL-Pr inhibitors belonging to different drug-classes. Notably, mutations in RdRp or 3CL-Pr modulate positively or negatively the binding-affinity of these drugs. Among them, P323LRdRp reduces the binding-affinity of specific compounds including remdesivir while increases the binding-affinity penciclovir and tenofovir, suggesting potential hypersusceptibility. Finally, specific mutations (including Y541CHel+H504CHel) strongly hamper Class-I/II epitopes recognition, while D614GSpike profoundly alters the structural-stability of a recently identified B-cell epitope target of neutralizing-antibodies (aa:592-620). Third, the analysis identified four specific NS5A polymorphisms significantly correlated with HCC: S3T, T122M, M133I, and Q181E. Result confirmed by multivariable analysis. ShannonEntropy revealed other three residues were more variable in HCC: the first residue is allocated in iv highly conserved N-terminal C13R/S, F127L/S, N137D/K, and an enrichment of additional amino acid mutation is observed at residue Q181E/G/H/P. Polymorphisms at these residues may affect NS5A capability to promote maturation of viral particles, favouring an intracellular accumulation of HCV proteins, a process known to induce cellular stress. Some of these mutations correlated with higher viremia in HCC patients. These mutations induced novel phosphorylation residues by crucial kinases and altered NS5A stability thus increase NS5A interacting vital cellular proteins regulating cell growth and apoptosis. Fourth, the analysis of HBx sequences revealed F30V was the only HBx-mutation correlated with HCC. In vitro, F30V determined a significant reduction in pgRNA and core-associated HBVDNA compared to wt, paralleling with a significant decrease of HBx-binding to cccDNA and decreased HBx-stability compared to wt. F30V also decreased percentage of apoptotic-cells compared to wt, and increased the probability of HBx-Ser31 to be phosphorylated by the kinase PI3KAkt. This suggests that F30V may promote hepatocytes survival, thus potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Summery, overall findings obtained in this thesis highlight the importance of viral genetic variability in having an important role on the course of the diseases. The genetic analysis of SARSCoV-2 and other human pathogenic coronaviruses provides a comprehensive and specific map of conserved regions across human coronavirus proteins which are essential for virus replication and thus with no or very limited tendency to mutate. Hence, these represent key druggable targets for novel compounds against this virus family. A close and continuous monitoring of SARS-CoV-2 mutational patterns is crucial to ensure the effectiveness of antiviral treatments and vaccines as well as the accuracy of diagnostic assays worldwide, in addition to further our understanding of disease immunity. Identifying viral genetic markers associated with HCC in HBV and HCV is crucial to identify patients at higher HCC-risk deserving more intense liver evaluation and/or early antiviral treatment