Emerging and re-emerging infectious viral diseases have consistently posed a substantial threat to public health worldwide. In particular, coronaviruses such as severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) and arboviruses such as Zika virus (ZIKV) and Dengue virus (DENV) have severely impacted global health, and the socioeconomy over the last several decades. To date, limited antiviral agents are available to treat viral infections caused by these pathogens, highlighting the urgent need for innovative therapeutic strategies. Understanding the molecular mechanisms of virus-host interactions and essential steps involved in the viral life cycle is crucial for identifying potential antiviral targets with enhanced efficacy and selectivity for therapeutic interventions. In addition, investigating antiviral compounds that target specific molecular events during viral infections holds promise for developing broad-spectrum antiviral therapies to combat emerging viral threats.

The first part of this thesis focuses on investigating the molecular mechanisms of SARS-CoV-2 entry depending on host receptor angiotensin-converting enzyme 2 (ACE2), especially critical amino acid residues of ACE2 that influence virus binding and entry. The interactions between the SARS-CoV-2 spike (S) protein and its primary receptor ACE2 orthologs across various animal species with different susceptibilities were comprehensively analyzed, illuminating the in vitro and in silico interactions between the two proteins. Subsequently, critical amino acid substitutions in human ACE2 that affect virus binding and entry were identified using predictive structural modeling and mutagenesis studies of ACE2 variants coupled with cell-based syncytia formation and pseudovirus entry assays. This study provided additional insights into host ACE2 variants interacting with SARS-CoV-2, which could further enhance our understanding of the molecular mechanisms underlying receptor recognition, virus entry and infection, as well as potential therapeutics targeting coronavirus entry.

Second, the study focuses on identifying small-molecule entry inhibitors of SARS-CoV-2. A SARS-CoV-2 lentiviral pseudovirus screening platform was established and used to screen an antiviral compound library containing 948 compounds. Ziresovir and TMC353121 were identified as SARS-CoV-2 entry inhibitors with low cytotoxicity and antiviral activity at micromolar levels. The mechanistic studies indicated that these two compounds targeted the early entry of SARS-CoV-2, especially S-mediated cell-cell fusion. In vitro and in silico protein-protein interaction studies revealed that they could bind to the fusion core of the SARS-CoV-2 S protein, disrupting the six-helix bundle (6HB) formation and membrane fusion. Remarkably, Ziresovir and TMC353121 exhibited broad-spectrum antiviral activities against other coronaviruses, including SARS, human coronaviruses (HCoVs) such as NL63, 229E, and OC43, as well as other animal coronaviruses like feline infectious peritonitis virus (FIPV) and mouse hepatitis virus (MHV). In summary, identifying and characterizing these inhibitors could provide potential therapeutic candidates against a spectrum of coronaviruses.

Third, the mechanism of action for the novel 1,2,4-oxadiazole derivative, KR26827, was examined. This compound had previously been identified as demonstrating a broad-spectrum antiviral activity against emerging flaviviruses, including ZIKV and DENV. Here, the antiviral activity of KR26827 was first confirmed in ZIKV-infected human hepatic cells, where it demonstrated inhibition of progeny virus production as well as the synthesis of viral proteins and RNA. Notably, time-of-addition experiments suggested that KR26827 targeted early steps in the ZIKV life cycle, particularly the initial post-entry stages. Further assessment utilizing flavivirus replicon systems demonstrated a broad-spectrum antiviral activity extending to DENV and West Nile virus (WNV) replication. Importantly, mechanistic studies suggested that KR26827 influenced the MAPK signaling pathway, which potentially delayed cell proliferation and host DNA synthesis, thereby affecting ZIKV replication. Additionally, KR26827 treatment enhanced cytokine-cytokine receptor interactions and modulated the immune response, collectively establishing an antiviral state that interfered with viral replication. These findings underscore the potential of KR26827 as a promising broad-spectrum therapeutic agent against flavivirus infections.

In conclusion, systematic analyses of ACE2-S interactions across various animal species and identification of critical amino acid substitutions in host ACE2 influencing viral entry advanced our understanding of virus-host interactions during SARS-CoV-2 infection. Additionally, identifying novel targets for therapeutic intervention and characterizing specific compounds with antiviral properties laid the foundation for innovative strategies against emerging coronaviruses and flaviviruses. Collectively, the data presented in this thesis shed light on the molecular intricacies of viral infections and offer a promising approach for developing broad-spectrum antiviral therapies.