UC Merced

The global pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has underscored the urgent need for effective therapeutic strategies to inhibit viral replication and transmission. Although the severity of Coronavirus disease 2019 (Covid-19) has diminished, the SARS-CoV-2 virus is still circulating and evolving, meaning that there is still a distinct risk that a potent Variant of Concern (VOC) could arise at any time. This thesis summarizes our efforts in investigating the efficacy of several prophylactic treatments that were being investigated by the scientific community, and subsequently describes our efforts in probing the molecular mechanisms governing SARS-CoV-2 infection to identify novel vulnerabilities to exploit against the virus.The first portion of this dissertation orients the reader in understanding the origins and features of SARS-CoV-2 and coronaviruses in general. We then move into briefly presenting key structural features of the SARS-CoV-2 virion before introducing the Spike protein and its role in facilitating infection. We cover these topics with a focus on the structural aspects of the Spike protein, including its glycosylation pattern as well as its conformational changes as it engages the human Angiotensin Converting Enzyme 2 (hACE2) receptor. We finish by describing how SARS-CoV-2 virus can interact with proteins on the surface of cells to modulate its ability to infect different kinds of cells, both in vivo and in vitro. The second phase of the thesis focuses on summarizing our work in searching for overlooked epitopes for the development of antiviral prophylactics. Specifically, we describe our work in assessing the role of SARS-CoV-2 Spike S1 domain N-linked glycans in retaining viral infectivity and mediating viral entry into susceptible cells. In addition, we assess whether certain glycans are important in mediating viral capture and presentation by the C-type lectin receptor DC-SIGN. To quantify DC-SIGN capture, we developed a cell novel cell assay whereby DC-SIGN would deliver virions to susceptible cells, thereby facilitating infection-in-trans (referred to as Trans-infection in this document). Overall, our studies indicated that the glycans at N17 and N122 were important in retaining SARS-CoV-2 pseudotyped viral infectivity. On the other hand, removal of the glycan at N234 appeared to enhance viral infectivity. Drawing from insights gained in performing these assays, we then identified and tested clusters of glycans to ascertain if they were able to act as viable “cold spots” for the design and development of viral entry inhibitors. We conclude by assessing whether SARS-CoV-2 Spike protein from different strains altered the ability of Spike pseudotyped virions in infecting cells and in being captured by DC-SIGN. We then move into assessing the efficacy of the broadly-antiviral lectin Griffithsin against SARS-CoV-2 Spike pseudotyped viruses. We tested the importance of several variables that were relevant in the context of Covid-19, including the role of the additional SARS-CoV-2 structural proteins in mediating DC-SIGN recognition, the efficacy of Griffithsin against pseudoviral strains that expressed additional structural proteins, and the differences in inhibitory capability of several Griffithsin variants. We finish by describing several additional attempted assays that made use of our newfound expertise in SARS-CoV-2 pseudoviral production. This included several collaborations, ranging from assessing the extent to which gaseous ozone could sterilize SARS-CoV-2 pseudovirus-contaminated fomites, to troubleshooting the parameters to attempt to detect pseoudotyped lentiviral binding to functionalized Biolayer Interferometry (BLI) probes. Ultimately, this thesis contributes to the growing body of knowledge surrounding SARS-CoV-2 inhibition and prophylactic treatment by elucidating vulnerabilities in critical viral infection processes. These findings serve as the first steps to possibly develop novel prophylactic inhibitors that mitigate Covid-19 disease progression.