SARS-CoV-2 entry mechanisms and antiviral strategies
Year: 2024
Type: dissertation
Abstract: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus disease 2019 (COVID-19), is an enveloped, positive-strand RNA virus. The virus binds to and infects cells through the interaction of the viral spike (S) proteins with the host cell surface receptor angiotensin-converting enzyme 2 (ACE2). For the release of the viral mRNA-like genome into the cytoplasm of the target cell, the trimeric S proteins need to go through proteolytic cleavages mediated by host proteases either at the cell surface or inside endo/lysosomes following endocytosis of the viral particle. This ACE2-mediated mechanism of cell entry and infection is shared by SARS-CoV, the coronavirus highly related to SARS-CoV-2 that caused a much smaller epidemic in 2002. However, while SARS-CoV-2 caused a global pandemic leading to more than 7 million deaths and hundreds of millions of infections worldwide, SARS-CoV caused 800 deaths and less than 8000 infections. Given that the two viruses use the same ACE2 receptor, why is SARS-CoV-2 so much more infectious than its predecessor SARS-CoV? In the first part of my thesis, I present an alternative and efficient infectious cell entry route for SARS-CoV-2. Similarly, to most zoonotic viruses, SARS-CoV-2 uses endocytic routes to enter host cells. I have uncovered that the genetic depletion of all dynamin isoforms, a class of cellular enzymes needed for the final stage of some endocytic processes, strongly inhibited SARS-CoV-2. However, the infection was restored by increasing viral loads in a dose-dependent manner demonstrating that, although less favorable, a dynamin-independent entry mechanism can be used by the virus to enter and infect cells. The result showed that both virus entry mechanisms, particularly the dynamin-independent one, required a functional actin cytoskeleton. In addition to SARS-CoV-2 and its variants of concern, the study also showed that numerous animal viruses can efficiently use this incompletely characterized dynamin-independent and actin-dependent endocytic pathway. The second part of my thesis outlines the importance of the cellular protein receptor Neuropilin-1 (NRP-1) in SARS-CoV-2 infection. NRP-1 is known to bind furin-cleaved substrates. The S protein of SARS-CoV-2 contains a furin cleavage site that is absent in the spikes of SARS-CoV. I have demonstrated that, unlike SARS-CoV, SARS-CoV-2 uses NRP-1 receptors in addition to ACE2 to potentiate infection. In vitro experiments showed that cells expressing both ACE2 and NRP-1 are more susceptible to infection as compared to cells expressing only ACE2. A control virus lacking the furin cleavage site did not depend on NRP1 for infectivity. Thus, NRP-1 is a host cell entry factor that facilitates SARS-CoV-2 but not SARS-CoV infection. The last part of my thesis depicts combinatorial antiviral strategies against SARS-CoV-2 infection. The rationale behind the development of this antiviral cocktail is that by blocking the main entry routes of SARS-CoV-2 with combined inhibitors of cellular proteases and of the phosphoinositol-5-kinase, PIKfyve, an enzyme involved in endosome maturation, synergistic block of infection is achieved with remarkably low doses of the inhibitors, suggesting a therapeutic strategy based on the combined use of these inhibitors. This approach suggests a therapeutic strategy that capitalizes on the combined use of these inhibitors to enhance antiviral efficacy against SARS-CoV-2. As a complementary strategy to block the host’s entry factors and subsequent viral infection, an extended, unmodified version of the heptad repeat 2 (HR2), longHR2_42, peptide was developed. HR2 targets a very conserved region of the SARS-CoV-2 trimeric spike protein and inhibits an intermediate step during the conformational changes induced during the fusion of viral and cellular membranes, required for the delivery of the viral genome into the cytoplasm of the target cell. Despite being only 40 amino acids in length, the extended HR2 displayed ∼100-fold more potent antiviral activity than all previously published shorter, unmodified HR2 peptides. These results suggest that cost-effective, easy-to-synthesize peptide-derived therapeutics for SARS-CoV-2 and its variants could be used as an alternative, or in combination, to other therapies.
Source: Työväentutkimus Vuosikirja
Author: Ojha, Ravi Kant
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7197402680