Elucidating virus-host interactions and viral immune evasion strategies using genetic editing and screening technologies

Abstract

This thesis examines DNA and RNA viruses using advanced genetic tools: 1) a high throughput CRISPR/Cas9 library screen to identify new host genes involved in HSV-1 infection; 2) biochemical methods to uncover a new regulator of heparan sulfate chain length; 3) CRISPR/Cas9 system application to generate single amino-acid substitutions in clinical viral strains; 4) next-generation sequencing to study mixed infections of drug-resistant HSV-1 variants; and 5) an arrayed SARS-CoV-2 cDNA library to identify a new immune evasion strategy by an RNA virus. Chapter 1 introduces key topics essential for understanding the thesis. It provides an overview of HSV-1, heparan sulfate, and SARS-CoV-2, detailing their biological significance. The chapter discusses heparan sulfate biosynthesis and its interactions with HSV-1, influencing viral infection. It reviews antiviral strategies against HSV-1, highlighting CRISPR/Cas9 technology in viral genome editing and the relationship between MHC class I molecules and viral immune evasion. Chapter 2 maps host genes essential for HSV-1 infection using a genome-wide CRISPR/Cas9 screen. The screen identified genes involved in heparan sulfate biosynthesis and transport, confirming its importance as an HSV-1 attachment factor. Notably, DIA1 (C3orf58) was identified as a new gene impacting cell-surface heparan sulfate expression. Additionally, BPTF, part of the NURF complex, was found essential for HSV-1 infection. DIA1 specifically contributes to elongating heparan sulfate chain length, opening opportunities for future research on DIA1-mediated biosynthesis. Chapter 3 focuses on the antiviral agent acyclovir (ACV), the treatment of choice for HSV-1. Long-term ACV therapy can lead to ACV-resistant HSV-1. A novel ACV-resistant HSV-1 mutation, F289S, was identified. Using CRISPR/Cas9-mediated HSV-1 genome editing, reverting F289S to the wild-type sequence resulted in an ACV-sensitive phenotype, while introducing F289S in an ACV-sensitive strain led to ACV resistance. This demonstrated that CRISPR/Cas9 is a powerful tool to assess clinical ACV-resistant mutations. Chapter 4 presents an immunocompromised patient with multi-resistant HSV-1 reactivation caused by viruses with mutations in the viral DNA polymerase or TK gene. Next-generation sequencing revealed these drug-resistant mutations were present before antiviral treatment onset. Chapter 5 explores SARS-CoV-2's molecular behavior during the pandemic. SARS-CoV-2 was found to interfere with antigen presentation, evading immune surveillance. Infection of monkey and human cell lines led to reduced cell-surface MHC class I expression. The accessory protein ORF7a was identified as responsible, interacting with HLA class I molecules in the ER, impairing their trafficking to the Golgi, and reducing surface expression. This action may impair cytotoxic T cell activation. A single amino acid mutation in SARS-CoV-1 ORF7a to the SARS-CoV-2 sequence induced a gain-of-function in HLA class I downregulation, aiding immune evasion. Chapter 6 summarizes and discusses the findings: CRISPR/Cas9 is a powerful tool for identifying host factors in HSV-1 infection. DIA1 regulates cell surface heparan sulfate expression, specifically elongating chain length. CRISPR/Cas9-mediated genome engineering is promising for modifying clinical viral isolates. Next-generation sequencing is valuable for diagnosing viral infections. SARS-CoV-2 ORF7a aids viral immune evasion by impairing anti-viral cytotoxic T cell activity, impacting COVID-19 pathology.