Understanding Epstein-Barr virus infection through genetic screens

To establish lifelong infection in the human host, EBV manipulates and hijacks numerous cellular processes using both viral proteins and miRNAs. Most host proteins that are hijacked by EBV or downregulated by virus-encoded miRNAs remain elusive. Unraveling these hostpathogen interactions could provide new insights into EBV biology, cellular host pathways, and possibly lead to the development of new antiviral drugs. In the research described in this thesis, we optimized and applied genetic techniques with the aim to identify new hostpathogen interactions that promote infection. We focused our studies on immune evasion properties of EBV miRNAs and host genes crucial for virus entry during primary infection in B cells. To understand the biological function of viral miRNAs, it is important to develop systems to specifically block miRNA expression during virus infection. In chapter 2, we generated and optimized Tough Decoy (TuD) miRNA inhibitors for all EBV miRNAs and discovered that the thermodynamic features of these inhibitors dictate their potency. In chapter 3, we applied an optimized TuD-based screen to study potential BART miRNA involvement in innate immune evasion. We identified miR-BART16-5p as novel negative regulator of type I IFN signaling by repressing CBP expression, a transcriptional activator. In recent years, the CRISPR/Cas9 technique has revolutionized the field of targeted genome editing in human cells. In chapter 4, we introduce the CRISPR/Cas9 technology and we review on the therapeutic potential of anti-viral CRISPR/Cas9 to interfere with human virus infections. Efficient multiplexed genome engineering may be applied to disrupt viral genomes at multiple sites, thereby limiting the chance to develop viral escape mutants in patients. In chapter 5, we present a lentiviral CRISPR/Cas9 system that allows for multiplexed genome editing in hard-to-transfect cells. This platform allows for efficient multiplexed gene disruption, and the ability to remove exons or large gene clusters from human cells. Next, in chapter 6, we performed a genome-wide CRISPR library screen to identify novel host genes involved 1 in primary infection of human B cells. We identified ±20 genes, including KPNA1, PDCL and GNAS, that were important for EBV infection in human B cells. Moreover, we characterized a novel transcriptional regulator (FBXO11) and two posttranscriptional regulators (ALG3 and OTUD5) of CD21, the main attachment receptor utilized by EBV. Finally, chapter 7 summarizes the findings discussed in this thesis and debates future perspectives regarding the usage of genetic approaches to study or eradicate human viruses from cells