Understanding Epstein-Barr virus infection through genetic screens

Summary

Introduction
Epstein-Barr virus (EBV) is one of the nine known human herpesviruses and establishes lifelong persistent infections in approximately 95% of the worldwide adult population. Generally, EBV infection is asymptomatic in humans, however when primary infection occurs in adolescents, the virus can cause infectious mononucleosis (IM; also known as Pfeiffer’s disease). Besides IM, EBV has potent transforming capacities and is therefore associated with a wide range of malignancies, including nasopharyngeal carcinoma (NPC), Burkitt’s lymphoma (BL) and post-transplant lymphoproliferative disorders (PTLD). PTLD remains a major clinical problem in immunocompromised patients such as transplant recipients and HIV patients.

EBV is transmitted from carrier to seronegative individuals through saliva. Upon transmission, the virus establishes a lytic infection in permissive epithelial cells within the oropharynx. Next, EBV spreads to naïve B cells that reside within nasopharyngeal lymphoid tissue (e.g. tonsils). Herein, EBV establishes a sequence of latency stages and mimics cellular differentiation pathways to mature into memory B cells. EBV enters epithelial and B cells within the nasopharyngeal region in a distinct manner, using alternative known host entry receptors.

During latency, viral protein expression is strictly limited, however approximately 45 mature micro(mi)RNAs, which are not detected by the host immune system, are produced. EBV miRNAs expression additionally occurs in EBV-associated tumors and derivative cell lines. Thereby, the miRNAs of concern are thought to play a role in cell proliferation, apoptosis and immune evasion. During recent years, studies have reported on direct host mRNA targets that are regulated by EBV miRNAs, which fulfill roles within the abovementioned cellular processes.

Scope of this thesis
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 host-pathogen 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 host-pathogen 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 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.