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Project

Understanding gamma-herpesvirus pathogenesis and development of novel treatment strategies.

At this moment, two human γ-herpesviruses have been identified i.e. Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated virus (KSHV). EBV was discovered in 1964 in an endemic Burkitt's lymphoma tissue. The first report of a Kaposi sarcoma dates back from 1872, though only in 1994 KSHV was identified to be the etiological agent.

An EBV primary infection is mostly known to cause infectious mononucleosis when it occurs in adolescence or early adulthood. A KSHV primary infection is less well defined and symptoms can include fever, lymphadenopathy and maculopapular rashes. In immunocompetent people, infection with human γ-herpesviruses leads to an equilibrium between the host immune system and the virus, resulting in the survival of the virus without disease development. However, in immunocompromised patients, latent infection can be associated with the development of various lymphoproliferative disorders and epithelial/endothelial malignancies.

Seeing the high burden of γ-herpesvirus-associated diseases, there is need for further basic research on human γ-herpesviruses, but also for new therapeutic strategies such as development of vaccines, virus specific cancer treatments and antiviral drugs. However, the development of EBV- and KSHV-targeted treatment strategies has been hampered by the lack of in vitro cell culture system permissive for efficient lytic viral replication. To overcome this, γ-herpesviruses are frequently studied using surrogate models such as the murine γ-herpesvirus-68 (MHV-68).In this thesis, we investigated multiple aspects of γ-herpesvirus antivirals. Using MHV-68, we studied antiviral resistance development to known nucleoside, nucleotide and pyrophosphate analogues and evaluated the impact that drug resistance mutations can have on herpesvirus replication capacity. Moreover, using different EBV-infected B-cell lines from Burkitt’s lymphoma origin, we determined different factors influencing the activation of a cyclic nucleoside phosphonate, cyclic-HPMPC.

First, we investigated the impact of specific thymidine kinase (TK) or protein kinase (PK) mutations on the viral replication capacity. The mutations in this chapter were previously identified in our laboratory. The viral replication capacity was determined using dual infection competition assays in which wild-type and drug-resistant virus compete in absence or presence of antivirals and the composition of the mixed viral population was analyzed using next-generation sequencing.

Second, we investigated the mechanism of MHV-68 antiviral resistance to nucleoside (ganciclovir), nucleotide (cidofovir, HPMP-5azaC, HPMPO-DAPy) and pyrophosphate (foscarnet) analogues and evaluated the impact of these drug resistance mutations located in the viral DNA polymerase (DP) on viral fitness. Two drug-resistant viral clones, linked to the DP amino acid changes C297W and C981Y, were identified to be mutator phenotype viruses. The mutator phenotype caused by C297W in MHV-68 DP was validated using a CRISPR/Cas9 reverse transfection/infection approach.

Lastly, we evaluated the effects of a single nucleotide polymorphism leading to the heterozygote amino acid change Q207R on the ability of the cellular enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) to metabolize cyclic-HPMPC (cyclic-cidofovir) and identified a relationship between EBV latency protein expression and CNPase protein expression and ability to metabolize cyclic-HPMPC. CNPase is part of the 2′,3′-cAMP-adenosine pathway and catalyzes the hydrolysis of nucleoside 2′,3′-cyclic monophosphates to nucleoside 2′-monophosphates in vitro, though in vivo, the function of cellular CNPase is largely unknown and the physiological substrate is still unclear. Besides this, we demonstrated the heterogeneity of the BL cell line Jiyoye by identifying a subpopulation with an altered EBV latency type caused by a 14 kb deletion of the viral genome and that is integrated in the host cell genome.

Since drug resistance is inherent to viruses, research on the mechanisms of antiviral resistance and impact of resistance mutations on the viral fitness is valuable. Our results allowed to compare mutation patterns among different herpesviruses, and provided a deeper understanding of the impact of drug resistance and antiviral pressure on viral fitness. Besides this, we highlighted how cellular and/or viral factors can affect the activation of cyclic-prodrugs.

Date:1 Aug 2014 →  3 Dec 2020
Keywords:gamma-herpesvirus, pathogenesis, treatment strategies
Disciplines:Microbiology, Systems biology, Laboratory medicine
Project type:PhD project