The gammaherpesvirus thymidine kinase
Lead Research Organisation:
University of Cambridge
Department Name: Virology
Abstract
Herpesviruses are ubiquitous infections and cause many clinical problems in immunosuppressed patients. The most effective treatment to date has been anti-viral drugs such as aciclovir, which exploit a viral enzyme called thymidine kinase (TK). The gamma-herpesviruses have this enzyme, but it is a much weaker enzyme than that of alpha-herpesviruses, against which aciclovir was developed. We suspect that the gamma-herpesvirus TK has different functions, and might therefore best be attacked through different types of drugs.
The human gamma-herpesviruses only infect primates, making many experiments impractical. We are therefore defining what the gamma-herpesvirus TK does using a related murine virus, called MHV-68. We know that the MHV-68 TK is important for viral replication in mice. We aim now to work out why, specifically whether it works in the same way as the alpha-herpesvirus TK, or whether it has very different functions that would be best inhibited in different ways. By understanding how the MHV-68 TK works, we should be able to provide a basis for effective TK-based therapies in gamma-herpesvirus infections.
The human gamma-herpesviruses only infect primates, making many experiments impractical. We are therefore defining what the gamma-herpesvirus TK does using a related murine virus, called MHV-68. We know that the MHV-68 TK is important for viral replication in mice. We aim now to work out why, specifically whether it works in the same way as the alpha-herpesvirus TK, or whether it has very different functions that would be best inhibited in different ways. By understanding how the MHV-68 TK works, we should be able to provide a basis for effective TK-based therapies in gamma-herpesvirus infections.
Technical Summary
Herpesviruses are major causes of disease. Thymidine kinase (TK)-based therapies have proved very effective against alpha- and beta-herpesviruses. Analogous therapies, combined with lytic cycle induction, are therefore being developed against gamma-herpesviruses. However, it is unclear whether gamma-herpesvirus TKs are equivalent to those of alpha-herpesviruses. They are larger than alpha-herpesvirus TKs, differently distributed in cells, and much less active in nucleoside phosphorylation. Because gamma-herpesviruses do not establish latency in terminally differentiated cells, they may have less need to phosphorylate nucleotides directly for their replication, allowing TK to evolve other functions.
Viral escape mutants inevitably limit the efficacy of drug treatments. It is therefore important for TK-based therapies to understand what role TK plays in gamma-herpesvirus pathogenesis. We also need to improve our basic understanding of these ubiquitous and complex pathogens. Human gamma-herpesviruses lack good experimental lytic replication systems. We and others have therefore developed MHV-68 - a murid gamma-herpesvirus - as a model system for analyzing conserved gamma-herpesvirus lytic gene functions in infected cells and infected animals. TK-deficient MHV-68 is severely attenuated for host colonization. Preliminary data suggest that this reflects a role for TK not in DNA replication in quiescent cells but in viral capsid transport, especially on entry into myeloid cells. This finding has important implications both for how TK-directed therapies might work and for how gamma-herpesviruses normally colonize their hosts.
Our aim now is to define a molecular basis for gamma-herpesvirus TK function, comparing EBV, KSHV and MHV-68. We will focus mainly on virus-infected cells by generating BAC-based MHV-68, EBV and KSHV TK mutants. We will identify how the large N-terminal domain shared by all gamma-herpesvirus TKs contributes to its function, particularly what cellular proteins it interacts with and how its function relates to nucleoside phophorylation. Using MHV-68, we will establish how the molecularly defined TK function explains the dramatic in vivo attenuation of TK-deficient mutants. Our overall aim is to establish a general model of how gamma-herpesvirus TKs work, which should both inform an important area of anti-viral therapy and provide significant new insights into basic gamma-herpesvirus biology.
Viral escape mutants inevitably limit the efficacy of drug treatments. It is therefore important for TK-based therapies to understand what role TK plays in gamma-herpesvirus pathogenesis. We also need to improve our basic understanding of these ubiquitous and complex pathogens. Human gamma-herpesviruses lack good experimental lytic replication systems. We and others have therefore developed MHV-68 - a murid gamma-herpesvirus - as a model system for analyzing conserved gamma-herpesvirus lytic gene functions in infected cells and infected animals. TK-deficient MHV-68 is severely attenuated for host colonization. Preliminary data suggest that this reflects a role for TK not in DNA replication in quiescent cells but in viral capsid transport, especially on entry into myeloid cells. This finding has important implications both for how TK-directed therapies might work and for how gamma-herpesviruses normally colonize their hosts.
Our aim now is to define a molecular basis for gamma-herpesvirus TK function, comparing EBV, KSHV and MHV-68. We will focus mainly on virus-infected cells by generating BAC-based MHV-68, EBV and KSHV TK mutants. We will identify how the large N-terminal domain shared by all gamma-herpesvirus TKs contributes to its function, particularly what cellular proteins it interacts with and how its function relates to nucleoside phophorylation. Using MHV-68, we will establish how the molecularly defined TK function explains the dramatic in vivo attenuation of TK-deficient mutants. Our overall aim is to establish a general model of how gamma-herpesvirus TKs work, which should both inform an important area of anti-viral therapy and provide significant new insights into basic gamma-herpesvirus biology.