Targeting the RNA helicase, UAP56: understanding KSHV RNA processing mechanisms to novel antiviral approaches

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus required for the development of Kaposi's sarcoma (KS) and is also associated with two lymphoproliferative disorders; primary effusion lymphoma and multicentric Castleman's disease. At present, there are no specific KSHV antivirals or vaccines and current treatments for KSHV-associated diseases are not targeted relying on rebuilding the immune system and using cytotoxic agents. As KS is an AIDS-defining disease, controlling HIV/AIDS using regulated immune reconstitution has been investigated as a possible treatment for KS. Although, even with effective Antiretroviral Therapy (ART) and well-controlled HIV infection, many patients still develop progressive KS. Consequently, specific, efficacious anti-KS therapies are still urgently needed. These antivirals would significantly enhance current treatments by (a) working in combination with ART, (b) inhibiting KSHV replication associated with transplant immunosuppression or (c) clearing the latently-infected reservoir by reactivating the virus and simultaneously blocking lytic infection.

Like all herpesviruses KSHV has two distinct life cycles, a persistence life-long infection (latency) and infectious productive cycle (lytic replication). KSHV lytic replication plays an important part in the pathogenesis of KSHV infection. Therefore, it is essential to study the molecular mechanisms which regulate lytic replication to fully understand KSHV pathogenesis. Moreover, inhibiting KSHV lytic replication may provide an opportunity to develop novel antiviral strategies to inhibit KS formation. We have exciting data demonstrating a novel approach to inhibit KSHV lytic replication. We have identified a small molecule which disrupts an essential virus-host cell interaction, the KSHV ORF57-cellular hTREX interaction, required for the correct processing of viral mRNAs.

The ability to specifically disrupt the ORF57-hTREX interaction using this small molecule now presents a novel opportunity to investigate other roles of the ORF57-hTREX interaction in KSHV lytic replication, in particular determining the role of the ORF57-hTREX interaction in enhancing the correct processing of a subset of cellular RNAs, which are also bound by the viral ORF57 protein, and are thought to enhance virus replication. Therefore, we will assess the global changes in cellular mRNA abundance and protein production, using transcriptomic and proteomic approaches, during KSHV latent versus lytic replication in the absence or presence of the small molecule. This will identify cellular transcripts which are stabilised and processed by the ORF57-hTREX interaction. Further analysis will then be performed to examine how ORF57 recognises and binds this subset of cellular transcripts and determine if these cellular transcripts are essential for virus replication.

In addition, we wish to further investigate the antiviral potential of the small molecule which disrupts the ORF57-hTREX interaction. We have shown that the small molecule termed, CCT018159, can inhibit KSHV lytic replication and infectious virion production in a cell culture system. The next stage will to be to explore the inhibitory activity of CCT018159 in an appropriate small animal model for human gamma-herpesvirus infection. However, although CCT018159 can be used in this setting, the small molecule has a relatively high metabolic turnover which may reduce its efficacy. Therefore, we will modify the small molecule to enhance its antiviral activity and pharmacokinetic properties using medicinal chemistry approaches. The small molecule and derivatives will then be tested for their ability to inhibit the replication of the murine gamma-2 herpesvirus, murine Gammaherpesvirus 68, in an in vivo setting.

The KSHV ORF57 protein orchestrates the formation of vRNPs by recruiting the cellular hTREX complex onto viral mRNAs. We now demonstrate that vRNP formation is driven by an ATP-cycle-dependent remodelling event of hTREX, required for ORF57 binding. Excitingly, this ATP-dependent formation of vRNPs can be prevented by a small molecule, CCT018159, which inhibits the ATPase activity of the hTREX component, UAP56. Further results have demonstrated the effective CCT018159-mediated inhibition of KSHV vRNP formation, lytic replication and infectious virion production.

The ability of CCT018159 to specifically disrupt the ORF57-hTREX interaction now presents an ideal opportunity to firstly investigate how KSHV ORF57 manipulates cellular mRNA processing. We present data showing that ORF57 sequestration of hTREX drastically inhibits bulk cellular mRNA nuclear export. However, ORF57 has also been reported to associate with a subset of cellular transcripts, as well as viral mRNAs. We therefore hypothesize that KSHV ORF57 binds this subset of cellular transcripts, enabling hTREX recruitment, to form ORF57-induced cellular RNPs, overriding virus-induced host cell shutoff mechanisms. In turn, we believe these ORF57-processed cellular transcripts probably enhance KSHV replication. As such, our small molecule inhibitor is an ideal tool to test this hypothesis and identify which ORF57-dependent cellular mRNAs are essential for KSHV lytic replication. Secondly, as the ORF57-hTREX interaction is essential for KSHV lytic replication, CCT018159 is a potentially exciting therapeutic reagent for KSHV infection. We have demonstrated the ability of CCT018159 to inhibit KSHV lytic replication and infectious virion production in cell culture-based experiments. We now aim to further develop CCT018159 to enhance its antiviral activity and pharmacokinetic properties prior to assessing its antiviral potential in an appropriate small animal model for human gamma-herpesvirus infection.

Herpesviruses are responsible for a range of debilitating acute and recurrent diseases, including a number of malignancies. Current treatments are limited to targeting herpesvirus DNA polymerases, but with emerging viral resistance and little efficacy against the oncogenic herpesviruses, there is an urgent need for new antiviral strategies. The research outlined in this interdisciplinary proposal therefore has the potential to benefit multiple different sectors.

Academic scientists: As outlined in the academic beneficiaries section, this project has the potential to increase our fundamental knowledge into virus-mediated mechanisms of RNA processing events which are essential for herpesvirus replication. In addition it will provide new impetus to investigate the role of dynamic RNA modifications in virus infection.

Industry: The work conducted in this project will be of direct interest to companies engaged in drug development. Research findings may lead to the identification of new therapeutic targets for herpesvirus infection. Moreover, by studying how to inhibit virus infection this project may also facilitate the new development of bio-pharmaceuticals for herpesviruses and other virus infections.

General public and clinicians: Engagement activities will increase the understanding of a range of herpesvirus-associated diseases by the general public and promote the need for research into these common worldwide infections. In the longer term, the development of new therapeutics for herpesvirus infections, which are urgently needed, will benefit the general public and the medical sector.

Young and future scientists: The training of PDRAs and a technician in interdisciplinary science will produce highly skilled scientists. Younger scientists, working as summer students, will also benefit from working in cutting edge science laboratories. Moreover, virus infections and related diseases are fascinating topics for public engagement activities to promote science and scientific careers to school children.