Systematic identification of host proteins involved in Human Cytomegalovirus replication, assembly and egress.

Human cytomegalovirus (HCMV) is an important human pathogen, causing significant problems in transplant patients, where infection can increase the speed and severity of organ rejection as well as causing sever disease in its own right due to immunosuppression of patients. It is also the leading infectious cause of birth defects.

There is currently no effective vaccine and available antiviral therapies have significant issues including side effects and development of resistance. The development of new antiviral treatments against HCMV is therefore a high priority for effective control in transplant and immunodeficient patients.

Very few antivirals have been developed for use against HCMV since the licensing of Ganciclovir and, of these, the same viral genes are targeted, reducing the likely usefulness of these drugs against resistant strains. An alternative strategy for the development of novel antivirals involves targeting of host genes required by the virus for successful replication. The relatively low stability of viral genomes results in rapid development of resistant strains against antiviral drugs that directly target viral genes. Even before treatment, virus populations often contain strains with mutations that render them resistant to such drugs. In contrast developing resistance against drugs that target host genes would be far more complex, as the virus would need to gain mutations that would compensate for the loss of a required cellular gene. In many cases such mutations may not exist.

By inhibiting individual host genes we have identified multiple host genes that are involved in human cytomegalovirus replication. We have shown that one of these host genes, VCP, is essential for one of the earliest steps in HCMV replication. Inhibition of this gene by a drug called NMS-873, resulted in complete inhibition of virus replication, validating this strategy for antiviral discovery and suggesting NMS-873 may be a potent future treatment option for HCMV infections.

In this application we aim to further characterise how VCP is involved in HCMV replication as well as characterising additional host factors that may be potential drug targets for HCMV treatment. These findings will have important implications for the development of novel antiviral treatments, understanding the role of host factors in HCMV replication as well as understanding the function of host factors themselves.

Disease outcome from virus infection depends on complex host-pathogen interactions. Rational design of therapies, vaccines and disease control depends on understanding these interactions at the molecular level. Identification of host virus interactions provide important potential avenues for novel therapeutics as development of resistance to drugs targeting host genes is less likely than with virus targets. Furthermore, small molecule inhibitors are already available for many host genes.

Genome wide siRNA screens have proven a useful tool for deciphering which cellular genes are required for virus replication and which genes have antiviral properties. As well as contributing to our understanding of virus host interactions, siRNA screens are a powerful approach for the identification of antiviral drug targets. The majority of screens performed measure virus replication through the expression of a reporter construct. Although this identifies entry and replication phenotypes, the crucial parameter of virus production is not considered.

We have devised a novel siRNA screen that measures both primary replication but crucially, also measures infectious progeny. Using a library of more than 160 siRNA pools against host genes involved in membrane organisation, we have identified multiple genes required for HCMV virus replication, assembly and egress. Crucially we have shown that the correlation between primary replication and infectious progeny is far lower than would be expected, indicating that measurement of primary replication alone may be misleading, especially when attempting to identify host targets for antiviral therapy. Further characterisation reveals the top hit from our screen, VCP/p97, is required for expression of the crucial viral trans-activator IE86. Despite being co-expressed, IE72 protein levels are unaffected. In addition, we show that a small molecule inhibitor of VCP is a potent anti-HCMV drug.

There are three major areas in which this research will have significant impact. Clearly, the identification of a novel and potent inhibitor of HCMV replication has important implications for the treatment of individuals in which HCMV causes potentially life-threatening complications. Although HCMV is not an acute problem in the majority of the population, in transplant patients it is still a major factor in health and quality of life. Current antivirals have significant drawbacks including side effects and there is a lack of options when resistance develops to the currently available drugs. If NMS-873 proved to be a viable drug therapy for HCMV it would be a major step forward in treating infections in susceptible populations.

This work also provides support for the identification of antiviral candidates using high throughput techniques. Besides the lower risk for the development of resistance, the other major benefit of identifying potential cellular targets for therapy is the extensive libraries of inhibitors already developed against cellular factors. For example, NMS-873 was originally developed as an anti-cancer therapy. We will test additional small molecule inhibitors of cellular factors identified in this screen, where they exist, against HCMV replication. We will also test these drugs against other viruses to determine whether they have broad activity.

Our data demonstrates a lack of correlation between primary replication and infectious progeny. This data will have a major impact on other studies involving high throughput screens of pathogens. In most cases such screens only measure infectious progeny on candidates identified initially through primary replication levels. Such strategies fail to identify assembly and egress factors and can result in false positive antiviral candidates. The approach we have developed could also be broadly applied to other pathogens for the identification of antiviral targets and host factors involved in assembly and egress.

Finally, the protocols, reagents and strategies being developed as a result of this project will be of use to others at the Roslin institute for the development of high throughput projects with other pathogens. In addition, funding of this application would have a major impact on the consolidation and on going research and training of a new investigator.