An analysis of wild type human cytomegalovirus

Cytomegalovirus is closely related to the herpes virus that causes cold sores. Like herpes, CMV is carried for life and can reactivate at any time to cause disease. Most people worldwide are infected with CMV, yet do not know. In hospitals , however, CMV is well known and recognized to be an important pathogen. CMV can cause severe multi-organ disease in individuals whose immune system is suppressed by drugs (bone marrow, heart or kidney transplant recipients), compromised by HIV-AIDS and also when the virus crosses the placenta to infect the foetus. CMV is the largest and most complex of any human virus, estimated to have some 184 genes. CMV researchers have increasingly been concerned about the viruses they have been using in the laboratory. CMV has evolved with us to grow in us. Before the virus will grow in vitro it mutates and changes; always the same genes first: RL13 and UL128 first. We already know a fair bit about UL128. Counterintuitively, RL13 suppresses the growth of CMV in all cell types. Why would a virus do that? We suspect it is to allow the virus to persist and spread in our tissue without alerting the immune response. Since this gene has a major effect on virus growth, it is crucial to work out what it is doing and how. Moreover, it is important to work out how to grow and work with the virus that causes clinical disease. Up until now, our understanding of CMV has been almost exclusively based on work with mutant viruses.

What do these ~184 genes do? Surprisingly only a minority are needed by the virus to grow, the majority appear to be there to control us and our immune system! As most of us carry the virus, this is a major concern. CMV is known to alter the repertoire of out white blood cells even in healthy carriers. We have nearly finished cloning all 184 genes into a different (replication-defective/disabled) virus, so we can study all CMV?s genes individually. Using this gene library and high throughput screening systems we seek to identify characterise as many of the key immune regulatory genes in this virus as possible. The more we know about this virus, the better we can manage disease and develop antiviral strategies.

Genetic changes in the human cytomegalovirus (HCMV) genome during growth in cell culture are associated with loss of virulence and immunomodulatory functions. To address this issue, low passage clinical isolate Merlin was cloned into a bacterial artificial chromosome (BAC). By passages 1-3 in fibroblasts, mutations in RL13 and UL128 had emerged and were then captured in the BAC. These defects were seamlessly repaired to generate (uniquely) an infectious clone of genetically-intact HCMV. Significantly, the repaired virus, and independent clinical isolates, exhibit severe growth impairment until mutants at this locus (re-)emerge. We seek to investigate the molecular mechanisms responsible for RL13 repression of replication, and (ii) whether RL13 expression is compatible with efficient virus replication and secretion in vivo. In order to be able to produce homogeneous RL13+ virus stocks, we engineered HCMV to provide conditional RL13 expression. RL13 is hypervariable and encodes a highly glycosylated virion envelope protein, making gpRL13 a potential target for neutralising antibody. A recent monovalent gB vaccine study demonstrated that humoral immunity can prevent HCMV transmission. In this context, there is an urgent need to identify the full complement of proteins/antigens presented on the surface of the wild-type virus. Having developed the capacity to propagate phenotypically wild-type (RL13+) virus, we now seek to define the proteome of such virions.

Our chief objective in accurately defining HCMV gene usage was to provide a reliable basis for studying HCMV immune evasion and modulation. In this context, we seek to systematically investigate the effect of HCMV productive infection on 244 cell surface markers in three cell types. The most significant changes in cellular gene expression will be analysed in detail. Previously whenever HCMV has been demonstrated to modulate a cellular function, there was no obvious means to identify the gene responsible. With MRC support, we have been cloning all 184 predicted HCMV genes into an adenovirus vector as the basis for a high throughput screening system. Interferons and TNF receptor family members together provide extremely effective barriers to virus infection. Remarkably, while HCMV clearly targets multiple member of the TNFR superfamily, the genes responsible have not been identified. While HCMV interferon resistance genes have been mapped, the list may well be incomplete. We therefore seek to screen the complete HCMV genome for novel immunomodulatory functions taking advantage of the luciferase reporter to facilitate high throughput assays.