An analysis of wild type human cytomegalovirus

Lead Research Organisation: Cardiff University
Department Name: School of Medicine

Abstract

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.

Technical Summary

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.

Publications

10 25 50

publication icon
Gatherer D (2011) High-resolution human cytomegalovirus transcriptome. in Proceedings of the National Academy of Sciences of the United States of America

publication icon
Murrell I (2017) The pentameric complex drives immunologically covert cell-cell transmission of wild-type human cytomegalovirus. in Proceedings of the National Academy of Sciences of the United States of America

publication icon
Patel M (2018) HCMV-Encoded NK Modulators: Lessons From and Genetic Variation. in Frontiers in immunology

publication icon
Seirafian S (2014) Human cytomegalovirus suppresses Fas expression and function. in The Journal of general virology

publication icon
Stacey MA (2011) IL-10 restricts activation-induced death of NK cells during acute murine cytomegalovirus infection. in Journal of immunology (Baltimore, Md. : 1950)

publication icon
Wang EC (2012) On death receptor 3 and its ligands. in Immunology

publication icon
Wang EC (2011) CD4+ T cells, human cytomegalovirus and end-stage renal disease. in Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

publication icon
Wang ECY (2018) Suppression of costimulation by human cytomegalovirus promotes evasion of cellular immune defenses. in Proceedings of the National Academy of Sciences of the United States of America

publication icon
Wilkinson GW (2015) Human cytomegalovirus: taking the strain. in Medical microbiology and immunology

publication icon
Wilkinson, GW, Aicheler, R.J. And Wang, E.C.Y. (2013) Cytomegaloviruses from Molecular Pathogenesis to Intervention

 
Title HCMV gene bank 
Description Replication deficient adenovirus vector expressing all 170 HCMV open reading frames. 
Type Of Material Database/Collection of Data/Biological Samples 
Year Produced 2006 
Provided To Others? Yes  
Impact The bank was completed using this grant, but initiated previously. Novel HCMV encoded NK cell modulatory functions have been identified and characterised using this resource. 
 
Description HCMV genome analysis (Univ Glasgow) 
Organisation University of Glasgow
Department MRC - University of Glasgow Centre for Virus Research
Country United Kingdom 
Sector Academic/University 
PI Contribution We have a long standing collaboration with Professor Andrew Davison's group in the MRC Centre for Virus Research which involves viral genomics, transcriptomics and primary investigation in to HCMV pathognesis. A major output has been the characterisation and subsequent development of HCMV Merlin as the prototype clinical HCMV strain in worldwide use, also adopted by WHO to provide an international diagnostic standard. We have provided a BAC clone of the HCMV strain Merlin genome and a range of recombinant viruses.
Collaborator Contribution The group in Glasgow are world leaders in herpesvirus genomics and have developed a series of ultra efficient technologies for rapid whole genome sequencing of the HCMV genome and transcriptional analysis. While initially developed for studying HCMV strains cultured in vitro, the technologies are now established to look at clinical samples. Thus all HCMV mutants and variants generated in Cardiff have been sequenced in Glasgow (whole genome). High resolution transcriptional analysis has been performed on the HCMV strain Merlin genome. Both groups are working together to understand the clinical significance of HCMV sequence variation. Both group are contributing to a Wellcome Trust Collaborative Grant studying HCMV pathogenesis in renal transplant patients, headed by Prof Paul Griffiths (UCL) and initiated 2018.
Impact Cunningham, C., Gatherer, D., Hilfrich, B., Baluchova, K., Dargan, D. J., Thomson, M., Griffiths, P. D., Wilkinson, G. W., Schulz, T. F. and Davison, A. J. (2010). Sequences of complete human cytomegalovirus genomes from infected cell cultures and clinical specimens, J Gen Virol. 91, 605-15. Davison, A.J., Akter, P. Cunningham, C., Dolan, A., Addison, C., Dargan, D., Hassan-Walker, A.F., Emery, V.C. Griffiths P.D. and Wilkinson, G.W.G. (2003). Homology between the human cytomegalovirus RL11 gene family and human adenovirus E3 genes. J. Gen Virol. 84, 657-663. Akter, P., Cunningham, C., McSharry, B.P., Dolan, A., Addison, C., Dargan, D.J., Hassan-Walker, A.F., Emery, V.C., Griffiths P.D., Wilkinson G.W.G. and Davison A.J. (2003). Two novel spliced genes in human cytomegalovirus. J. Gen Virol. 84, 1117-1122. Dolan, A., Cunningham, C., Hector, R. D. Hassan-Walker, A.F., Lee, L., Addison, C., Dargan, D.J., McGeoch, D.J., Gatherer, D., Emery, V.C., Griffiths P.D., Sinzger, C., McSharry, B.P., Wilkinson G.W.G. and Davison A.J. (2004). Genetic content of wild type human cytomegalovirus. J. Gen Virol. 85, 1301-1312. Jacob, S.C., Davison, A.J., Car, S., Bennett, A.M., Phillpotts, R. and Wilkinson, G.W.G. (2004) Characterization and manipulation of the human adenovirus 4 genome. J. Gen Virol. 85, 3361-3366. Bradley, A.J., Kovacs, I.J., Gatherer, D., Dargan, D.J., Alkharsah, K.R., Chan, P.K., Carman, W.F., Dedicoat, M., Emery, V.C., Geddes, C.C., Gerna, G., Ben-Ismaeil, B., Kaye, S., McGregor, A., Moss, P.A., Pusztai, R., Rawlinson, W.D., Scott, G.M., Wilkinson, G.W., Schulz, T.F. and Davison, A.J. (2008). Genotypic analysis of two hypervariable human cytomegalovirus genes. J. Med. Virol. 80, 1615-23. Bradley, A. J., N. S. Lurain, P. Ghazal, U. Trivedi, C. Cunningham, K. Baluchova, D. Gatherer, G. W. Wilkinson, D. J. Dargan, and A. J. Davison. (2009) High-throughput sequence analysis of variants of human cytomegalovirus strains Towne and AD169. J Gen Virol. 90, 2375-80 Dargan, D.J. Douglas, E., Cunningham, C. Jamieson, F., Stanton,R.J., Baluchova, K., McSharry, B, Tomasec, P, Emery, V.C., Percivalle, E., Sarasini, A., Gerna, G., Wilkinson, G,.W. and. Davison A. J. (2010). Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture. J. Gen. Virol. 91, 1535-1546. Stanton, R.J*, Baluchova, K*, Dargan, D.J., Cunningham, C., Sheehy, O, Seirafian, S., McSharry, B.P., Neale, M.L., Davies, J.A., Tomasec, P., Davison, A.J. and Wilkinson, G.W.G. (2010). Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication. J. Clin Invest. 120, 3191-208. Gatherer D, Seirafian S, Cunningham C, Holton M, Dargan DJ, Baluchova, K., Hector, R. D., Galbraith, J., Herzyk, P., Wilkinson, G. W. and Davison, A. J. (2011). High-resolution human cytomegalovirus transcriptome. Proc Natl Acad Sci U S A 108: 19755-19760. Prod'homme, V., P. Tomasec, C. Cunningham, M. K. Lemberg, R. J. Stanton, B. P. McSharry, E. C. Wang, S. Cuff, B. Martoglio, A. J. Davison, V. M. Braud, and G. W. Wilkinson. (2012). Human Cytomegalovirus UL40 Signal Peptide Regulates Cell Surface Expression of the NK Cell Ligands HLA-E and gpUL18. J Immunol. 188, 2794-804. Fielding, C. A., Aicheler, R.. Stanton, R. J., Wang, E. C., Han, S., Seirafian, S., Davies, J., McSharry, B. P., Weekes, M. P., Antrobus, P. R., Prod'homme, V., Blanchet, F. P., Sugrue, D., Cuff, S., Roberts, D., Davison, A. J., Lehner, P. J., Wilkinson, G.W.G. and Tomasec, P. (2014) Two novel human cytomegalovirus NK cell evasion functions target MICA for lysosomal degradation. PLoS Pathogens, 10, e1004058. Stanton, R.J., Prod'homme, V., Purbhoo, M.A., Moore, MA, R.J., Heinzmann, M., Bailer, S.M., Haas, J., Antrobus, R., Weekes, M.P., Lehner, P.J., Vojtesek, B., Miners, K.L., Man, Wilkie, G.S. Davison, A.J., Wang, E.C.Y. Tomasec, P., and Wilkinson, G.W.G. (2014) Human Cytomegalovirus UL135 elicits efficient protection against natural killer and CD8+ cytotoxic T lymphocytes. Cell Host Microbe. 16, 201-14. Hsu, J-L., van den Boomen, D.J.H., Tomasec, P., Weekes, M.P., Antrobus, R., Stanton, R.J., Ruckova, E., Sugrue, D., Davison, A.J., Wilkinson, G.W.G. and Lehner, P.J. (2015). Plasma membrane profiling defines an expanded class of cell surface proteins selectively targeted for degradation by HCMV US2 in cooperation with UL141. PLoS Pathogens 11, e1004811. Fielding CA, Weekes MP, Nobre LV, Ruckova E, Wilkie GS, Paulo JA, Chang C, Suarez NM, Davies JA, Antrobus R, Stanton, RJ, Aicheler, RJ, Nichols, H, Vojtesek, B, Trowsdale, J, Davison, A J, Gygi, S P, Tomasec, P, Lehner, PJ and Wilkinson, GW (2017). Control of immune ligands by members of a cytomegalovirus gene expansion suppresses natural killer cell activation. eLife 6:e22206 DOI: 10.7554/eLife.22206 Suarez, N. M., Lau, B., Kemble, G. M., Lee, R., Mocarski, E. S., Wilkinson, G. W., Adler, S. P., McVoy, M. A. & Davison, A. J. (2017). Genomic analysis of chimeric human cytomegalovirus vaccine candidates derived from strains Towne and Toledo. Virus Genes. Wang, E.C.Y., Pjechova M., Nightingale K., Vlahava, V.-M., Patel. M., Ruckova, E., Forbes, S., Nobre,L., Antrobus, R., Roberts, D., Fielding,C.A., Seirafian, S., Davies, J., Murrell, I., Lau, B., Wilkie, G.S., Suárez, N.M., Stanton, R.J., Vojtesek, B., Davison, A., Lehner, P.J., Weekes, M.P. Wilkinson, G.W.G., Tomasec, P. (2018). Suppression of co-stimulation by human cytomegalovirus promotes evasion of cellular immune defenses. PNAS. 115, 4998-5003. Nightingale K., Lin, K.-M., Ravenhill, B.J., Ruckova, E., Davies, C., Nobre, L., Fielding, C.A., Fletcher-Etherington, A., Soday, L., Nichols, H., Sugrue, D., Wang, E.C.Y., Moreno, P., Umrania, Y., Antrobus, R., Davison, A.J, Wilkinson, G.W.G., Stanton, R.J., Tomasec, P. and Weekes, M.P. (2018). High definition analysis of protein stability during cytomegalovirus infection informs on cellular restriction. Cell Host Microbe 24, 447-460.
 
Description School students invied to laboratory for workexperience 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Primary Audience Schools
Results and Impact The students (6 groups of three) undertook a laboratory research project that examined the effect of intrinsic immune defences on expression from an adenovirus vector.

Many Questions were asked. Formal feed back from students was extremely positive.
Year(s) Of Engagement Activity 2011