Modulation of Natural Killer Cell By Human Cytomegalovirus

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

Abstract

Cytomegalovirus (CMV) is 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. However, in hospitals, CMV is well known and a major problem. CMV can cause severe multi-organ disease in individuals whose immune system has been compromised by infections (e.g. HIV AIDS), suppressed by drugs (for bone marrow, heart or kidney transplants) and also when it infects unborn children in the womb. CMV is the most complex of any human virus with 167 genes. What do they all do? Surprisingly, most of are not needed by virus to grow, rather they may be there to attack our immune system.

Natural Killers (NK) cells are crucial in fighting infections (CMV in particular) and cancers. NK cells detect signals on the surface cells that indicate whether things are OK or not. If they are not, NK cells are fierce ? rapidly both killing the problem cell and triggering the immune system to respond. Only recently have we begun to understand what signals NK cells are responding to ? and there are many. Surprisingly, CMV has helped tell us how NK cells work.

Eight CMV genes have been identified that prevent NK cells from killing. These CMV genes affect the expression of cellular proteins recognised by NK cells. Our laboratory has been heavily involved in this work. Because HCMV has an enormous genome, finding these genes is been tough. We know the virus encodes many more NK evasion functions, although not how many. Over the past 5 years, we have worked hard to develop a hyper-efficient system to screen the whole genome, and now this system is ready.

We want to identify and characterise as many NK evasion genes as possible. How these genes work, will tell us how NK cells work. There is also good evidence in a mouse model that these genes are essential for the virus to cause disease. With scientists in the US, we intend to determine whether this is true in primates. This study is important to understanding how CMV causes disease. However, by identifying and knocking these NK evasion genes out of the virus, it may yet be possible to develop a CMV vaccine.

Technical Summary

Human cytomegalovirus (HCMV) has the largest genome (~236Kb) of any characterised human virus encoding 167 putative open reading frames (ORFs). Although this pathogen is carried by majority of the world?s population, the much of the HCMV genome remains uncharacterised. The complexity of the genome makes the mapping of even recognised viral functions to specific HCMV genes extremely problematical. Natural Killer (NK) cells are crucial in controlling cytomegalovirus infections. A substantial proportion of the HCMV genome acts to suppress NK recognition; to date eight HCMV NK evasion functions have been characterised even though only a fraction of the genome has been sampled. We propose exploiting DNA recombineering technology to generate a bank of 167 replication-deficient adenovirus (Ad) vectors each encoding a discrete, epitope-tagged HCMV ORF. Both Ad recombinants and HCMV proteins expressed in a prokaryotic system will be used to generate specific antibodies to support expression studies. A compete HCMV gene bank of Ad recombinants will be an invaluable resource for the HCMV research community. Our specific goal is to perform functional NK assays, using CD107 mobilisation and intracellular interferon-gamma staining, to identify HCMV gene function capable of promoting and suppressing NK recognition. Constructs encoding rhesus CMV homologues of HCMV NK evasion functions will also be evaluated to facilitate studies in the closest tractable in vivo model for HCMV.

Publications

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Bradley AJ (2008) Genotypic analysis of two hypervariable human cytomegalovirus genes. in Journal of medical virology

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Clark SR (2013) Characterization of platelet aminophospholipid externalization reveals fatty acids as molecular determinants that regulate coagulation. in Proceedings of the National Academy of Sciences of the United States of America

 
Description MRC I3-IRG PhD Studentship
Amount £50,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2006 
End 09/2009
 
Description Mrs John Nixon Scholarship (PhD studentship)
Amount £50,000 (GBP)
Organisation Cardiff University 
Sector Academic/University
Country United Kingdom
Start 10/2008 
End 09/2011
 
Description Programme Grant
Amount £1,062,032 (GBP)
Funding ID WT090323 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2010 
End 09/2015
 
Description Project Grant
Amount £445,567 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2008 
End 09/2011
 
Title Adenovirus Vector Bank of HCMV ORFs 
Description Adenovirus vectors encoding nearly all HCMV open reading frames have been generated 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact We have characterised the expression of nearly all ORFs RL1-UL46. We have identified 4 candidate NK modulatory functions. 
 
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 Investigations into HCMV gene usage 
Organisation Medical Research Council (MRC)
Department MRC Virology Unit
Country United Kingdom 
Sector Public 
PI Contribution We have provided HCMV clinical isolates, HCMV BAC vector and Ad recombinants to assist in studies in Glasgow. We have hosted a researcher in our laboratory to facilitate technology transfer.
Collaborator Contribution There has been a major problem in the HCMV field. The virus genome degenerates whenever it is propagated in vitro. Consequently, the gene content of HCMV genome was ill-defined. We have addressed this issue together. HCMV strain Merlin, a Cardiff isolate, was sequenced and annotated by Dr Davison's group in Glasgow. HCMV strain Merlin proved the first complete sequence of an HCMV clinical isolate, and consequently became the prototype strain on GenBank. In followup studies a number of additional HCMV clinical isolates have been sequenced. Such analyses it has been revealed that a number of HCMV genes are hypervariable, RL13 and UL128 locus mutate in cell culture and that some viruses present in clinical isolates have mutant genes. We cloned the complete genome of strain Merlin in a bacterial artificial chromosome (BAC). Defect that had arisen by minimal passage in vitro were identified and corrected by reference to the original clinical sample. For HCMV to replicate efficiently in human fibroblasts it has two acquire mutations in two loci. We are in the process of characterising the RL13 gene in collaboration with Dr Davison's laboratory. A basic understanding of HCMV gene usage has been crucial for our studies of virus gene function.
Impact Wilkinson G.W., Tomasec P., Stanton R.J., Armstrong, M., Prod'homme, V., Aicheler, R., McSharry, B.P., Rickards, C.R., Cochrane, D., Llewellyn-Lacey, S., Wang, E.C., Griffin, C.A. and Davison, A.J. (2008). Modulation of natural killer cells by human cytomegalovirus. J. Clin. Virol. 41, 206-12. 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. Cunningham, C., D. Gatherer, B. Hilfrich, K. Baluchova, D. J. Dargan, M. Thomson, P. D. Griffiths, G. W. Wilkinson, T. F. Schulz, and A. J. Davison. 2009. Sequences of complete human cytomegalovirus genomes from infected cell cultures and clinical specimens. J Gen Virol. 91, 605-15. 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