Project 1: Novel role for neutrophils in CMV dissemination and reactivation
Andrew Cowburn1, Matthew Reeves2, Edwin Chilvers1
Divisions of 1Respiratory Medicine and 2Infectious Diseases, Department of Medicine, University of Cambridge School of Clinical Medicine
Human cytomegalovirus (hCMV) remains a major cause of viral disease in transplant recipients, patients who are critically ill and those with late stage HIV infection. To date, the interaction between neutrophils and hCMV remains contentious and, as such, no precise role for neutrophil function in hCMV infection has been defined. Our recent studies however have shown clear evidence for hCMV binding to, and activation of, human neutrophils, which triggers a profound survival response in these cells and also the production of a soluble factor(s) that results in monocyte chemotaxis, differentiation, and strikingly, hCMV reactivation in latently infected cells. Furthermore, the observation that these events occur in the absence of any lytic viral gene expression suggests that the interaction at the point of virus entry is an important mediator of this response. This establishes the hypothesis that during primary infection, binding of hCMV to neutrophils (which are themselves recruited via hCMV-induced epithelial CXCL-1) triggers a cytokine response that promotes monocyte recruitment and differentiation, which in turn supports more efficient virus dissemination. In the context of latent infection, hCMV reactivation and disease is common in patients who are exposed to other primary infections and thus we hypothesise that neutrophil activation by an alternative viral or bacterial infection, could also result in the production of a cytokine milieu that promotes hCMV reactivation in recruited myeloid cells harbouring latent hCMV genomes.
This project, which represents a new collaboration between the ID (Reeves/Sinclair) and Respiratory (Cowburn/Chilvers) labs, will: (1) define the precise neutrophil-derived factor(s) responsible for hCMV reactivation in human monocytes, (2) address if other viral, bacterial or pathogen-derived molecules including inflammatory cytokines can initiate a similar neutrophil response, (3) examine patients with sepsis to see if this causes a generic expansion of the circulating myeloid-derived suppressor cells (MDSC) subset and determine the extent of hCMV carriage in these cells, and (4) explore whether the neutrophil-derived factor(s) identified can, ultimately, reactivate naturally latent hCMV from blood and tissue (BALF) monocytes/dendritic cells. This project will give broad based and specific training in several aspects of neutrophil cell and molecular biology, hCMV infection and cellular immunology.
This project is supported by a collaboration with Professor JH Sinclair, Professor of Molecular Virology, Department of Medicine.
Project 2: RNA biology of non-coding RNAs in Cytomegalovirus Infection
Herpesviruses are large DNA viruses which, upon primary infection, establish a life-long, latent infection leaving the infected individual at risk of subsequent reactivation and disease. Over millions of years of co-evolution with their animal and human hosts they have mastered host-cell modulation to aid their needs. Thus, they provide ideal tools to study many fundamental cellular processes.
Recently, the identification of a new class of regulatory RNA molecules termed microRNAs (miRNAs) has resulted in a paradigm shift in our understanding of how cellular gene expression is regulated. MicroRNAs are small, single stranded RNA molecules of ~22 nucleotides that affect gene expression at post-transcriptional level. They regulate gene expression of ~30% of our genes and are key regulators in virtually all cellular processes. There is now increasing evidence that the expression of miRNAs is strongly regulated, and that these molecules are not as stable as they were initially thought to be. They appear to be part of a sophistically tuned, complex reciprocal interaction network with the mRNA targets they regulate.
Herpesviruses not only utilize the miRNA machinery to express their own set of miRNAs, but also alter the expression of cellular miRNAs to match their needs. As such, we just identified a novel highly abundant transcript of the murine cytomegalovirus (MCMV), m169, which efficiently targets two cellular miRNAs, namely miR-27a and b, for degradation. This is mediated by binding of the m169 transcript to miR-27 and involves 3’-miRNA tailing and trimming and is important for efficient virus replication in vivo (Marcinowski et al., PLoS Pathogens 2012, in press). The underlying mechanism, however, is only poorly understood.
Although the m169 transcript accounts for ~5% of all transcripts in an MCMV infected cell at late stages of infection, it is only poorly translated. This block in translation is mediated by its 5’-UTR. This apparently contains a small upstream ORF (uORF), deletion of which results in an over 100-fold increase in m169 protein levels (unpublished data). Due to its massive abundance and uORF-mediated regulation we propose the m169 transcript to represent the functional ortholog of the human cytomegalovirus β2.7 transcript.
Although uORFs are found in ~50% of all human genes, the regulatory molecular mechanisms involved are mostly unknown. At least in parts, this is due to lack of proper techniques to study complex RNA-protein interactions. This problem can now be overcome by combining PAR-CLIP (photo-activated ribonucleotide-enhanced UV cross-linking followed by immunoprecipitation) with SILAC (selective isotope labeling of amino acids in cell culture).
In summary, the aim of this project is to combine PAR-CLIP and SILAC to identify novel RNA binding proteins involved in a) target-mediated miRNA degradation and b) uORF mediated post-transcriptional regulation of MCMV m169 and HCMV β2.7. In addition, reverse virus genetics will be employed to characterize their functional role in the regulation of gene expression in vitro and in vivo.
Project 3: Genetic and functional mechanisms of Primary Immunodeficiencies
Primary Immunodeficiencies are a heterogeneous group of more than 200 diseases caused by Mendelian mutations in the immune genes. Primary Immunodeficiencies manifest as severe recurrent infection and often can be life-threatening. Mutations in some patients have been identified previously using linkage analysis and candidate gene approach. These discoveries proved to be extremely informative for understanding of the human immune system. Nevertheless, genetic causes in the majority of patients with Primary Immunodeficiencies remain unknown.
The aims of the proposed PhD project are to discover new causative mutations in patients with Primary Immunodeficiencies, to investigate functions of the affected proteins and to uncover new mechanisms of susceptibility to infection. Initially, advanced methods of human genetics will be employed, such as sequencing of exomes and whole genomes of patients, focusing primarily on those suffering from mycobacterial infections. Then a variety of molecular and cell biology techniques will be used, including quantitative PCR, Western blotting, cytokine secretion analyses, immunofluorescence confocal microscopy, electron microscopy, RNA interference, gene cloning and in vitro infection models. These techniques are established in the laboratory and training will be provided. Discovery of new genetic and functional mechanisms of Primary Immunodeficiencies will improve understanding of the immune system and will allow to design new diagnostic assays and new therapeutic approaches that will save patients’ lives.
Project 4: The role of the KCNJ5 K-channel in aldosterone release and aldosterone-dependent hypertension
It has recently been reported that a rare monogenic syndrome with hypertension and massive adrenal adenomas (type III familial hyperaldosteronism) is caused by germ line mutations in the KCNJ5 K channel Science 2011;331(6018):768-72. It also transpires that sporadic Conn’s adenomas frequently carry similar somatic KCNJ5 mutations. We have now confirmed this in a large cohort of sporadic aldosteronomas from Cambridge and Brisbane Australia (Hypertension 2012, in press copy attached and further submission). However, it is not clear how these mutations in the selectivity filter of the KCNJ5 channel affect aldosterone release and whether they are important in the molecular pathogenesis of Conn’s tumours. The project will directly address these issues making extensive use of an established adrenal cortical cell line H295R (although we are looking at developing a transgenic mouse if BHF funding applications are successful). We have worked extensively with the H295R and are currently looking at the patch clamp behaviour and aldosterone release from H295R cells expressing mutant KCNJ5 channels. The student would build on this work and also look at the response of these mutant channels to channel inhibitors. We have show early data suggesting they are relatively resistant to tertiapin, which is consistent with the proximity of its binding site to the mutated inner pore region of the mutant KCNJ5 channels. They will also investigate the possibility that the KCNJ5 mutants cause a direct growth stimulus by Ca-loading of the adrenal cortical cells. Evidence of activated growth pathways will also be sort by expression profiling cells stably expressing mutant KCNJ5 channels. We have preliminary evidence that tumours with KCNJ5 mutations have a definite phenotype-genotype correlation (loss of the postural response to standing and ZF-predominant cell type histology). The project will follow this lead looking for evidence of a ZF gene expression signature in these stable cell lines. Overall, this area is evolving rapidly and is certain to add very significantly to our molecular understanding of the Conn’s tumour. It is no longer seen as a rare curiosity but a treatable form of hypertension in probably 1-2 % of essential hypertensives.
Project 5: Elucidation of the molecular pathways responsible for human cytomegalovirus reactivation as a paradigm for therapeutic intervention
A major cause of disease in a number of immune-compromised populations, the molecular mechanisms responsible for human cytomegalovirus (HCMV) reactivation are still not fully understood. Currently, the working model for HCMV is that latency is established in a pluripotent pool of haematopoietic cells resident in the bone marrow. Despite the pluripotency of the progenitor, HCMV carriage is observed predominantly in the cells of the myeloid lineage with reactivation occurring upon terminal differentiation of these cells. As such, my current research is aimed at identifying the dual role myeloid cell differentiation and concomitant activation of inflammatory signalling plays in HCMV reactivation in dendritic cells (DCs).
The pivotal event (at a molecular level) for HCMV reactivation is the transition of the major lytic promoter (MIEP) from a silenced to an active state and current data suggests the activation of cellular signalling is crucial for this. It is hypothesized that this is not a linear event and this project seeks to address the contribution of different signalling pathways on HCMV reactivation and the cross-talk that occurs between them in the context of HCMV but with potentially wider implications for cell biology. As well as using models of HCMV latency in the laboratory to address these pathways using established techniques other specific aspects will include the use of recombinant HCMVs in which the CREB/ATF, NF-kB and IFN response elements have been deleted from the MIEP to address the relative impact of downstream molecular targets of the signalling pathways that trigger HCMV reactivation. A second aspect will take advantage of our recent observations that subsets of myeloid DCs respond differentially to reactivation signals depending on maturation status. Suggestive that the presence (or absence) of specific cellular proteins are required for signalling pathways to be pro-reactivation which will be addressed using comparative proteomics which, in itself, could yield new insight into the pathways analysed as well as furthering the specific aims of the research. Overall this project would expose the student to a wide range of techniques and will provide training in the areas of virology, molecular biology, cell signalling and DC immunology.