Human Cytomegalovirus (HCMV) and Adenovirus Virology
This group has exploited replication-deficient adenovirus (Ad) vectors extensively over the last 20 years to study gene function, generate immune responses and detect immune responses.
The unit is based in the division of Infection and Immunity within the School of Medicine and has a primary focus on two distinct areas: Cytomegalovirus and Adenovirus vectors.
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HCMV is a clinically important pathogen with high prevalence worldwide. It is the leading infectious cause of congenital malformation, is associated with life-threatening disease in immunocompromised individuals (e.g. AIDS sufferers and transplant recipients), and is a causative agent of hepatitis, colitis, post-transplantation arteriosclerosis and infectious mononucleosis. As a result, the US Institute of Medicine has designated HCMV as a highest priority (Level I) vaccine target.
We have major interests in the basic biology of the virus, the development of therapeutics and diagnostics, the way in which the virus interacts with the immune system, and using the virus to understand how the immune system functions in both healthy and diseased states.
This group has exploited replication-deficient adenovirus (Ad) vectors extensively over the last 20 years to study gene function, and as vaccine agents. We have developed a new adenovirus cloning system, the AdZ system, which enables us to rapidly and easily modify the Ad backbone, and to rapidly clone multiple transgenes into Ad vectors.
Recently, in collaboration with the Oncolytic Adenovirus Virology group, our research has extended to developing vectors based on multiple different serotypes, and modifying vectors to alter tropism in order to therapeutically target tumours.
Genetics of HCMV
HCMV has the largest genome of any characterised human virus. The complete DNA sequence for the laboratory strain AD169 determined 20 years ago has proved invaluable in HCMV research. However, strain AD169 and other lab strains have accumulated major defects during propagation in vitro.
This video is of Dr Richard Stanton and Dr Ian Humphreys discussing the significance of research into HCMV.
Many genes, including predicted NK evasion functions, are non-essential for in vitro replication and have been deleted or mutated. It has therefore been important to define the intact HCMV genome. In collaboration with Dr Andrew Davison’s laboratory (University of Glasgow), the complete sequence of an HCMV clinical isolate - strain Merlin - from Cardiff was sequenced and annotated.
Merlin is now the international reference strain in NCBI Refseq, and we have subsequently cloned the Merlin genome a bacterial artificial chromosome (BAC). In collaboration with others, we have studied the changes that occur during in vitro passage of clinical HCMV (Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture), and also the changes that occur when viruses derived from BACs are passaged in vitro (Genetic Stability of Bacterial Artificial Chromosome-Derived Human Cytomegalovirus during Culture In Vitro). As a result of this work we are now able to analyse pathogenesis, immune evasion, tropism and virus genome evolution using virus that closely represents the clinical agent, and does not mutate in vitro.
The Merlin BAC has already provided insight into clinical HCMV by allowing us to identify the HCMV gene RL13 as a potent inhibitor of viral replication in vitro. More recent work has shown that when a virus containing a complete clinical genome spreads between cells, it does so in a very different way to viruses that have been grown in the lab, and this has major consequences to the ways in which we develop vaccines and therapeutics. See The pentameric complex drives immunologically covert cell-cell transmission of wild-type human cytomegalovirus.
We have also been working with collaborators to use cutting edge proteomics technology to understand the way in which HCMV affects the entire cell when it infects them. This led to the first ever description of the way in which the virus modulates hundreds of proteins on the cell surface, and several thousand within the cell, across an entire timecourse of infection. See Quantitative temporal viromics: an approach to investigate host-pathogen interaction.
We have developed a number of infectious BACs containing defined, full length HCMV genomes. They are based on the Merlin strain - Merlin is the NCBI reference sequence for HCMV, and in collaboration with the world health organisation (WHO), we have developed it as the international diagnostic standard for HCMV.
We have BACs containing the full HCMV genome, as well as some containing genomes which express green fluorescent protein (GFP) to allow easy monitoring of infection.
We also have BACs with certain defined mutations - HCMV contains two gene regions that inhibit growth in vitro, and therefore reproducibly mutate when grown in the lab. The gene RL13 always mutates first, no matter which cell type is used. Then the UL128 locus (UL128, UL130, UL131A) mutates if virus is grown in fibroblasts.
Viruses with both gene regions mutated are very easy to handle, and grow to high titres in fibroblasts. However, if you want to infect other cell types (leukocytes, endothelial cells, epithelial cells), then you need to use a virus that is wildtype in UL128L, however these viruses grow to very low titres.
To solve this problem, we use tet repression to selectively repress/express UL128L as needed. Virus is grown in a cell type that represses UL128L, enabling the stable production of high titre virus. A single round of replication can then be done in cells that allow expression of UL128L. This produces wildtype virus, but because UL128L is only turned on when needed, the chance of mutation is severely limited.
A recombinant adenovirus (Ad) vector with zero cloning steps
- s complete Ad5 vector is carried on a Bacterial Artificial Chromosome (BAC)
- the DNA insert is transformed directly into cells carrying the AdZ BAC.
The insert can be:
- synthetic oligonucleotides (e.g. encoding shRNAs)
- PCR product
- synthesized gene
- conventional plasmid clone (e.g. from an expression library).
Recombineering is performed:
- the transgene replaces dual selectable markers
- positive clones are identified without need for screening
- BAC DNA is purified & transfected into cells
- AdZ recombinant grows.
- cloning genes into the AdZ vectors and making virus - for inserting genes with any tag into the AdZ vectors
- growing RAds - for reconstituting RAds from the AdZ BACs, and growing them up
- titering viruses - a simple immunofluorescence protocol to get titres
- general recombineering - for inserting the sacB cassette in order to modify the backbone.
Vector maps for the AdZ vectors
The following maps are all just the expression cassettes - the remaining sequence is identical to pAdZ5-CV5 above:
- pAdZ5-CV5 for adding C terminal V5 tags
- pAdZ5-NV5 for adding N terminal V5 tags
- pAdZ5-NGFP for adding N terminal GFP tags
- pAdZ5-CGFP for adding C terminal GFP tags
- pAdZ5-CmCherry for adding C terminal mCherry tags
- pAdZ5-mIR155 for cloning shRNAs
- pAdZ5-CStrep2 for adding C terminal Strep-2 or strep-3 tags
- pAdZ5-CV5-NT for adding C terminal V5 tags, promoter lacks tet operators
- pAdZ5-CGFP-NT for adding C terminal GFP tags, promoter lacks tet operators
- pAdZ5-Ctrl is empty vector control, containing just a V5 tag inbetween the promoter & polyA