David Evans, PhD
Dept. of Medical Microbiology & Immunology
University of Alberta
Faculty of Medicine & Dentistry
6-020 Katz Group Centre
T6G 2E1 Edmonton, AB
Office Phone : (780) 492-2109
Lab Phone: (780) 492-2106
Fax : (780) 492-7521
- Professor, Dept. of Medical Microbiology & Immunology
- Director, Alberta Institute for Viral Immunology
- Member, WHO Advisory Committee on Variola (Smallpox) Virus
My lab studies poxviruses. These are large DNA viruses, best known for causing the diseases smallpox and myxomatosis in humans and rabbits, respectively. We are currently studying three different aspects of poxvirus biology using funding provided by CIHR, NSERC, the Alberta Cancer Board, and from funds generated by my commercial research activities. How you could participate will change as these projects evolve and people come and go. Please contact us for the latest news.
1.0 Poxvirus replication and recombination – Poxviruses appear to encode nearly all of the genes they need to catalyze virus DNA replication and the closely linked process of virus recombination. We have shown that virus recombination is catalyzed by the virus-encoded DNA polymerase and have commercialized an application of that discovery for use in high-throughput gene cloning. Four different projects currently fall under this heading:
1.1 Antiviral drugs – We are studying how acyclic nucleoside phosphonate drugs (ANPs) inhibit poxvirus replication. These drugs target the virus DNA polymerase, but unlike most polymerase inhibitors, most ANPs are not classical chain terminators. Our studies suggest that ANPs get incorporated into DNA, and then partially inhibits both primer extension and DNA synthesis across sites where the drugs have been incorporated into the template strand (1). Our discovery of this last drug property has been described as creating a “paradigm shift” in the current understanding of ANP therapeutics. We are using these observations, and drug-resistant viruses, as a tool for investigating the mechanism of ANP action in vivo (2). We are also studying how HIV reverse transcriptase behaves when presented with templates containing ANPs.
1.2 New reagents for studying virus replication and recombination – We have generated monoclonal antibodies against the vaccinia virus single-strand DNA binding protein (I3), the DNA polymerase (E9), and the small subunit of the virus-encoded ribonucleotide reductase (F4). We are using these reagents, as well as viruses encoding mutations in these and other virus replication genes, as tools for investigating the composition of the vaccinia virus-replication machinery and the mechanism of poxvirus recombination. We have devised a working hypothesis for how nucleotide biosynthesis might regulate virus recombination and are also using these reagents as tools for studying some predictions arising from this model.
1.3 Novel methods for imaging virus replication and recombination – We have fused a gene for green fluorescent protein to one encoding the phage lambda cro protein, and then expressed the resulting fusion protein in mammalian cells. Cro encodes a high-affinity DNA binding protein and thus this method provides a way of tagging viruses encoding cro-binding sites. We are using this as a tool for following the dynamics of virus entry, DNA replication, factory movement, and factory fusion using live cell fluorescence imaging methods (see the movie below).
MPEG movie of poxvirus replication in a host cell. Poxviral DNA is stained green by a fluorescent green DNA binding protein, while the host cell nuclear DNA is stained in blue (Image data courtesy of Dr. James Lin).
MPEG movie showing virus replication in a mitotic cell. At the start you see the EGFP-bright middle cell go through mitosis, you then see two virosomes appearing, growing and fusing in the daughter cell on the bottom left (Image courtesy of Dr. James Lin)
2.0 Oncolytic viruses – It has been suggested that poxviruses might be used as therapeutic agents for killing tumour cells. One approach has involved crippling vaccinia virus so as to reduce the capacity to replicate in normal human cells. A second approach exploits the fact that some animal poxviruses, like the Leporipoxviruses, can’t replicate in normal human cells, but will replicate in human tumour cells. We are studying this subject in collaboration with colleagues at the Cross Cancer Institute in Edmonton and the Tom Baker Cancer Institute in Calgary. My lab’s particular focus concerns transferring vaccinia genes into myxoma virus, with the goal of improving the capacity of the modified virus to disseminate to metastatic sites.
3.0 Host factors required for poxvirus infection – Poxviruses exhibit an extraordinary capacity to manipulate host cell functions for their own advantage. For example my departmental colleague, Dr. M. Barry, has extensively characterized the way in which poxviruses suppress immune defenses by targeting apoptosis and ubiquitin pathways. Our own virus-host studies fall into three areas of research:
3.1 The role of topoisomerases in virus replication – It has long been thought that poxviruses are “self-sufficient” when it comes to encoding the enzymes they need for replication and assembly. However, we have shown that vaccinia virus recruits cellular topoisomerase II from the nucleus out to sites where poxviruses replicate in the cytoplasm (3). It does this by forming protein-protein complexes involving the topoisomerase and the virus-encoded DNA ligase. We are currently using a variety of methods to gain further insights into how topoisomerases are recruited from the nucleus and what they do once they have entered sites of virus replication.
3.2 The role of ribonucleotide reductases (RR) in virus replication – A few poxviruses encode both the large and small subunits of the RR enzyme, most encode just the small subunit, and a few don't encode either. We are studying the properties of viruses encoding different combinations of these enzymes to see what role protein-protein interactions play in ensuring replicating poxviruses are supplied with an abundance of DNA precursors.
3.3 High-throughput siRNA screens – We have been using small interfering RNAs (siRNA) as a tool for suppressing the activities of both virus (I3L) and cellular (ribonucleotide reductase, topoisomerase) genes. As we gain familiarity with this exciting new technology, we are starting to use high-throughput screens and large commercial siRNA libraries to explore what cell systems are critical for poxvirus replication. We are currently optimizing the transfection and recombinant virus reporter systems (LacZ, GFP, and luciferase are all options) now that we have completed installation of new CFI-funded robotic systems.
Methods: These projects provide training in cell culture, virus molecular genetics, imaging, molecular and cell biology, basic immunology, and enzymology.
Background preparation: A solid undergraduate training in molecular cell biology (biochemistry, cell biology, genetics, microbiology, or immunology) would be a great asset. We can teach virology to nearly anyone!
Useful lab equipment: In addition to much shared instrumentation, the lab is equipped with an Amersham Akta HPLC, centrifuges (Beckman Avanti J-E, Allegra X-22R, and L8-80 ultracentrifuge), thermal cyclers (Biometra T-gradient [regular PCR] and Bio-Rad MJ mini [real-time PCR]), spectrophotometers (Beckman DU640 and Thermo Nanodrop 1000), LiCor Odyssey infrared reader (we’ve gone all digital), Beckman LS6500 scintillation counter, and microscopes (Applied Precision Delta Vision [deconvoluting with live cell stage] and Zeiss Axioscope 2+).
Contact information: Please contact Dr. Evans at (780) 492-2309 or by e-mail at email@example.com. The laboratory can be contacted at (780) 492-1966.
- Megan Desaulniers (Graduate Student)
- Chad Irwin (Graduate Student)
- Patrick Paszkowski (Graduate Student)
- Li Qin (Graduate Student)
- Wondim Teferi (Graduate Student)
- Nicole Favis (Technician)
- Qin, L., Upton. C., Hazes, B., and EVANS, D.H. (2011) Genomic analysis of the vaccinia virus strain variants found in Dryvax vaccine. J. Virol. 85:13049-13060.
- Julien, O., Beadle, J.R., Magee, W.C., Chatterjee, S., Hostetler, K.Y., EVANS, D.H., and Sykes, B.D. (2011) Solution structure of a DNA duplex containing the potent anti-poxvirus agent cidofovir. J. Am. Chem. Soc. 133:2264-2274.
- Gammon, D.B., Gowrishankar, B., Duraffour, S., Andrei, G., Upton, C., and EVANS, D.H. (2010) Vaccinia virus-encoded ribonucleotide reductase subunits are differentially required for replication and pathogenesis. PLoS Pathogens. 6(7):e1000984.
- Lin, Y.-C., and EVANS, D.H. (2010) Vaccinia virus particles mix inefficiently in co-infected cells and in a way that would restrict viral recombination. J. Virology 84: 2432-43. (JV Cover, November, 2010)
- Gammon, D.B., and EVANS, D.H. (2009) Vaccinia virus recombination requires a 3'-to-5' exonuclease activity encoded by the viral DNA polymerase. J. Virology 83: 4236-50.
- Lin, Y.-C., Li, J., Irwin, C.R., Jenkins, H., DeLange, A., and EVANS, D.H. (2008) Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly. J. Virology 82: 5922-32.