Molecular Virology Research and Services
The Molecular Virology Laboratory provides essential services and research support in the areas of antiviral mechanism of action, gene cloning, DNA sequencing, protein production, expression and purification, in situ hybridization, polymerase chain reaction technologies, and enzymology. Specifically, we can support your work through a variety of applications.
Biochemical Mechanism of Action
Our Molecular Virology Laboratory support mechanism of action studies for all of our drug development areas. The virus replication pathway can be dissected into its component steps and assays are designed to evaluate the formation of these products using both cell-based and biochemical assays. Currently available assays include viral polymerase/reverse transcriptase, protease, integrase, binding/fusion, and transcriptional activation. Through close interactions with our HTS facility, it becomes possible to efficiently migrate low-throughput, benchtop assays to our robotics platform for purposes of screening large numbers of compounds rapidly.
Site-directed mutagenesis is a powerful tool that can be used to explore the effect of any mutation engineered into a gene on the eventual expression and function of that gene product. Our primary use of this technology has been to introduce specific amino acid changes into viral proteins and to explore the effect of these changes on drug and vaccine sensitivity. During the genotypic analysis of drug-resistant viruses, a number of amino acid changes are often found in important viral structural and regulatory proteins. The role of these mutations in conferring drug-resistant phenotypes to the wild-type virus can be investigated directly via site-directed mutagenesis. Using these approaches, we have created extensive panels of drug and vaccine-resistant virus strains that are now available for use with products submitted for evaluation. As the emergence of drug-resistant viruses continues to be a major problem in the clinical setting, the availability of defined drug-resistant viruses will be invaluable in assisting future antiviral drug discovery and development efforts.
Automated DNA sequencing capabilities exist for the rapid completion of your sequencing project. We offer core sequencing services that range from simple sequence data acquisition (you provide the templates or sequencing reactions) to large-scale cloning and sequencing/analysis projects where we provide all necessary services. In either case, the data can be provided rapidly to the sponsor by email or Internet access (ftp or telnet). Our ABI sequencers (ABI 310 and 377) have been used primarily to evaluate viral nucleic acid sequences, especially with drug- and vaccine-resistant viral isolates. In the near future, our HTS robotics laboratory will provide high-throughput genotyping services to complement our drug-sensitivity phenotyping services.
Polymerase chain reaction technology has advanced at a steady pace over the past years and we have remained on the cutting edge of this technology. Presently, we are utilizing real-time quantitative PCR instrumentation (Applied Biosystems Prism 7700) to evaluate plasma virus load for clinical trial purposes as well as an extremely sensitive endpoint detection methodology for a variety of in vitro and in vivoassays. Highly sensitive and reproducible assays have been developed and validated for HIV-1, SIV, HBV, HCV, EBV, CMV, and HHV-8; other assays of interest can be rapidly developed and implemented if desired.
Gene and Protein Expression
Our Molecular Virology laboratory can support your requirements for the production of recombinant proteins using E. coli and baculovirus expression systems. We have proven experience with small- and large-scale production/purification of biologically-active viral enzymes and proteins. Protein purification methods include salt fractionation, column chromatography (ion-exchange, size-exclusion, and affinity), and HPLC/FPLC. We are prepared to work closely with the sponsor to customize purification procedures as appropriate.
Other specific core capabilities of our Molecular Virology laboratory include:
- Plasmid DNA purification/characterization
- Genomic DNA and RNA isolation
- Genomic DNA and cDNA cloning
- PCR diagnostics
- Recombinant virus construction
- Transformation/transduction protocols
- Southern, Northern, Western blot analysis