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Craig CameronPaul Berg Professor of Biochemistry and Molecular Biology 201 Althouse Laboratory, University Park, PA 16802 B.S. in chemistry and mathematics, Howard University Cameron Lab Web Site |
RNA Polymerases and RNA-binding Proteins in Viral Infection and Mitochondrial Disease
Since its inception, the primary goal of this laboratory has been development of strategies to treat or to prevent infections by RNA viruses. We have used poliovirus and hepatitis C virus as our primary model systems. Our expertise in virology, biochemistry and mechanistic enzymology brings a unique combination of intellectual and technical resources to the study of RNA viruses. Our initial focus was the viral RNA-dependent RNA polymerase (RdRp). In particular, we were interested in the kinetic, thermodynamic and structural basis for fidelity of nucleotide incorporation, a topic of considerable importance not only for accurate maintenance, transmission and expression of genetically encoded information but also for targeting the RdRp for antiviral therapy. These studies have led to exciting discoveries that have moved the lab into many new areas, including enzyme dynamics, vesicular trafficking, innate immunity, vaccine development and mitochondrial molecular biology. The projects discussed below have been selected to illustrate the breadth, depth and impact (current and future) of our research programs.
RNA-dependent RNA polymerase mechanism
Over the past ten years, the world has witnessed the emergence of SARS, the spread of West Nile encephalitis, and the fear of a global flu pandemic. These diseases are caused by RNA viruses. In addition, the threat of intentional release of RNA viruses as weapons or agents of terror has increased substantially. The long-term goal of this research program is to develop strategies to treat and/or prevent RNA virus infection by targeting the RdRp. Our program has employed a prototypical RNA virus, poliovirus (PV), and its RdRp (3Dpol) as our model system. We have obtained new insight into the chemical mechanism for nucleotidyl transfer. We discovered a link between RdRp incorporation fidelity and pathogenesis. We discovered a connection between RdRp dynamics and incorporation fidelity. Together, our studies lead to the very provocative hypothesis that RdRp incorporation fidelity is a target for antiviral and vaccine development. Our current research objectives are: (1) Elucidate additional roles for the general acid in polymerase function; (2) Identify novel determinants and mechanisms of polymerase fidelity; and (3) Establish dynamics-function relationships for the RdRp. Our studies of RdRp dynamics include: molecular dynamics simulations, performed in collaboration with Coray Colina (Materials Science and Engineering Department, PSU); hydrogen deuterium exchange mass spectrometry, performed in collaboration with Patrick Wintrode (Case); and NMR spectroscopy, performed in collaboration with David Boehr (Chemistry Department, PSU).
Viral attenuation and vaccine development
RNA viruses represent an existing and emerging threat to human health and include human pathogens like hepatitis C virus, SARS coronavirus, West Nile virus, dengue virus, influenza virus, measles virus and ebola virus, to name only a few. RNA viruses also represent a threat to animal health and include veterinary pathogens like foot-and-mouth disease virus that infects cattle. Vaccination is the only known approach to prevent viral infection, with the most efficacious vaccines being live, attenuated virus strains. Current approaches for development of vaccine strains are random and slow and preclude a rapid response to natural, unintentional or intentional outbreaks caused by viruses. We have discovered a polymerase-mechanism based strategy for viral attenuation and vaccine development that can be extrapolated to any RNA virus. In collaboration with Avery August (Veterinary and Biomedical Sciences Department, PSU), we are using mouse models to characterize the immune response to our vaccine candidates. In addition, we are expanding this program to include other viruses, including West Nile Virus, performed in collaboration with Maria Rios (FDA), and Respiratory Syncytial Virus, performed in collaboration with Michael Teng (Biochemistry and Molecular Biology Department, PSU) and scientists at MedImmune (Mountain View, CA).
Picornavirus genome replication
Picornaviruses represent an existing and emerging threat to US public health and also serve as important model systems for positive strand RNA viruses in general. Although protein factors and genetic elements required for picornavirus genome replication are known and appear to be conserved, a clear understanding of the mechanisms employed to produce picornaviral RNA is lacking. The long-term objective of this program is to reconstitute picornavirus genome replication in vitro from purified components. In collaboration with Jim Hogle (Harvard Medical School), we have solved the first crystal structure for a picornaviral 3CD protein. In collaboration with Mark Foster (Ohio State University), we developed the technology to study 3C-RNA interactions by using NMR spectroscopy. Finally, we discovered that the 3CD protein has both pre- and post-replication functions. Our current effort continues our studies of picornavirus genome replication as well as explores our newly discovered function for 3CD by pursuing the following objectives: (1) Define the mechanism of assembly and structural organization of the picornavirus genome replication initiation complex by using molecular genetic, biochemical and biophysical approaches; (2) Define the molecular basis for sequence- and structure-specific RNA recognition by 3C by using NMR spectroscopy; and (3) Elucidate the function of 3CD in formation of replication complexes, especially the effects of 3CD on vesicular trafficking. In collaboration with Cheng Kao (Indiana University), we are using three-dimensional reconstructions of images captured by using negative stain electron microscopy to view the structure of the replication initiation complex. Our studies of the subversion of the vesicular trafficking pathway by picornaviruses are being pursued in collaboration with Ellie Ehrenfeld (NIAID/NIH) and Jesse Hay (University of Montana).
HCV NS3 and NS5A proteins: Biochemical mechanisms and biological functions
At least 3% of the world's population is infected with hepatitis C virus (HCV); over 50% of infections never resolve, resulting in persistent virus carriage. Over time, this chronic infection can lead to liver fibrosis and, progressively, to severe and fatal diseases including liver cirrhosis and primary liver cancer. In the US, hepatitis C is an emerging disease, with 1.7 million individuals already chronically infected and 30,000 more infected every year. The economic cost of this medical burden is estimated at approximately $1.0 billion per year.
Our studies emphasize two proteins required for HCV genome replication: non-structural proteins 3 (NS3) and 5A (NS5A). NS3 is a RNA helicase. HCV NS5A is a phosphoprotein comprised of three domains (I-III). In collaboration with Kevin Raney (University of Arkansas for Medical Sciences), we have established a unique system to study NS3 biochemistry that has provided an unprecedented level of mechanistic insight. We are exploiting this insight to develop tools to uncover the role(s) of the RNA helicase activity in the lifecycle of HCV.
Our laboratory was the first to purify NS5A. We have shown that NS5A is a GU-rich RNA-binding protein. In collaboration with Paul Bohjanen (University of Minnesota), we have shown that NS5A antagonizes the decay of mRNAs controlled by GU-rich elements. We are pursuing the link between this observation and mechanisms of oncogenesis. We have produced the first biochemical evidence supporting a direct, inhibitory interaction between NS5A and the double-stranded RNA activated kinase (PKR), an effector of innate immunity. Our studies of NS5A-PKR interaction are performed in collaboration with Phil Bevilacqua (Chemistry Department, PSU) and James Cole (University of Connecticut). It is now clear that an interaction between NS5A and cyclophilins is required for HCV multiplication. Importantly, this interaction can be disrupted by a variety of drugs that target cyclophilins, thus revealing a novel strategy for development of anti-HCV therapeutics. Our studies of NS5A-cyclophilin interaction are performed in collaboration with Philippe Gallay (Scripps). We are currently pursuing the hypothesis that interactions of NS5A with host factors such as PKR are controlled by a phosphorylation code.
Mitochondrial transcription and disease
Interest in all aspects of mitochondrial molecular biology and biochemistry has increased over the past decade as data accumulate implicating mitochondrial dysfunction in a variety of human diseases and the aging process. The scope of pathologies now suspected of having a basis in altered mitochondrial function includes certain cancers, neurodegenerative disorders, muscular dystrophies and cardiac diseases. A primary goal of mitochondrial medicine is the assignment of specific defects in mitochondrial molecular biology to particular disease states. We are interested in understanding how defects in transcription of the mitochondrial genome contribute to disease and aging, an important, emerging area of research.
We are the first to reconstitute human mitochondrial transcription in vitro from purified components produced solely in bacteria. This advance defines a new era for the field. This system is now marketed by Enzymax, LLC (Lexington, KY). We are studying the enzymology and regulation of human mitochondrial transcription initiation, elongation and termination. We have established a network of collaborations to exploit our unique capabilities. We are working with Hasan Koc (Biochemistry and Molecular Biology Department, PSU) to use mass spectrometry to define interactions between the core RNA polymerase and transcription factors. In collaboration with Katsu Murakami (Biochemistry and Molecular Biology Department, PSU), we are using X-ray crystallography to achieve a structural perspective of the different stages of mitochondrial transcription. In collaboration with Cheng Kao (Indiana University), we are using three-dimensional reconstructions of images captured by using negative stain electron microscopy as an additional approach to view the structures of the various transcription complexes. In collaboration with Emine Koc (Biochemistry and Molecular Biology Department, PSU) and Gerry Shadel (Yale), we are studying factors that may facilitate coupling of transcription to translation. In collaboration with scientists at Gilead Sciences (Foster City, CA), we are testing the hypothesis that the mitochondrial RNA polymerase is an off-target for antiviral ribonucleosides that are being developed for the treatment of HCV infection. Finally, in collaboration with Carlos Moraes (University of Miami), we are establishing a mouse transcription system that will ultimately permit us to determine the impact of transcriptional defects on development, health and aging.
Lethal Mutagenesis as an Antiviral Strategy
The quasispecies nature of RNA viruses permits these viruses to resist challenges by the host that would otherwise kill the virus population. Poliovirus, and likely most RNA viruses, have optimized population diversity. In the case of poliovirus, each member of the population differs from another by a few single nucleotide changes. Our studies of the broad-spectrum, antiviral ribonucleoside, ribavirin, demonstrated that this compound is a lethal mutagen of the poliovirus genome and functions by increasing the number of differences between members of the population to an extent that does not permit the population to be sustained. These studies defined lethal mutagenesis as a clinically tractable mechanism for antiviral drug development. In collaboration with Marco Vignuzzi (Pasteur) and Raul Andino (UCSF), this project has transitioned to a study that focuses on identifying the properties of RNA viruses that determine sensitivity to lethal mutagens. In collaboration with Blake Peterson (University of Kansas), we are working to develop, synthesize and validate antiviral ribonucleosides that function by exploiting the promiscuity of the RdRp. Lethal mutagens represent a subset of these antiviral compounds.
Representative Publications:
Journal Articles
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Oh, H.S., Pathak, H.B., Goodfellow, I.G., Arnold, J.J., and Cameron, C.E. (2009). Insight into poliovirus genome replication and encapsidation obtained from studies of 3B-3C cleavage site mutants. J. Virol. 83, 9370-9387.
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Castro, C., Smidansky, E.D., Arnold, J.J., Maksimchuk, K.R., Moustafa, I., Uchida, A., Götte, M., Konigsberg, W. and Cameron, C.E. (2009). Nucleic acid polymerases employ a general acid for nucleotidyl transfer. Nat. Struct. Mol. Biol. 16, 212-8.
- Arias, A., Arnold, J.J., Sierra, M., Smidansky, E.D., Domingo, E. and Cameron, C.E. (2008). Determinants of RNA-dependent RNA polymerase (in)fidelity revealed by kinetic analysis of the polymerase encoded by a foot-and-mouth disease virus mutant with reduced sensitivity to ribavirin. J. Virol. 82, 12346-55.
- Pathak, H.B., Oh, H.S., Goodfellow, I.G., Arnold, J.J., and Cameron, C.E. (2008). Picornavirus genome replication: Roles of precursor proteins and rate-limiting steps in oriI-dependent VPg uridylylation. J. Biol. Chem. 283, 30677-88.
- Amero, C., Arnold, J.J., Moustafa, I., Cameron, C.E. and Foster, M.P. (2008). Identification of the oriI-binding site of PV 3C by using NMR spectroscopy. J. Virol. 82, 4363-70. Epub 2008 Feb 27.
- Graci, J.D., Too, K., Smidansky, E.D., Edathil, J.P., Barr, E.W., Harki, D.A., Galarraga, J.E., Bollinger, J.M., Jr., Peterson, B.R., Loakes, D., Brown, D.M. and Cameron, C.E. (2008) Lethal mutagenesis of picornaviruses with N6-modified purine nucleoside analogues. Antimicrob. Agents Chemother. 52, 971-9. Epub 2008 Jan 7.
- Nallagatla, S.R., Hwang, J., Toroney, R., Zheng, X., Cameron, C.E. and Bevilacqua, P.C. (2007) 5’-Triphosphate-dependent activation of PKR by RNAs with short stem-loops. Science 318, 1455-8.
- Castro, C., Smidansky, E., Maksimchuk, K.R., Arnold, J.J., Korneeva, V.S., Gotte, M., Konigsberg, W., and Cameron, C.E. (2007). Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases. Proc. Natl. Acad. Sci (USA). 104, 4267-72.
- Vignuzzi, M., Stone, J.K., Arnold, J.J., Cameron, C.E., and Andino, R. (2006). Genomic diversity in a viral population determines fitness, tissue tropism and pathogenesis. Nature 439, 344-8. Epub 2005 Dec 4.
- Crotty, S., Maag, D. Arnold, J.J., Zhong, W., Lau, J.Y.N., Hong, Z., Andino, R., and Cameron, C.E. (2000). The broad-spectrum antiviral ribonucleoside, ribavirin, is an RNA virus mutagen. Nature Medicine 6, 1375-1379.
Review Articles
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Graci, J.D. and Cameron C.E. (2008) Therapeutically targeting RNA viruses via lethal mutagenesis. Future Virology 3, 553-566.
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Castro, C., Arnold, J.J., and Cameron, C.E. (2005). Incorporation fidelity of the viral RNA-dependent RNA polymerase: A kinetic, thermodynamic and structural perspective. Virus Res. 107, 141-149.
Book Chapters
- Smidansky, E., Arnold, J.J., Sholders, A., Peersen, O.B., and Cameron, C.E. (2008) Nucleic acid polymerase fidelity and viral population fitness. In Origin and Evolution of Viruses. Domingo, E., Parrish, C., and Holland, J.J. eds. Academic Press (Elsevier), London, pp. 135-160.
- Ng, K.K., Arnold, J.J., and Cameron, C.E. (2008). Structure-function relationships among RNA-dependent RNA polymerases. In Current Topics in Microbiology and Immunology. Paddison P., and Vogt, P., eds. Springer Publishers, NY, pp. 137-156.
Books
- Cameron, C.E., Götte, M., and Raney K.D. (2009). Viral Genome Replication. Springer Publishers, NY.
Search the MEDLINE database at PubMed for articles by C Cameron