Craig Cameron Paul Berg Professor of Biochemistry and Molecular Biology 201 Althouse Laboratory, University Park, PA 16802 Phone: (814) 863-8705 Fax: (814) 863-7024 E-mail: cec9@psu.edu B.S. in chemistry and mathematics, Howard University Ph.D. in biochemistry, Case Western Reserve University Cameron Lab Web Site

RNA Virus Genome Replication

My laboratory is interested in defining the molecular details of genome replication in positive-strand RNA viruses and identifying components of this process suitable for antiviral drug development.

During the past seven years, my laboratory has focused on understanding the molecular mechanisms governing specific and efficient replication of poliovirus genomes by its RNA-dependent RNA polymerase (RdRP), 3D^pol. Initially, our effort was directed on development of the technology necessary to study the RdRP. In particular, we developed an expression system for production of polymerase in Escherichia coli that contains an authentic amino terminus, primer/template substrates for kinetic and thermodynamic analysis of RdRP-catalyzed nucleotide incorporation, and a structural model for the RdRP in complex with substrates: primer/template and nucleotide. Together, these technological advances have permitted the elucidation of the kinetic mechanism for nucleotide incorporation, an understanding of the kinetic and structural basis for RdRP fidelity—that is, selection for the correct base and correct ribose configuration, and discovery of novel strategies to modulate RdRP function in a manner that inhibits virus multiplication.

In spite of the safeguards engineered into the poliovirus RdRP for faithful replication of viral RNA, the steady-state level of transition mutations in progeny genomes is approximately two per genome. 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, exist on the threshold of error catastrophe—that is, a subtle, 4-fold increase in the mutation frequency of the virus is sufficient to decrease virus viability by at least 20-fold. Our studies of the broad-spectrum, antiviral ribonucleoside, ribavirin, demonstrate that this compound is a lethal mutagen of the poliovirus genome and functions by forcing the virus into error catastrophe. These studies benefit from a collaboration with Dr. Raul Andino, University of California, San Francisco.

Because ribavirin is currently used in combination with interferon-a to treat hepatitis C virus (HCV) infection, we were interested in determining whether lethal mutagenesis might be the operative mechanism of action for ribavirin with this virus. In order to address this issue, we required a derivative of the RdRP from HCV that could be studied by using the technology developed by us to study the poliovirus polymerase. This was not a simple task; however, in collaboration with scientists at ICN Pharmaceuticals, we succeeded in developing a suitable HCV polymerase derivative. Consistent with observations made for poliovirus, our data show that the antiviral activity of ribavirin can be mediated by the HCV polymerase, causing lethal mutagenesis of the HCV genome. In addition, our data suggest that the presence of ribavirin in HCV RNA may decrease the capacity of this RNA to serve as a template for production of full-length, plus- and/or minus-strand RNA. Together, our studies of the anti-poliovirus and anti-HCV activity of ribavirin suggest that lethal mutagenesis is a viable antiviral strategy.

Currently, we are continuing to use our structural model for the ternary complex of 3Dpol to guide mutational analysis of this enzyme. In particular, we are defining the path of the nucleic acid over and through the enzyme and identifying residues important for stable association with nucleic acid and local unwinding of structured RNA in the path of an elongating enzyme. Regarding lethal mutagenesis, in collaboration with Dr. Blake Peterson, Department of Chemistry, we are currently synthesizing novel, “universal” ribonucleosides and evaluating the antiviral activity of these compounds. In an effort to uncover mechanisms viruses might employ to develop resistance to lethal mutagens, we are selecting mutant polioviruses that are capable of replicating efficiently in the presence of grossly imbalanced cellular nucleotide pools. It is likely that some mutations will map to polymerase-coding region and produce enzymes with enhanced fidelity. These mutant polymerases may not utilize mutagenic ribonucleosides as efficiently as the wild-type enzyme, thereby conferring resistance to this class of antiviral agents.

For several years my laboratory has worked with Dr. Kevin Raney, University of Arkansas for Medical Sciences, to define physical and functional interactions between HCV nonstructural proteins. My laboratory has focused and will continue to focus on the kinetics and mechanism of HCV polymerase (NS5B) while Raney’s laboratory has focused on the kinetics and mechanism of HCV helicase (NS3-4A). We have developed an in vitro system that requires both activities for production of full-length RNA. Interestingly, the efficiency of RNA synthesis can be modulated by HCV NS5A, a protein without a known function in genome replication. Interestingly, we have recently discovered that this protein is a sequence-specific RNA-binding protein. We are using a variety of approaches to map the sites of interaction between the HCV non-structural proteins, identifying those interactions that are required for function in the in vitro system and verifying the biological significance of these interactions by evaluating the effect of disruptive mutations on the replication of subgenomic replicons.

The newest project in the laboratory is directed towards understanding how cellular genes involved in RNA metabolism impact replication/multiplication of RNA viruses, in particular hepatitis C virus. This work is being performed in collaboration with Dr. Joseph Reese, Department of Biochemistry and Molecular Biology. This project has significant implications clinically, ranging from development of tools for non-invasive methods of staging liver disease to development of novel therapeutics.

Representative Publications:

Journal Articles

• Arnold, J.J. and Cameron, C.E.. (2000). Poliovirus RNA-dependent RNA polymerase (3D^pol): Assembly of stable, elongation-competent complexes by using a symmetrical primer/template substrate (sym/sub). J. Biol. Chem. 275, 5329-5336.
• Gohara, D.W., Crotty, S., Arnold, J.J., Yoder, J.D., Andino, R., and Cameron, C.E. (2000). Poliovirus RNA-dependent RNA polymerase (3D^pol): Structural, biochemical and biological analysis of conserved structural motifs A and B. J. Biol. Chem. 275, 25523-25532.
• 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.
• Crotty, S., Cameron, C.E., and Andino, R. (2001) RNA virus error catastrophe: Direct molecular test by using ribavirin. Proc. Natl. Acad. Sci. (USA) 98, 6895-6900.
• Maag, D., Castro, C., Hong, Z., and Cameron, C.E.. (2001). Hepatitis C virus RNA-dependent RNA polymerase as a mediator of the antiviral activity of ribavirin. J. Biol. Chem. 276, 46094-46098.
• Harki, D.A., Graci, J.D., Korneeva, V.S., Ghosh, S.K.B., Hong, Z., Cameron, C.E., and Peterson, B.R. (2002) Synthesis and antiviral evaluation of a mutagenic and non-hydrogen bonding ribonucleoside analogue: 1-b-D-ribofuranosyl-3-nitropyrrole. Biochemistry 41, 9026-9033.
• Arnold, J.J. and Cameron, C.E. (2004). Poliovirus RNA-dependent RNA polymerase (3D^pol): Pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of magnesium.
• Biochemistry 43, 5126-5137. Arnold, J.J., Gohara, D.W., and Cameron, C.E. (2004). Poliovirus RNA-dependent RNA polymerase (3D^pol): Pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of manganese. Biochemistry 43, 5138-5148. Gohara, D.W. and Cameron, C.E. (2004). Conserved structural motifs A and B of poliovirusRNA-dependent RNA polymerase (3Dpol) are required for nucleotide selection. Biochemistry 43, 5149-5158.

Book Chapters

• Cameron, C.E., Gohara, D.W., and Arnold, J.J. (2002). Poliovirus RNA-dependent RNA polymerase (3Dpol): Structure, function and mechanism. In Molecular Biology of Picornaviruses. Semler, B.L. and Wimmer, E., eds. ASM Press, Washington, D.C., pp. 255-267.
• Huang, L., Gledhill, J., andCameron, C.E. (2003). The RNA-dependent RNA polymerase. In Gene Silencing. Hannon G., ed. Cold Spring Harbor Press, Cold Spring Harbor, NY., pp. 175-203.
• Korneeva, V., Gohara D.W., and Cameron, C.E. (2003). The RNA-dependent RNA polymerase: Structure, function and mechanism. In Mechanisms of Replication and Transcription of RNA Viruses. Zhang, X., ed. Research Signpost, Kerala, India, pp. 17-36

Review Articles

• Graci, J.D. and Cameron, C.E.. (2002). Quasispecies, error catastrophe and the antiviral activity of ribavirin. Virology 298, 175-180.
• Hong, Z. and Cameron, C.E. (2002). Pleiotropic mechanisms of ribavirin antiviral activities. Prog. Drug Res. 59, 41-69.
• Graci, J.D. and Cameron, C.E. (2004). Challenges for the development of ribonucleoside analogues as inducers of error catastrophe. Antivir. Chem. Chemother. 15, 1-13.
• Freistadt, M.S., Meades, G.D., and Cameron, C.E. (2004). Lethal mutagens: Broad-spectrum antivirals with limited potential for development of resistance? Drug Resist. Updat. 7, 19-24.

Search the MEDLINE database at PubMed for articles by C Cameron