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Viruses: Developing Strategies to Prevent and Treat Infections

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Over the past decade, the world has observed the emergence of SARS, the spread of West Nile, and the fear of a global flu pandemic; besides being viruses, the shared link between each of these diseases is RNA.

30 May 2013

cameron labThe threat of intentional release of RNA viruses as biological weapons has increased substantially as well, motivating scientists to develop innovative strategies to produce vaccines to prevent infection and new antiviral therapies. Viruses have not only become a threat to human health, but have impacts on other parts of society, such as food supply and the economy.

Craig E. Cameron, a professor of biochemistry and molecular biology, and holder of the Eberly Chair in Biochemistry and Molecular Biology, studies viruses and looks for ways to cure diseases caused by them. The viruses he studies all contain a common bond—RNA is their genetic material. Cameron’s research focuses on RNA polymerases and RNA-binding proteins required for viral replication or mitochondrial function. His work has contributed to the conceptual and practical development of strategies to treat and to prevent viral infection.

cameron jpgCraig E. Cameron

Cameron knew that although he wanted a career where he could help people using science, there were paths aside from being a doctor. “I was a student during the AIDS epidemic of the 1980s. HIV was discovered during that time, as well as the first drugs to treat HIV/AIDS. Watching the devastation caused by this viral parasite inspired me to enter a career that would facilitate annihilation of viral pathogens. I saw research as a viable way to help people,” he said. “Viruses were clearly a menace, and we just weren’t equipped to deal with the challenges associated with combatting them.”Although Cameron is now a research scientist and faculty member, that was not always the plan. “When I first went to undergraduate school, I really thought I wanted to become an M.D.,” Cameron said. “I spent a lot of time volunteering in hospitals but discovered that dealing with aging, death, and dying on a daily basis just wasn’t for me.”

cameron effect 1 jpgThis image illustrates a human cell during a normal viral infection. Credit: Craig Cameron lab, Penn State University

Cameron’s studies of the chemical mechanism of the RdRp have led to the first universal, polymerase mechanism-based strategy for viral attenuation and vaccine development in which one amino acid substitution produced a poliovirus vaccine with the efficacy and stability of the Sabin vaccine strains. He discovered that the mitochondrial RNA polymerase (POLRMT) is an off-target for therapeutic ribonucleosides, especially those developed to treat hepatitis C virus infection that exhibited adverse effects during clinical trials. The FDA now expects evaluation of the effect of therapeutic ribonucleosides on POLRMT prior to consideration of investigational new drug applications.The long-term goal of Cameron’s RNA research program is to develop strategies to treat and/or prevent RNA virus infection by targeting the RdRp. Using poliovirus, and its RdRp (3Dpol) as their model system, the team has

The Cameron group’s work also focuses on another emerging threat to public health, picornaviruses, which are small, RNA-containing viruses of the family Picornaviridae, including poliovirus and the rhinoviruses that cause the common cold. The objective of their work with picornaviruses is to reconstruct picornavirus genome replication in vitro from purified components. In collaboration with Jim Hogle, a professor of biochemistry at Harvard Medical School, Cameron’s team has solved the first crystal structure for a picornaviral 3CD protein. Additionally, working with David Boehr, assistant professor of chemistry at Penn State, and Mark Foster, professor of biochemistry at Ohio State University, they developed the technology to study 3C-RNA interactions by using NMR spectroscopy. Cameron doesn’t plan to stop with those discoveries; his team continues to study picornavirus genome replication as well as exploring newly

cameron effect 2 jpg
This image illustrates an anti-viral drug causing adverse effects. The skull and crossbones, which represent the antiviral drug, illustrate that the drug gets not only into the viral RNA, but also into the healthy mitochondrial RNA, causing side effects and problems. Credit: Craig Cameron lab, Penn State University
discovered functions for 3CD protein.development; picornavirus genome replication; biochemical mechanisms and biological functions of HCV NS3 and NS5a proteins; mitochondrial transcription and disease; and, lethal mutagenesis as an antiviral strategy. Although

the effect of viruses on the body is often complex, the way they work is pretty simplistic: infect a cell, make more viruses and then break out of the cell to infect more cells. Viruses multiply quickly with the help of an enzyme, polymerase, which makes more copies of the viral genetic material. Once a virus infects a cell, the immune system kicks in and triesobtained new insight into the chemical mechanism for nucleotidyltransfer. They have discovered a link between RdRp incorporation fidelity and pathogenesis and a connection between RdRp dynamics and incorporation fidelity. These discoveries have led Cameron and his team to hypothesize that RdRp incorporation fidelity is a target for antiviral and vaccine development.Cameron and his team, consisting of postdoctoral scholars, research assistants, graduate students, and undergraduate students, are currently working on severalprojects, including: RdRp mechanism; viral attenuation and vaccine to control the spread. If the immune system is unsuccessful at stopping the spread of the virus, it can cause disease or even death. However, if the body has been exposed to a vaccine – a weakened form of the virus – the body can respond more rapidly when it is exposed to the virulent strain. The key to developing vaccines is finding the mutation that will prime the immune system without causing disease. Since its inception, the primary goal of Cameron’slaboratory has been development of strategies to treat or to prevent infections by RNA viruses, using poliovirus and hepatitis C virus (HCV) as primary model systems. Cameron’s

cameron effects 3This image illustrates an antiviral drug without adverse effects. The ideal situation is to design a drug that would get into the viral RNA only, but not into the mitochondrial RNA. Credit: Craig Cameron lab, Penn State University

initial research focus was viral RNA-dependent RNA. “It’s nice to be able to see that your work has practical outcomes that could prevent people from getting sick,” said Cameron. “Creating thatfor 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 RNA-dependent RNA polymerase (RdRp) for antiviral therapy.polymerase. He studied the kinetic, thermodynamic and structural basis need to be cleared in order to show that it is a promising strategy for the future.” Viral attenuation and vaccine development is an important aspect of Cameron’s research. Vaccination is the only known approach to prevent viral infection, with the most effective vaccines being live, weakened virus strains. Current approaches for development of vaccine strains are random, slow, and prohibit a rapid response to natural, unin knowledge is exciting. But of course, there are numerous hurdles that tentional or intentional outbreaks caused by viruses. Cameron’s team has discovered a polymerase-mechanism based strategy for viral attenuation and vaccine development that can be extrapolated to any RNA virus. They are currently using mouse models to characterize the immune response to vaccine candidates. Because of their success to date with the mouse models, the team is expanding the program to include other viruses, including West Nile Virus and Respiratory Syncytial Virus.

cameron stock photo jpg

Cameron’s most recently published work involves replicating the adverse side effects of certain hepatitis C medications in the lab. “The new method not only will help us to understand the recent failures of hepatitis C antiviral drugs in some patients in clinical trials,” said Cameron. “It also could help to identify medications that eliminate all adverse effects.” The team’s findings, published in the journal PLOS Pathogens, may help pave the way toward the development of safer and more-effective treatments for hepatitis C, as well as other pathogens such as SARS coronavirus and West Nile virus.

Unlike hepatitis A and B, there is currently no vaccine to prevent hepatitis C infection. 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 United States, 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 billion per year. Because of this, it’s critical for researchers to develop effective treatments, and perhaps a vaccination, for the disease.

Jamie Arnold, a research associate in Cameron’s lab at Penn State, explained that the HCV, which affects over 170,000,000 people worldwide, is the leading cause of liver disease and, although antiviral treatments are effective in many patients, they cause serious side effects in others. “Many antiviral medications for treating HCV are chemical analogs for the building blocks of RNA that are used to assemble new copies of the virus’s genome, enabling it to replicate,” he said. “These medications are close enough to the virus’s natural building blocks that they get incorporated into the virus’s genome. But they also are different in ways that lead to the virus’s incomplete replication. The problem, however, is that the medication not only compromises the virus’s genetic material, but also the genetic material of the patient. So, while the drug causes damage to the virus, it also may affect the patient’s own healthy tissues.”

A method to reveal these adverse side effects in the safety of a laboratory setting, rather than in clinical trials where patients may be placed at risk, has been developed by the research team, which includes Cameron; Arnold; Suresh Sharma, a research associate in Cameron’s lab; other scientists at Penn State; and researchers from other academic, government, and corporate labs.

“We have taken anti-HCV medications and, in Petri dishes and test tubes, we have shown that these drugs affect functions within a cell’s mitochondria,” Cameron explained. “The cellular mitochondria – a tiny structure known as ‘the powerhouse of the cell’ that is responsible for making energy known as ATP – is affected by these compounds and is likely a major reason why we see adverse effects.” Cameron noted that scientists have known for some time that certain individuals have “sick” mitochondria. Such individuals are likely more sensitive to the mitochondrial side effects of antiviral drugs.

“We know that antiviral drugs, including the ones used to treat HCV, affect even normal, healthy mitochondria by slowing ATP output,” Arnold added. “While a person with normal mitochondria will experience some ATP and mitochondrial effects, a person who is already predisposed to mitochondrial dysfunction will be pushed over the ‘not enough cellular energy’ threshold by the antiviral drug. The person’s mitochondria simply won’t be able to keep up.”

Cameron added that the next step for his team is to identify the genes that make some individuals respond poorly to these particular antiviral treatments. “By taking blood samples from various patients and using the new method to test for toxicity in the different samples, we hope to discover which individuals will respond well and which will experience mitochondrial reactions, based on their genetic profiles,” he said. “That is, we hope to use this method as a step toward truly personalized medicine, opening the door to pre-screening of patients so that those with mitochondrial diseases can be treated with different regimens from the start.”

The team members also hope their method will be a means to study toxicity and side effects in other diseases. “Specifically, our technology will illuminate toxicity of a particular class of compounds that interrupts viral RNA synthesis,” Cameron said. “While this class of compounds currently is being developed for treatment of HCV, a wide range of other RNA viruses, including West Nile virus, Dengue virus, SARS coronavirus, and perhaps even the Ebola virus, could be treated using this class of compounds as well.”

Lethal mutagenesis is another antiviral strategy that Cameron is pursuing with his research. 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, has optimized population diversity. In the case of poliovirus, each member of the population differs from another by a few single nucleotide changes. The previous studies done by the team of the broad-spectrum, antiviral ribonucleoside, ribavirin, demonstrated that the 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 other institutions around the world, this project has transitioned to a study that focuses on identifying the properties of RNA viruses that determine sensitivity to lethal mutagens. Additionally, the team is 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. Although significant efforts have been made in developing effective therapies for viral infections, the number of approved antiviral drugs is limited. Cameron and his lab are hopeful that their work with lethal mutagenesis will someday allow for the development of antiviral drugs that are more effective and available for more ailments.

In addition to interest in creating vaccinations and treatments for RNA viruses, Cameron also focuses on mitochondrial dysfunction in human diseases. Scientists and the medical community suspect that altered mitochondrial function include 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.

invertedvirus.jpgCameron’s team was the first to reconstitute human mitochondrial transcription in vitro from purified components produced solely in bacteria. The system, marketed by Indigo Biosciences, defines a new era for the field. Cameron is now focusing on the enzymology and regulation of human mitochondrial transcription initiation, elongation, and termination. To achieve this, the team has established a network of collaborations to utilize their capabilities, which includes researchers at Penn State, in addition to scientists at other universities and industries across the country. These collaborations involve using mass spectrometry to define interactions between the core RNA polymerase and transcription factors; employing X-ray crystallography to achieve a structural perspective of the different stages of mitochondrial transcription; developing 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; studying factors that may facilitate coupling of transcription to translation; testing the hypothesis that the mitochondrial RNA polymerase is an off-target for antiviral ribonucleosides being developed for the treatment of HCV infection; and pursuing the hypothesis that mutations in mtDNA regulate mitochondrial transcriptional output and are involved in the transformational process of certain cancers. By combining their abilities with the capabilities of their collaborators, Cameron and his team are hoping to better understand how defects in transcription of the mitochondrial genome contribute to disease and aging.

The threat of disease and viral pandemics continues to rise each year, encouraging scientists like Cameron to develop vaccines and effective treatments. Creating new vaccines and expanding access to people across the world can eradicate viruses that threaten our society, extending life expectancy. Treatments for these viruses can also improve the quality of life for those affected. Cameron and his team continue forging ahead with their research, aspiring to contribute to improving lives around the globe with antiviral discoveries.