Scientists streamline method for making key virus

By devising a novel way to package the genome of a common human tumor virus – the virus that causes common warts, genital warts and that is implicated in prevalent cancers – scientists have paved the way for making the pathogen far more accessible to biomedical science.

The work, reported today (June 13) in the online edition of the Proceedings of the National Academy of Science (PNAS), promises to accelerate the search for an effective cancer vaccine and treatments for cervical, head, neck and some skin cancers.

The new study on human papillomavirus by a team of scientists from the Howard Hughes Medical Institute, the Institute for Molecular Virology and the McArdle Laboratory for Cancer Research at UW–Madison, describes a method for making large quantities of the virus in the lab.

The feat could help scientists overcome long-standing hurdles to understanding the basic biology of a major human pathogen: how the virus replicates, infects host cells and evades the immune system. It also promises to speed development of therapeutic drugs and new vaccines, including live, attenuated vaccines, according to Dohun Pyeon, the lead author of the report.

“This new approach offers dramatic advantages,” says Paul Ahlquist, a UW–Madison virologist and the senior author of the PNAS paper. “It increases virus yield over a thousand fold, speeds production ten-fold, and lets us make and test virus mutants that weren’t possible before.”

Papillomavirus can be transmitted non-sexually, but is also one of the most common sexually transmitted diseases in humans, with more than 5 million new infections reported each year in the United States alone. It is perhaps best known as the cause of genital warts, although some forms of the more than 100 subtypes of the virus are known to cause cervical cancer, and cancers of the head, neck and skin.

In nature, the virus lurks in skin cells, where it uses a “stealth strategy” to evade the immune system. It hides, initially, in undifferentiated skin cells, the only type of cell that can be infected by the virus. When the undifferentiated cells begin to develop into mature skin cells, the virus switches gears, acquires a protein coat for its DNA, and begins to churn out particles to infect other undifferentiated cells.

“The human papillomavirus life cycle is unique,” says Ahlquist. “It is closely linked to skin cell differentiation. In the basal cells, the virus just sits there quietly. Once differentiation occurs, a whole new program kicks in.”

The virus’s use of the skin cell differentiation process to jump start infection was an obstacle in the lab, says Pyeon, and prevented scientists from making large quantities of the virus for research. Established methods were cumbersome, labor and time intensive, and yielded only small amounts of infectious particles.

Obtaining useful amounts of the virus have greatly limited studies on many aspects of papillomavirus biology, says Ahlquist, who conducted the new work with Wisconsin colleagues Pyeon and Paul F. Lambert.

The team devised a way to encapsulate the complete genome of the virus in a protein coat by adding the key components of the virus – its DNA and the protein molecules that make the virus capsid or coat – to a culture of undifferentiated skin cells.

“When the capsid protein is in the cell, it will assemble the capsid” around the genome of the virus, says Pyeon. “This takes just two days.”

The advantages of the new system, in addition to greatly increasing the volume of infectious virus particles and the speed with which they can be assembled, is that is can be used to culture any of the 100 or so subtypes of the papillomavirus as well as genetic mutants created in the lab.

“There is absolutely no problem in packaging the virus, and we can make any mutant virus we want with this technique,” Pyeon explains.

The ability to make large quantities of the virus and genetically manipulate it means that scientists can bring to bear the many tools at their disposal to unravel the biological mysteries of a clinically intractable pathogen, says Ahlquist.

“This system provides major benefits for studying the virus in the early phases of its life cycle,” he says.

Moreover, the technique can provide enough of the virus to begin to experiment with and develop live, attenuated vaccines. At present, there are two human papillomavirus vaccines in trials, but they depend only on the empty protein coat of the virus to prompt a limited immune response.

“There is good reason to believe that those vaccines will be very valuable,” says Ahlquist. “Nevertheless, this new approach offers important opportunities to activate a larger set of immune responses against a larger set of viral gene products – responses that could be crucial if you want to attack the reservoir of the virus.”

In addition, the ability to culture large amounts of the virus means that high-volume drug screens can be developed to speed the search for drugs that can treat human papillomavirus infections. The cultured virus also can be used to test vaccines now in development, and genetically manipulated viruses could potentially be used as gene therapy vectors to ferry introduced genes to diseased cells.

The study was supported by a grant from the National Cancer Institute.