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Princeton Viral Escape Hatch Could Be Treatment Target for Hepatitis E

Researchers at Princeton and Rutgers universities have found that the hepatitis E virus — an emerging liver virus historically found in developing countries but now on the rise in Europe — uses a technique to spread infection that scientists could in fact exploit to treat the disease. (Image courtesy of Alexander Ploss, Department of Molecular Biology)

The technique that the hepatitis E virus — an emerging liver virus historically found in developing countries but now on the rise in Europe — uses to spread could present a weak spot scientists can exploit to treat the disease, according to a new study.

Princeton University-led researchers report in the Proceedings of the National Academy of Sciences that when the hepatitis E virus infects a cell, it makes proteins that poke holes in the cell’s membrane to allow newly made virus particles to escape and spread. Known as viroporins, these proteins create channels that allow the inflow and outflow of charged particles called ions that disrupt the infected cell’s physiology and lead it to release the infectious particles.

Yet the very proteins the virus uses to escape a host cell and spread infection could in fact provide an avenue for developing antivirals that target hepatitis E, the researchers said. They suggest that curtailing the virus’ ability to produce viroporins may prevent the pathogen from being able to burst forth from infected cells to attack other cells. Drugs that target viroporin production have already been developed for other viruses that employ the proteins to aid infection, including hepatitis C virus, HIV-1 and influenza A.

“These proteins cause changes in the cell membrane that allow the newly made hepatitis E virus particles to spread,” said senior author Alexander Ploss, a Princeton assistant professor of molecular biology. “If we can prevent the release of viral particles, we could slow the spread of infection and give the immune system time to clear the virus from the body.”

Viruses multiply by taking over an infected cell’s reproductive machinery so that they can make millions of viral copies. These copies then exit the infected cell, usually by bursting it open. Hepatitis E virus, the researchers found, is among a subset of viruses that prime the cell membrane for bursting by using viroporins. Exactly how the transmission of ions through viroporin channels causes the host cell to burst is an active area of research. It may be that ions entering through the pores draw in so much water that the cell explodes like a water balloon, Ploss said.

The available treatments for hepatitis E are not specific and have side effects. Believed to be uncommon in the United States, the virus has become prevalent in Europe in the last few decades where it is contracted primarily via the consumption of undercooked pork and foods such as raw pork-liver sausage. In developing nations, the disease is primarily spread through contaminated food and water. Globally, the virus causes about 20 million cases per year.

In most cases, the virus causes only mild, short-term symptoms such as fever and jaundice. For pregnant women and people with compromised immune systems, however, the disease can be much more severe. Among women in the third trimester of pregnancy, fatality rates as high as 25 percent have been reported, according to the World Health Organization. Immunocompromised individuals, including those infected with HIV and organ-transplant patients taking immunosuppressant drugs, can develop a persistent infection that can eventually destroy their liver.

The researchers discovered that within the virus’s genomic material — which in this virus is RNA — a section known as open reading frame 3 (ORF3) contains the “instructions” the virus uses to make viroporins. Qiang Ding, a postdoctoral fellow in Princeton’s Department of Molecular Biology, performed a series of experiments aimed at finding the function of the protein made from ORF3. Ding’s colleagues at Princeton included research specialist Brigitte Heller, graduate students Bokai Song and Ila Nimgaonkar, and research specialist Gabriela Hrebikova, all in molecular biology.

During these experiments, Ding and his colleagues noted several pieces of evidence indicating that the ORF3 protein could span the cell’s membrane. They noticed that the protein collected on one of the cell’s internal membranes surrounding an organelle known as the endoplasmic reticulum. The researchers also noticed that the proteins formed small clusters that resembled ion channels.

To find out whether these membrane-spanning proteins were needed for letting virus particles escape the cell, Ding and his colleagues removed the ORF3 RNA from the hepatitis E virus. They found that without ORF3, the virus could not exit the infected cell. When they substituted in RNA from influenza A virus — which also use viroporins — the researchers were able to restore the ability of the virus particles to exit the host cell.

A more direct test came with help from researchers at nearby Rutgers University. When ions exit or enter the cell, they cause measurable changes in the electrical currents traveling through the membrane. These currents can be measured with a technique called patch clamping, which involves measuring the currents inside and outside the cell.

To conduct these tests, Ploss contacted Jorge Contreras, an assistant professor at Rutgers New Jersey Medical School. Contreras and graduate student Juan Capuccino taught Ding how to do the experiments, which involved adding ORF3 RNA to cells isolated from Xenopus laevis, or the African clawed frog.

The researchers found that the frog cells expressing ORF3 RNA allowed ion fluxes across the cell membrane whereas X. laevis cells without the viral RNA did not. “We recorded the currents and if the protein was able to facilitate the movement of ions across the membrane down their concentration gradient, it was working as an ion channel,” Contreras said.

The researchers are now exploring the structure of ORF3 to look for areas that could become treatment targets, Ploss said. “We are working on identifying parts of the protein that could possibly prevent the formation of the ion channel or prevent the flux of ions through the channel,” Ploss said.

The paper, “Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles,” was published in the journal Proceedings of the National Academy of Sciences the week of Jan. 16. The research was supported by the Burroughs Wellcome Fund and the New Jersey Commission on Cancer Research.

Significant Contributor and Catalyst to N.J. Economy, Quality of Life

Princeton University has a substantial impact on the New Jersey economy, generating an annual total of $1.58 billion in economic output as an employer, research and innovation leader, sponsor of construction projects, purchaser of goods and services, and financial and civic contributor to local communities. That total supports an estimated 13,450 jobs with $970.7 million in earnings.

The economic and other benefits the University generates within the town of Princeton and neighboring communities, Mercer County and the state of New Jersey are presented in a new report, “Education, Innovation and Opportunity: The Economic Impact of Princeton University.” This study, conducted by Appleseed, an economic consulting firm, updates a 2008 analysis and provides a comprehensive look at the University’s direct and indirect effects on the vitality of the local, regional and state economy in the fiscal year ending June 30, 2015. The study also identifies resources, services and opportunities that the University contributes to enhance the quality of life in its neighboring communities and throughout the region.

“Princeton University takes pride in its longstanding partnership with the communities that surround it,” said University President Christopher L. Eisgruber. “Through on-campus learning opportunities, joint transportation and safety programs, community service projects, and initiatives that encompass the arts, entrepreneurship and many other areas, the University and its neighbors continue a vibrant tradition of cooperation founded on shared values, interests and histories.”

Consistently ranked as one of central New Jersey’s largest, most stable employers, the University employs a workforce of more than 6,900 people (excluding student employees). Of those employees, 58 percent live in the Princeton area or elsewhere in Mercer County and 80 percent reside in New Jersey. Princeton paid nearly $602 million in employee salaries and contributed an additional $24.8 million in New Jersey state income and unemployment insurance taxes and paid family leave insurance in fiscal year 2015.

The study also found that the University’s investments in research and entrepreneurship catalyze innovation throughout the state. As a major research institution, Princeton attracts hundreds of millions in federal research dollars to New Jersey each year, much of which is spent locally, to pursue solutions to pressing societal challenges. Research expenditures amounted to $457.6 million in fiscal year 2015 — a nearly 80 percent jump since 2007. Internal University resources allocated to research increased significantly, comprising 30 percent of the 2015 total.

Research collaborations with leading New Jersey companies such as Siemens and Bristol-Myers Squibb and academic institutions including Rutgers University and the Robert Wood Johnson University Hospital further extend the impact of Princeton’s research enterprise. In the last five years, the University has strengthened the state’s innovation ecosystem through the launch of the Intellectual Property Accelerator Fund to advance researchers’ proof-of-concept work, the Keller Center’s eHub as a focal point for entrepreneurship, and the Alumni Entrepreneurs Fund to support alumni startups.

“Princeton is committed to extending the benefits of its research capabilities and innovation beyond the campus, into companies and organizations that can translate ideas into practical solutions to improve social and economic well-being,” said Coleen Burrus, Princeton’s director of corporate engagement and foundation relations. “There are so many innovative companies right here in New Jersey, and the University is eager to continue to work with them to build research collaborations.”

Businesses across the region significantly benefit from University spending on goods, services and capital construction projects. In 2015, Princeton spent $453.6 million in goods and services to advance its education and research mission; of that total, nearly $137.3 million was paid to New Jersey vendors, including $35 million paid to Princeton-area businesses and $16.5 million paid to businesses elsewhere in Mercer County. During the same period, capital construction and renovation projects amounted to $318.1 million in University spending, 43 percent of which went to New Jersey contractors and vendors.

With the campus serving as a destination for academic, athletic and cultural events, the Princeton regional economy sees a steady infusion of visitor spending each year. An estimated 768,000 people visited the University in 2015, spending nearly $50 million and directly and indirectly accounting for $63.4 million in economic output. Local consumer spending was supplemented by an estimated $59.7 million that Princeton students spent in the community.

The University makes direct financial contributions to the towns and county in which it resides. In fiscal year 2015 it paid more than $16.3 million in property taxes, fees and voluntary contributions to local governments. This included a voluntary unrestricted contribution of $2.86 million to the town of Princeton under a seven-year agreement that provides for increases each year through 2020 as well as capital contributions valued at $2.9 million. The report notes that “the stabilizing presence of Princeton University” contributes to an Aaa bond rating for the town that leads to significant savings in its borrowing costs.

The University enhances quality of life through its financial and volunteer support to community and civic organizations; maintenance of area roads; volunteer, training and funding support to public safety agencies; commitment to sustainability; and provision of local affordable housing. In addition, community members of all ages enjoy the wide variety of educational, cultural and athletic programming that the University offers year-round, much of it at modest cost or free of charge. In 2014-15, for example, 1,165 people participated in the University’s Community Auditing Program to take regular university courses on a non-credit basis, with more than half of the participants coming from the Princeton area.

The Appleseed analysis forecasts that Princeton’s continued investment in research and innovation, renovation and construction of new facilities, and plans to expand student enrollment will lead to even greater contributions by the University to New Jersey’s economic strength in the years to come.