Conference Highlights Oncogenic Virus Research at UW

Starting in late July 2018, the five-day International Conference on EBV and KSHV melded the International Association for Research on Epstein-Barr Virus (EBV) and Associated Diseases’ biannual conference with the Kaposi’s Sarcoma Herpes Virus (KSHV) annual meeting. This event overlapped—for the first time—with the Molecular Biology of DNA Tumor Viruses Conference and was sponsored by several UW–Madison entities, the National Institutes of Health (NIH) and other supporters. A joint reception and scientific talks provided opportunities for a diverse group of researchers to interact for the greater good.

“I believe this is the largest group of DNA tumor virus researchers that’s ever been together in one place,” exclaimed Shannon C. Kenney, MD — the Wattawa Bascom Professor in Cancer Research at the UW School of Medicine and Public Health and co-leader of the Virology Program at the UW Carbone Cancer Center — when she welcomed participants to a keynote talk.

The conference covered several oncogenic viruses, including EBV, KSHV and human papillomavirus (HPV), with topics such as latency and reactivation, gene replication, epidemiology, vaccine development and therapeutic interventions. Speakers represented government agencies and academic medical centers from throughout North America and the world.

As president of the International Association for Research on EBV and Associated Diseases, Kenney co-organized the conference with Eric C. Johannsen, MD, associate professor, UW Departments of Medicine and Oncology, and Janet E. Mertz, PhD, the Elizabeth McCoy Professor of Oncology, Department of Oncology. They designed the conference around themes, rather than type of virus, to foster cross-sectional opportunities.

Johannsen, Kenney and Mertz, along with Bill Sugden, PhD — the James A. Miller Professor of Oncology and American Cancer Society Research Professor and associate director of career development and education for the Carbone Cancer Center — collaborate on EBV research at the McArdle Laboratory for Cancer Research, part of the cancer center, in the Wisconsin Institutes for Medical Research. Together, they have expertise in all aspects of EBV, including its biochemistry and molecular biology, pathogenesis, and the development of unique mouse models to test hypotheses and novel therapies. Kenney and Johannsen also provide patient care in infectious diseases at UW Health.

One of the most common human viruses and the first human virus discovered to have oncogenic properties, EBV is among eight known human herpesviruses. It’s found worldwide, and most people contract this virus at some point, often with no symptoms or only mild illness. Once infected, people host the latent (inactive) virus for life.

Spread most commonly through bodily fluids, including saliva, EBV is best known for causing infectious mononucleosis, but it can cause severe illnesses in individuals who have a weakened immune system. Several cancers are associated with EBV, including Burkitt’s lymphoma, nasopharyngeal carcinoma (NPC), gastric carcinoma, Hodgkin’s disease and non- Hodgkin’s lymphoma.

“Until recently, researchers had access to only a handful of EBV genomes, but in recent years, knowledge has vastly expanded. There now are hundreds of genomes available to study,” Kenney notes. “Because certain virus strains are more prevalent in specific tumors, it’s important to analyze variants.”

Research on mutations and various strains relies on next-generation sequencing, which maps massive amounts of RNA and DNA to determine the genomes from which the strands originate, explains Johannsen.

“This technology helps us know what is mutated and what genes are active to learn how to tailor treatments,” he says.

Paul F. Lambert, PhD ’85 — the Howard M. Temin Professor and Chair of Oncology at the School of Medicine and Public Health and director of the McArdle Laboratory for Cancer Research— notes that approximately 15 percent of human cancers globally are known to have a viral etiology, and this percentage likely will grow as researchers identify additional human viruses and investigate viral causes for more cancers. Thus, it’s an increasingly important global public health concern.

About the conference, Mertz says researchers from various nations — such as Korea, Japan, China and Germany — were drawn to attend because presentations explored virus-related cancers that are prevalent in distinct regions of the world, as well as techniques and characteristics that are similar across the spectrum.

“EBV-associated gastric carcinoma is common in Korea and Japan; and nasopharyngeal carcinoma due to the virus shows a high prevalence in southeast Asia,” she explains. “In central Africa — where EBV was discovered — the virus causes a high incidence of Burkitt’s lymphoma.”

Noting that countries tailor their screening strategies with the goal of diagnosing the most frequent diseases in their populations, Mertz says, “In Korea, for example, it’s common for a person’s annual physical to include screening for virus-induced stomach cancer. And in China, physicians routinely test for NPC. Also, countries ramp up their research funding and encourage global collaboration related to the diseases most likely to affect people in their regions.”

At McArdle, a large NIH program project grant, “Molecular Biology and Genetics of Human Tumor Viruses,” answers this call. Its principal investigator since 2007, Lambert, notes that UW–Madison, the School of Medicine and Public Health and the Carbone Cancer Center also contribute substantial funding.

Decades of Work

The program project grant’s legacy dates to 1977, when Howard M. Temin, PhD, first received the competitive NIH grant. Having joined the UW faculty in 1960, Temin continued important tumor virology work until he died, at age 59, from cancer in 1994.

“Dr. Temin established tumor virus research as a major strength at UW–Madison, with this program project grant as the central point of the effort,” notes Lambert, adding that Temin was among three internationally recognized researchers to earn the Nobel Prize in Physiology or Medicine in 1975 for their discovery of reverse transcriptase, which challenged the central dogma of genetics, that there is a unidirectional flow of information from DNA to RNA to protein.

He continues, “By discovering reverse transcriptase, Dr. Temin demonstrated that there also is a flow of information from RNA back to DNA. This seminal discovery led to drugs that today help keep HIV-infected patients alive, and it revolutionized our ability to study gene function.”

Temin’s dedication to rigorous scientific methods has carried through to the current program project grant, which earned a competitive five-year renewal in April 2018.

Mertz observes that McArdle teams often attract trainees and faculty members who hail from international locales, likely related to the global nature of the viruses they study.

Several investigators who work on the program project grant — now in its 41st year — worked with Temin. Among them are Lambert; Mertz; Sugden; Paul Ahlquist, PhD ’81, the Paul J. Kaesberg Professor of Oncology and Molecular Virology, investigator, Howard Hughes Medical Institute and Morgridge Institute for Research, associate director of basic research, Carbone Cancer Center, and professor, Department of Plant Pathology, UW College of Agricultural and Life Sciences; and Dan Loeb, PhD, professor, Department of Oncology.

“Dr. Temin was instrumental in attracting some of the grant’s current investigators to McArdle, including Drs. Mertz, Lambert and Loeb, and me,” says Sugden, who was the program project grant’s principal investigator from 1992 to 2007.

Loeb recalls joining the team following a memorable phone call, which he received while working as a post-doc at the University of California, San Francisco.

“My lab mate answered the phone and said, ‘Hey, Dan, it’s Howard Temin, and he wants to talk with you,’” Loeb shares. “To my astonishment, he invited me to apply for a research position, but instead of asking me questions, he shared selling points about the work at UW–Madison. I have my notes from that phone call hanging by my desk!”

Mertz began working with Temin after she had conducted research with another Nobel Prize winner, Paul Berg, PhD, at Stanford University in California.

“I published my first paper when I was a graduate student in Dr. Berg’s lab. The study showed how to make recombinant DNA using restriction enzymes and ligase, and it opened up the field for others to make recombinant DNA,” she says.

Project Expansion

Throughout the program project grant’s four-decades of continuous funding, it has expanded to include three core areas: administration/ statistics, instrumentation and virus production. Lambert has recruited additional faculty members, including Kenney and Nathan Sherer, PhD, associate professor of molecular virology and oncology, who brings his “world-class live cell imaging skills to bear on Hepatitis B (HBV) biology.”

The program project grant’s five projects are conducted by eight team members, as follows:

• Project 1: Molecular Genetics of HPV Infection and Oncogenesis, led by Lambert with Ahlquist as a co-investigator
• Project 2: Mechanisms of HBV Virus Replication, led by Loeb with Ahlquist and Sherer as co-investigators
• Project 3: Characterizing the Amplification Factories of EBV and Kaposi’s Sarcoma- Associated Herpesvirus, led by Sugden with Johannsen as a co-investigator
• Project 4: Controlling the Latent-Lytic Switch in EBV, led by Mertz with Kenney as a co-investigator
• Project 5: EBV Drivers of Oncogenesis and Novel Therapies, led by Kenney with Johannsen and Sugden as co-investigators

In line with the program project grant’s goals to “understand the life cycles of human tumor viruses, identify their contributions to the induction and maintenance of human tumors, and develop novel approaches to prevent and treat these tumors,” the team focuses on three types of human tumor viruses — papillomaviruses, hepadnaviruses and herpesviruses — which contribute to 19 of the 21 viral-induced human cancers.

In progress reports, Lambert describes the grant’s seminal contributions. For instance, the team has trained more than 140 post-doctoral fellows, many of whom have established distinguished careers. Also, over the past five years, program project grant team members:

• published more than 90 papers that contribute to the global understanding of human tumor virus biology and ways to prevent or treat viral-associated cancers;
• discovered the critical role of estrogen receptor alpha expression in the stroma in mediating estrogen’s carcinogenic effect on cervical epithelia in mice and women, and the cell non-autonomous effects of HPV oncogenes, expressed only in the cervical epithelia, on the stroma, thus defining new roles of the tumor microenvironment in cervical carcinogenesis;
• defined the recently identified mouse papillomavirus, MmuPV1, to be a highly informative model for high-risk cutaneous HPVs in causing skin disease and cancer, at the pathophysiological, molecular and genomic levels;
• developed high-resolution imaging strategies to view multiple steps in replication of the HBV genome;
• used new imaging capabilities to discover the novel association of HBV reverse transcriptase (Pol) with mitochondria, identifying a potentially novel role of Pol in virus-host interactions;
• used “visible” EBV, created by this program project grant, to discover that EBV remarkably reorganizes the architecture of the host nucleus to allow it to support the amplification of the EBV genome upon lytic activation; and
• developed “visible” KSHV and discovered that it replicates during latency differently than does the highly related EBV.

“Another important feature is that our team’s interactions go beyond the program project grant’s boundaries, impacting research across the country and the globe,” says Lambert. “More than half of our publications involve collaborations outside the grant.”

He adds, “By having investigators work on shared projects focused on one or more human tumor viruses, we aim to use advances in understanding one virus to foster experiments on others. These collaborations are aided by meetings at which faculty discuss findings, technical problems, breakthroughs and much more.”

More than a quarter of the team’s studies in the past five years involved two or more program project grant labs. For instance, Lambert — who has developed powerful mouse models to understand oncogenesis by the human papillomavirus HPV16 — works with Ahlquist to build on their findings that the tumor microenvironment plays critical roles in cervical carcinogenesis. And Loeb is working with Ahlquist and Sherer to define the replication steps of human HBV using high-resolution and live-cell imaging technologies that are shared with Johannsen and Sugden.

In other collaborative work, Mertz and Kenney’s laboratories focus on understanding the regulation of EBV’s switch from latent to lytic (replicating) states with the goal of developing beneficial therapies.

“We want to better understand what controls that switch by looking at cellular factors in strains of EBV. We then look for drugs — lytic-induction therapy — that can switch the virus from its dormant state into its replicating state, which it must be in for drugs to work. After we test drugs in a petri dish, we progress to testing the successful compounds in mouse models to see whether they effectively kill cancer cells without harming normal cells,” explains Mertz.

Kenney describes a recently published study in which the team found that a specific base pair change present in certain cancer-associated EBV strains has a large effect on the activity of a critical EBV gene promoter.

“This viral promoter, which drives the switch from latent to lytic infection, is more active in the cancer-associated EBV strains, making them able to produce lots of infectious virus,” she says. “We believe that the importance of this promoter alteration is that it allows the virus to reproduce more actively, particularly when patients are co-infected with malaria.”

Kenney says the presence of malaria is why EBV-positive Burkitt’s lymphoma appears primarily in the “malaria belt” in Africa but is uncommon in the United States. Researchers believe malaria infection is a co-factor in causing cancer in Africa, and she predicts other co-factors that preferentially activate the cancer-associated promoter variant could be responsible for southern China’s high incidence of NPC.

“Although almost everybody is infected with EBV, researchers don’t fully understand why it causes cancer in only a small percentage of people, nor why it causes different cancers regionally,” says Kenney.

Johannsen notes, “Immunocompetent people require more cellular mutations to develop EBV-associated tumors. Contracting EBV is one step in a multi-step process to get cancer, but what are the other steps? Our work aims to answer questions like this.”

In addition to investigative work, Carbone Cancer Center faculty members share their knowledge with the next generation of researchers through a grant-funded Virology Training Program, led by Lambert. Aimed at predoctoral and postdoctoral trainees interested in all aspects of virology, it has 19 faculty members, including all program project grant team members, who study a broad variety of viruses, including those that infect plants and insects, and others that cause human cancers. Also, some study viruses related to emerging diseases, such as Ebola and new strains of influenza.

Through research and teaching, McArdle Laboratory’s faculty members continually search for and share innovative discoveries and approaches in their quest to understand viruses, prevent illnesses and develop treatments for patients here and throughout the world.