University of Michigan Scientists Discover How Viruses Hide Inside Human Cells

ANN ARBOR --- What evil lurks in the hearts of cells? Erle Robertson and Murray Cotter know.

These University of Michigan scientists have discovered how some viruses can hide inside the nucleus of human cells for long periods of time---without producing symptoms or triggering an immune response---by attaching to host cell chromosomes. The virus survives by going dormant until a weakened immune system allows infected cells to begin multiplying wildly again.

In an article to be published in the Nov. 25, 1999, issue of Virology, Robertson and Cotter describe a series of experiments with Kaposi's sarcoma-associated herpesvirus or KSHV---a human virus associated with a type of cancer called Kaposi's sarcoma. In these studies, U-M scientists found a protein expressed by one gene on the virus that builds a biochemical docking station linking viral DNA to the chromosomes of lymphoma cells. The U-M study is the first to identify a specific tethering mechanism between a virus and its host cell.

KSHV, also known as human herpesvirus 8, is one of a family of gammaherpesviruses known to remain dormant in humans long after the initial infection is over. Other similar viruses include the Epstein-Barr virus, the human papilloma virus which causes cervical cancer, and viruses responsible for hepatitis B and hepatitis C.

"We've always suspected that latent viral DNA couldn't survive long term within cells without some type of tethering," said Robertson, Ph.D., assistant professor of microbiology and immunology in the U-M Medical School. "But the latency mechanism for these viruses has been a black box. Now we have a key that will get us in the front door."

Using cultures of lymphoma cells infected with KSHV, Cotter and Robertson identified a protein called the latency-associated nuclear antigen or LANA, which is expressed by one of approximately 80 genes encoded by the virus. They found that LANA binds to three regions of the KSHV genome, but is most likely to lock onto one specific region for tethering the virus to host chromosomes.

In addition to viral DNA, U-M scientists found that LANA also binds to histones---small proteins that link bundles of DNA called nucleosomes together to make chromatin fibers, which are folded and packed to form chromosomes.

"The results suggest a biochemical mechanism which binds elements of viral DNA to host chromosomes through the interaction of LANA, histone H1 and possibly other chromosomal proteins," Robertson said.

Robertson has evidence for a similar tethering mechanism in the Epstein-Barr virus, which infects immune system cells called B-lymphocytes. Associated with several varieties of cancer, including breast cancer, Epstein-Barr virus is found in more than 90 percent of the world's population. In most people, a healthy immune system keeps the virus suppressed. If something upsets the balance between virus and immune response, however, the virus can re-activate. The trigger that signals a dormant virus to begin multiplying and infecting new cells remains unknown, according to Robertson.

In previous studies with Epstein-Barr virus, scientists identified a protein called EBNA1, which binds to B-lymphocyte chromosomes. Since ENBA1 is expressed in all EBV-infected cells and LANA is expressed in all KSHV-infected cells, Robertson believes they may have similar functions. "We haven't linked all the pieces yet, but it is extremely likely that EBNA1 is part of a similar tethering mechanism for Epstein-Barr viral DNA," he said. "If there are two viruses with the same mechanism, then there probably are more."

HIV, the virus that causes AIDS, is a different type of virus and is unrelated to the U-M study, adds Robertson. However, Kaposi's sarcoma is a common cancer in people whose immune system has been suppressed by the AIDS virus.

In future research, U-M scientists will try to find LANA's exact binding site on Kaposi's viral DNA and on histone proteins. "Once we understand the biochemistry of the tethering site, we can start developing therapeutic agents to block it. Blocking the binding site could mean the difference between just treating symptoms and eradicating these viruses from the host population," added Murray A. Cotter II, a graduate student in the U-M Medical School.

Cotter also hopes to apply what he learns from KSHV biology to gene therapy research. "The central question in gene therapy is how do you stabilize foreign genes in a cell's nucleus, so they will express beneficial proteins over long periods of time," he said. "This tethering mechanism may give us clues that we could use to develop more effective gene therapy vectors."

The U-M has applied for a patent on the viral tethering mechanism. The research was supported by the National Cancer Institute, the American Heart Association, the Leukemia Society of America, and the U-M Health System's Comprehensive Cancer Center.