"We have uncovered a new target that could potentially lead to new antivirals. There is certainly a need because there are no specific human papillomavirus antivirals out there at this point," said Peter Howley, the Shattuck professor of pathological anatomy at HMS.
Unlike some viruses–such as influenza, HIV, and the common cold virus–which churn out copies within the confines of a single host cell that is then destroyed, human papillomaviruses invade proliferating epithelial cells and distribute their copies among subsequent generations. To maintain their integrity, and to ensure that the viral DNA is properly partitioned between daughter cells, human papillomaviruses have adopted the strategy of tying their genetic material, via the viral protein E2, to a protein found on the host's mitotic chromosomes. But the identity of that chromosomal protein has been elusive.
Howley, Jianxin You, HMS research fellow in pathology, and their colleagues have identified the missing chromosomal link in cells infected with a bovine form of papillomavirus. By blocking the protein, BRD4, they prevented papillomavirus-infected mouse cells from becoming cancerous. Perhaps most significant, BRD4 appears to tether human papillomavirus as well.
"This suggests that if one could come up with a small molecule or chemical that could inhibit the binding of E2 to BRD4, that could be a drug lead," said Howley. He and his colleagues are currently working with members of the Harvard University Department of Chemistry to identify such small molecules.
Human papillomavirus is a leading cause of sexually transmitted diseases in the United States. Though the genital warts resulting from such infections might be treated with human papillomavirus-specific therapies, the real beneficiaries would be women with virus-associated precancerous lesions of the cervix, said Howley. Combined with early methods of detection such as Pap smears, human papillomavirus specific antivirals could lower the rate of cervical cancer, currently the second most common cancer in women in this country and abroad.
"The real value of this approach for treating papillomavirus infections would be to treat precancerous cervical lesions so that they cannot establish a persistent infection and progress into cancer," said Howley.
The story of how papillomaviruses maintain infection in dividing cells got a boost in 1997 when researchers discovered that the viruses link their genetic material--a ring of DNA, or plasmid--to the host's chromosomes by means of the viral protein, E2. "What was missing is the identity of the cellular protein with which the viral protein E2 interacts," said Howley. Researchers tried a variety of genetic approaches such as expressing the E2 gene in yeast as a kind of bait for interacting partners, but the cellular half of the equation--the chromosomal tether--remained elusive.
As it turns out, E2 is an incredible multitasker. The viral protein is involved in replication, transcription, and cell growth, in addition to its tethering duties. Rather than try to isolate a single interacting partner, You decided to capture the entire package in vivo. Using a relatively new proteomic approach developed by Yoshihiro Nakatani, HMS professor of biological chemistry and molecular pharmacology at Dana-Farber Cancer Institute--she extracted E2 plus its many interacting partners and then set out to identify each member in the complex. It was during this experimental foray that she spotted BRD4. Though little was known about it, a mouse homolog had been identified as a mitotic chromosome-associated protein. "It immediately jumped out at us that this could very well be the mitotic tether that we had been looking for," said Howley.
The researchers performed a series of experiments, each of which would confirm the hunch. To begin, they used immunofluorescence staining to see where E2 and BRD4 resided in infected mouse cells. Both localized to the mitotic chromosomes. In fact, biochemical tests showed that the proteins did interact. Next, they blocked BRD4 with a competitive mutant, or dominant negative, version of the protein. Sure enough, upon immunofluorescence staining, E2 no longer appeared to be associated with the mitotic chromosomes.
Most exciting, the researchers found that when the dominant negative BRD4 competitors were introduced into mouse cells turning cancerous by a strain of bovine papillomavirus, the cells' rate of transformation to cancer declined by over 90 percent. "All the evidence pointed to BRD4 as the receptor for E2," said You.
That is probably true of human papillomavirus as well. You and colleagues found that human papillomavirus E2 interacts with BRD4. In fact, the region on the E2 protein that binds BRD4 is highly conserved in all species of papillomavirus. "That suggests to us that BRD4 is probably the common target for all the papillomavirus E2 proteins," said Howley.
As it turns out, papillomaviruses are not the only viruses that have the task of maintaining and partitioning their viral genome in a dividing cell. Epstein-Barr and Kaposi's sarcoma herpesviruses face a similar challenge. "To date, none of the mitotic receptors for any of these other viruses has been found," said Howley. He and his colleagues are currently exploring the possibility that BRD4 is the mitotic tether for these viruses as well--and that drugs targeting BRD4 could be used to treat an even broader array of infections.
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*Via EurekAlert: http://www.eurekalert.org/jrnls/cell/pages/pdf/cell/c1173you.pdf
*Contacting Heidi Hardman at Cell: hhardman@cell.com.
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Cell