Yale Cancer Center scientists have filled in a key gap in understanding the unusual route by which the Human papillomavirus (HPV) infects cells. Their findings, published online today in the journal Cell, may eventually help to broaden the scope of defenses against HPV and provide valuable clues for delivering drugs into cells.

HPV is a family of killers. Although there are effective vaccines against these viruses, they still cause about 5% of cancer deaths worldwide, including more than 250,000 women who die of cervical cancer each year.

“As viruses go, HPV is relatively simple,” said Daniel DiMaio, M.D., Ph.D., deputy director of YCC and professor of genetics, molecular biophysics and biochemistry, and therapeutic radiology at Yale School of Medicine. “HPV’s circular DNA is stored inside a protein capsule containing only two viral proteins. Most of this capsule is made up of a protein called L1, with smaller numbers of a protein called L2 largely buried in the L1 protein shell.”

This simple virus, however, follows a complicated path to get into a cell and then its nucleus.

In previous experiments, DiMaio and his colleagues showed that after HPV enters the cell in a membrane-bound sac called an endosome, the virus latches on to a cellular protein in the cytoplasm called retromer. The retromer protein carries the virus into a cellular transport mechanism known as the retrograde pathway, which then delivers the virus to the nucleus. The YCC team also found that retromer binds directly to the end of the HPV L2 protein.

But how does the L2 protein cross the endosome membrane into the cytoplasm in the first place, so that it can bind to the retromer?

In their latest study, the investigators discovered that a short segment of the L2 protein transfers the protein across the endosome membrane into the cytoplasm. The short segment turns out to be one of a group of peptides called cell-penetrating peptides (CPPs), named for their ability to carry cargoes into cells.

“CPPs were discovered 30 years ago, but we don’t have a clear idea what the vast majority of them do,” DiMaio said. “In our case, we’ve shown that this CPP carries a protein segment through an intracellular membrane and into the cytoplasm, where it can bind to another protein.”

“We are trying to exploit this new knowledge to develop novel ways to block HPV infection,” he said. As one important part of the work, his team developed an assay that can determine whether the L2 protein has crossed the endosome membrane. “You can imagine using the assay as a drug screen, to look for compounds that block that particular step of infection,” he noted.

Many researchers have been intrigued by the possibility of using CPPs to deliver proteins and other molecules into cells as drugs. DiMaio pointed out that genomic sequencing of about 350 types of HPV has identified hundreds of variants of the L2 CPP, all evolved for their membrane-penetrating role over millions of years.

“With this study, we have uncovered an entirely new universe of CPPs,” he said. “Some of them may have very interesting and useful properties as drug delivery tools.”

Pengwei Zhang is lead author on the Cell paper. Gabriel Monteiro da Silva, Catherine Deatherage and Christopher Burd are study co-authors. The research was supported by grants from the National Institutes of Health.