September 11, 2002

Scientists at NSLS discover how papillomavirus ‘unzips’ DNA

Finding may lead to drugs to prevent sexually transmitted disease and cervical cancer

Infection with the human papillomavirus (HPV) is the most common sexually transmitted disease in the U.S. According to the Centers for Disease Control and Prevention in Atlanta, Georgia, an estimated 20 million Americans are currently infected – but the vast majority does not know it.

Though HPV sometimes causes genital warts, in most cases, it infects people without causing visible symptoms. Women with persistent infections from certain types of HPV are at risk for cervical cancer, as 99 percent of cervical cancers around the world are associated with HPV infection.

Preventing papillomavirus from multiplying is one way of stopping the infection. Toward that goal, a team of scientists from Cold Spring Harbor Laboratory (CSHL) in New York, working at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory, have gained new insight into how papillomavirus – in this case, cow, or bovine papillomavirus, commonly used as a model system – starts to multiply, causing infection. This new understanding could be used to design drugs to stop HPV infection, which is of particular significance since no cure or vaccine are currently available, although vaccine development is underway.

“We know very little about how papillomavirus replicates,” says biologist Leemor Joshua-Tor, the Cold Spring Harbor team leader. “So we decided to look at the molecular details of how the replication mechanism is initiated, with the aim of helping to design drugs that act like monkey wrenches in the replication process.”

Like all viruses, a papillomavirus is an infectious agent that uses the cells it infects to reproduce itself. Replication of HPV does not kill the host cells, but can make them cancerous.

The infection process starts as follows: The papillomavirus first inserts its DNA – a double-stranded helix containing the virus’s genetic information – into the host cell. The virus hijacks the protein production machinery of the host cell to produce a viral protein called E1. By attaching to the viral DNA, E1 proteins can initiate the DNA replication process, so that more viruses can be formed, and later multiply further.

“The DNA double helix can be replicated only if it is ‘unzipped,’ which allows proteins called DNA polymerases to make copies of each strand,” Joshua-Tor explains. “The E1 protein is known to initiate the ‘unzipping’ process, but how it does it is not very well understood.”

To look carefully at how E1 proteins attach to viral DNA, Joshua-Tor and her postdoctoral associate, Eric Enemark, in collaboration with Arne Stenlund, a renowned papillomavirus expert at CSHL, grew crystals of E1 and papillomavirus DNA at two different stages of the attachment process, in which either two or four E1 proteins bind to DNA.

The researchers then used a technique called x-ray crystallography to determine the positions of the atoms making up the E1 proteins and DNA. X-rays produced by the NSLS were projected toward the crystals, and the positions of the atoms were determined by looking at how the x-rays scattered off the crystal.

To their surprise, Joshua-Tor and her colleagues observed that E1 uses two separate modules, one shaped like a loop and the other like a helix, to bind DNA, each one binding to a different DNA strand (Figure 1). “This is very unusual,” Joshua-Tor says. “We expected that both modules would bind the two strands simultaneously.” The scientists also noticed that the loop bind more tightly than the helix, giving loops a larger role in E1-DNA binding than helices.

When two E1 proteins attach the DNA, Joshua-Tor and her collaborators observed that the loops bind different strands (Figure 1). When four proteins bind to DNA, they form two pairs facing each other, with proteins in each pair binding the same DNA strand as the ones on the opposite side (Figure 2).

These results suggested a mechanism by which the double-stranded DNA might ‘unzip’ (Figure 3). “We already know that, ultimately, the unzipping process involves two bundles of six E1 proteins each, called hexamers, each ‘unzipping’ the DNA in opposite directions,” Joshua-Tor says. “So, we think that the initial assembly of the two hexamers from the four proteins shown in our structure is what causes the strands to separate by forming around the single strands.”

Joshua-Tor and her colleagues suggest that the four proteins separate into two pairs, each recruiting four additional E1 proteins, thus creating two hexamers that would move in opposite directions (Figure 3). Each hexamer would encircle either strand, and act like a little propeller that rotates around the strand, thus ‘unzipping’ it from its partner DNA strand along the way.

“If this is the way these proteins operate, it is pretty clever,” Joshua-Tor says. “This is the first time that it has been found that the two individual DNA strands bind to two separate protein modules prior to the DNA ‘unzipping’ process.”

While Joshua-Tor and her colleagues are still investigating the papillomavirus-induced DNA replication, they are also starting to test compounds that interfere with papillomavirus DNA replication. For example, Anitra Auster, a graduate student, is developing compounds that could interfere with the DNA replication induced by a high-risk type of human papillomavirus that can lead to cervical cancer.

“Understanding these binding mechanisms could significantly improve the treatment of this sexually transmitted disease,” Joshua-Tor says. “We can now design and test drugs aiming to prevent E1 proteins from attaching to the viral DNA, which is one of the first steps to making much-needed antivirals against HPV infections and HPV-induced cervical cancer.”

BEAMLINE
X26C

PUBLICATION
E. Enemark, A. Stenlund, and L. Joshua-Tor, "Crystal Structures of Two Intermediates in the Assembly of the Papillomavirus Replication Initiation Complex," The EMBO Journal, Vol. 21, No. 6, 1487-1496 (2002).

FOR MORE INFORMATION
Leemor Joshua-Tor
http://www.cshl.org/public/SCIENCE/joshua.html

SCIENCE WRITER: Patrice Pages