August 4, 2006
CONTACT: Karen Kreeger
karen.kreeger@uphs.upenn.edu
Penn Researchers Determine Structure
of Smallpox Virus Protein Bound to DNA
Structure Could Aid in Anti-Viral Drug Design
(Philadelphia, PA) - Researchers at the University of Pennsylvania
School of Medicine have determined the structure of an important
smallpox virus enzyme and how it binds to DNA. The enzyme, called a topoisomerase,
is an important drug target for coming up with new ways to fight smallpox.
The researchers present their findings in the August 4 issue of Molecular
Cell.
“This enzyme is one of the most closely studied DNA-modifying enzymes
in biology,” says Frederic D. Bushman, PhD, Professor
of Microbiology, one of the senior authors. “The structure of the
DNA complex has been long-awaited.” DNA-modifying enzymes bind to
specific sequences in the genetic code to aid in the many steps of DNA
replication.
The smallpox virus is one of the most easily transmissible infectious
diseases known to humans, resulting in up to 30 percent mortality. The
efficiency with which it spreads, combined with the deadly nature of the
disease, has raised fears that smallpox could be revived for use in bioterrorism.
Knowing the exact three-dimensional structure of smallpox virus proteins
could help researchers design antiviral agents, but few structures of
whole viral proteins exist.
Poxviruses are large viruses that contain two strands of DNA and replicate
themselves entirely in the cytoplasm of infected cells. Poxviruses do
not take over the genetic machinery inside the nucleus of the host cell,
as many viruses do. Because of this strategy, poxviruses encode many of
the enzymes they need to replicate their own genes, and hence reproduce.
One of these enzymes is a topoisomerase, which is used by the virus to
relieve the excessive twisting of DNA strands that normally occurs during
DNA replication and transcription of the viral genes. Upon initial infection,
the poxviruses come already equipped with some proteins, including topoisomerases,
to kick-start replication.
The structure was determined in a collaborative effort between the Bushman
lab and the lab of the other senior author Gregory D. Van Duyne,
PhD, Professor of Biochemistry and Biophysics and an Investigator
with the Howard Hughes Medical Institute (HHMI). Using purified topoisomerase
enzyme that had been expressed in bacterial cells, they bound the enzyme
to short segments of DNA that contained the viral topoisomerase’s
specific recognition sequence. They then determined the three-dimensional
structure of the topoisomerase-DNA complex using X-ray crystallography.
One of the primary differences between the viral topoisomerase enzyme
and the closely related human enzyme that functions in the nucleus of
all human cells is that the viral enzyme only relaxes supercoiled DNA
when it binds to specific DNA sequences. The structure of the poxvirus
topoisomerase-DNA complex provides some important clues about how this
recognition and activation mechanism works.
“The more the viral enzyme differs from the human nuclear enzyme,
the more likely it is that inhibitors could be developed that are specific
to the viral enzymes,” says Bushman.
Knowing the three-dimensional structure of the smallpox virus topoisomerase-DNA
complex will also facilitate the design of agents to combat poxvirus infections.
Topoisomerases are some of the most widely targeted proteins by drugs
that are intended to inhibit growth of the cell. Drugs that target topoisomerases
generally stabilize an intermediate of the enzyme’s reaction in
which one of the DNA strands is broken. If these breaks are not repaired,
the DNA cannot be replicated and the cell dies.
In the case of smallpox virus, the hope is that drugs targeted to the
viral topoisomerase enzyme will prevent viral replication through a similar
mechanism. The X-ray structure provides a template for designing small
molecules that could stabilize the broken DNA in the intermediate form,
thereby killing smallpox virus particles.
Study co-authors are Kay Perry and Young Hwang, both from Penn. The research
was supported by HHMI and the National Institutes of Health through the
Middle Atlantic Regional Center of Excellence.
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This release is available online at
http://www.uphs.upenn.edu/news/News_Releases/aug06/smlpxenz.htm