July 18, 2006
Host protein triggers infection by smallpox-related viruses
WEST LAFAYETTE, Ind. — For the first time, scientists have shown that a protein in the nucleus of victims' cells triggers progression of smallpox-related illnesses, a finding that could help prevent use of such viruses as bioterrorism weapons.
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"This protein is required for activation of the middle- and late-stage poxvirus genes," said Steven Broyles, a Purdue biochemistry professor. "In the past, we were just groping around. We now have a model for how the poxvirus growth process is orchestrated."
Their research results are published in the current issue of the Journal of Virology, a publication of the American Society for Microbiology. The study is currently available online.
Although the last naturally occurring case of smallpox was in Somalia in 1977, experts believe that the disease and related viruses could be used as biological weapons.
Though most potential biological weapons occur naturally, it's possible that bioterrorists could engineer viruses and bacteria to increase their virulence, make them resistant to currently used vaccines and drugs, or partner two biological agents to make a new, more lethal illness, according to the Centers for Disease Control and Prevention (CDC).
People no longer are vaccinated against or exposed to smallpox since it was eradicated. However, laboratory stockpiles of the virus still exist in the United States and other places around the world, CDC experts report. If some of the virus were released, either in its common or a mutated form, a health crisis could result.
Two types of the most common smallpox form, variola major, almost always are fatal, according to CDC statistics. Overall, the average death rate of all types of variola major is about 30 percent.
In order to guard against a terrorist-planted or spontaneous outbreak, major scientific efforts are under way to learn how smallpox and similar illnesses so effectively can halt normal cell activity in mammals, including humans. If scientists understand the biochemical changes that allow pox viruses to cause illness, and also the processes that allow the disease-causing organisms to mutate, it may be possible to create new vaccines and treatments that could be used should outbreaks occur.
Smallpox, monkey pox, cowpox and camel pox, all members of the virus genus Orthopox, are large brick-shaped molecules. Because the pox viruses have the largest genomes of all animal viruses, the finding that these diseases recruit a victim's proteins was surprising, Broyles said.
"Poxviruses are very adept at clamping down on the cell and not letting go," he said. "These viruses are proficient in shutting down everything the host cell does."
Some poxviruses are more dangerous for animals than for people, but all cause everything from blisters to death. Monkey pox, a rare member of this virus group that usually infects monkeys, rodents and children in central Africa, appeared in the upper Midwest United States in 2003. Monkey pox, though not as virulent as smallpox, produces many of the same symptoms, including fever, pus-filled blisters and respiratory problems.
The U.S. monkey pox cases were the first known human incidents in the Western Hemisphere. An imported rodent apparently spread the disease to some pet shop prairie dogs. The people who contracted the disease had direct contact with the animals.
In all, the CDC identified 72 cases. No one died in the U.S. outbreak, although in Africa the fatality rate is as high as 10 percent.
Another of the poxviruses, vaccinia, has a genome that is 97 percent identical to the smallpox genome. Because of this similarity, vaccinia has been used as a vaccine against smallpox.
Broyles and his research team used vaccinia to find out how poxviruses progress. They knew that specific vaccinia genes are expressed at specific stages of the disease, but scientists didn't know what activated these genes, causing a cascade of events culminating in the virus controlling the host cell.
The Purdue researchers studied vaccinia's genetic makeup at the intermediate and late stages of the biochemical process that leads to a takeover of victims' cells. The scientists analyzed the resulting data and discovered that the pox virus was grabbing the TATA-binding protein (TBP) from the host cell.
TBP acts as a timer switch to turn on poxvirus intermediate- and late-stage genes, Broyles said.
"We identified TATA-binding protein as being at the center of the ability to activate the promoters — the genes that make the virus' progression and control possible," he said. "The virus begins the process by grabbing the protein out of the host nucleus. The protein comes out and hits the trigger on the virus to start the chain of events that allows the virus to attack the cell."
Now the researchers are investigating what signals the virus' genes to grab the TBP and what signals TBP to hit the trigger.
The other researchers involved in this Purdue-funded study were Bruce Knutson, a Purdue biochemistry doctoral student; Xu Liu, now a researcher at U.S. Patent and Trade Office in Alexandria, Va.; and Jaewook Oh, now with the Department of Microbiology at the University of Pennsylvania School of Medicine.
Writer: Susan A. Steeves, (765) 496-7481, ssteeves@purdue.edu
Source: Steven Broyles, (765) 494-0745, broyles@purdue.edu
Ag Communications: (765) 494-2722;
Beth Forbes, forbes@purdue.edu
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Purdue Department of Biochemistry
PHOTO CAPTION:
Steve Broyles is part of a research effort that may help prevent terrorists from using smallpox and similar illnesses as biological weapons. Broyles, shown in his lab, is a professor of biochemistry at Purdue University. (Purdue Agricultural Communication photo/Tom Campbell)
A publication-quality photo is available at https://www.purdue.edu/uns/images/+2006/broyles-poxvirus.jpg
Vaccinia Virus Intermediate and Late Promoter Elements are Targeted by the TATA-Binding Protein
and Steven S. Broyles* — Department of Biochemistry,
Purdue University, West Lafayette, Indiana 47907
Vaccinia virus replicates in the cytoplasm of the host cell and encodes its own RNA polymerase and transcription factors. The proteins that target the poxvirus RNA polymerase to intermediate- and late-class promoters have not been identified. In this study, representatives of the intermediate and late promoters were characterized at the nucleotide level to identify essential motifs. Both intermediate and late viral promoters are shown to have an essential element suggestive of TATA boxes, which are potential targets for the TATA-binding protein (TBP). Several approaches were used to test for TBP requirement in vaccinia virus transcription, including overexpression of TBP, expression of a dominant negative mutant of TBP, RNA interference, and expression of adenovirus E1A protein, which inactivates TBP. In each case, the results support an essential role for TBP in vaccinia virus intermediate- and late-gene transcription. These findings indicate that poxviruses have integrated TBP as a central feature into an otherwise heterologous transcription system. A model for transcriptional switching, in which both intermediate and late promoter elements are targeted by TBP that recruits viral transcription factors to assemble a functional complex on their cognate promoters and a dysfunctional, repressed complex on the other class, is proposed.
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