National Center for Macromolecular Imaging

Houston researchers fight microscopic battles


Houston Chronicle Medical Writer

This story is copyright material from the Houston Chronicle

If you climb one flight of stairs and walk down the long third-floor hall across the front of Baylor College of Medicine, your trip spans a half century of science directed at understanding and defeating the smallest living organism -- the virus.

At one end of that short trek is the second-floor office of Joseph Melnick, a pioneer virus researcher. At the other is Wah Chiu, who uses computers to create live, three-dimensional pictures of those tiny microbes.

It is the age of viruses. One of the most devastating and obvious is the human immunodeficiency virus -- HIV, which has spawned a global AIDS epidemic. The deadly Ebola virus has unleashed its killing force in Zaire. Hantavirus, previously seen in Korea and China, made headlines when it dealt death in the Four Corners region of the U.S. Southwest two years ago. Sporadic cases are now reported every summer.

And scientists speculate there are other, as yet undiscovered viruses percolating in other corners of the Earth --viruses that inevitably will cause disease in man and animals.

Melnick and Chiu are not working on Ebola or Hantavirus. Those are more safely probed at places such as the Centers for Disease Control and Prevention in Atlanta, where there are special containment facilities.

But their work on herpes viruses, the rotavirus and the various viruses that infect bacteria will provide information that can be applied in other virus studies.

Melnick is a pioneer in viral research, one of those who identified whole classes of viruses. He developed biochemical techniques that provided the first real information about how viruses work and developed theories about the viral causes of human disease.

Chiu is the pioneer of the '90s, charting a new course in a field called structural biology. Using powerful microscopes, computers and mathematics, he is developing three-dimensional models on computer screens. It is a cutting-edge technique that approaches viruses as locks; if the lock can be properly seen and studied, perhaps a key can be created to open and destroy it.

The two bring different tools to the fight, but each respects the other's work.

"He was in at the beginning," says Chiu.

"What he's doing is amazing," says Melnick. "He's taking them (viruses) apart to find out how they work."

At 80, Melnick is a tall, solid figure in a white lab coat. Behind the frames of his dark glasses, his eyes sparkle when confronted with new ideas. He speaks slowly when explaining difficult concepts to the uninitiated.

Melnick received his doctorate in biochemistry from Yale at the dawning of the virology era in the 1930s.

As late as 1939, there was only one widely used anti-virus vaccine -- the smallpox immunization that dates from the late 1700s.

But by 1939, science was mustering its forces to take on a new enemy -- the polio virus that was maiming and killing the nation's youth -- and Melnick became a part of the battle.

The National Foundation for Infantile Paralysis that became the March of Dimes funded the first great collaborative effort aimed at eliminating a disease from the United States.

Melnick worked on the first research grant that grew out of the effort, beginning his scientific career in the company of fledgling giants such as Albert Sabin and Jonas Salk.

"When I first began working with viruses, most of them could not be seen under a microscope," Melnick says. With the crude light microscopes they used at the time, "Even the biggest -- vaccinia --were only little dots."

Melnick went on to help define an entire new class of viruses -- the enteroviruses. Polio is one of the enteroviruses most likely to infect the gastrointestinal tract. It is only when polio invades the nervous system that it causes paralyzing disease.

His work with rotaviruses helped explain how they cause the diarrhea that kills more than 1 million children around the world each year.

Melnick saw the science change. The work took a giant leap when scientists developed techniques to grow viruses in cultures of cells nurtured in fragile petri dishes in a laboratory.

With an electron microscope, he could bombard viruses with negatively charged subatomic particles and, for the first time, see the hazy outlines of his tiny foes.

. . .

Biochemical techniques allowed him to analyze and study the structure of viruses and to detect the presence of viruses that no one knew existed.

What Melnick's early work was to the age of test tubes, Chiu's is to the age of computers and video displays.

The former's research is part of the structure upon which the latter's work is built. Except for their common goal of stopping disease through viral research, the two men have different approaches and different styles -- professional and personal. While Melnick favors white lab coats, the 48-year-old Chiu prefers shirt sleeves or a suit when he is on the lecture circuit.

Unlike Melnick and many others in the field who studied biology, Chiu, who went to college in California, started out in physics.

In the late 1960s, the space race between the United States and the Soviet Union drew many young people into the sciences, and Chiu became one of them. He entered the University of California at Berkeley and studied physics.

But after finishing his bachelor's degree, Chiu decided he wanted to look into problems that affected people more directly. His graduate work was in biophysics -- a study that usually leads into cancer treatment.

"I was still more physics than biology," he says.

However, biology was what drove him. He studied the subject and "learned to talk like a biologist."

After receiving his doctorate in biophysics, he began to work with imaging technologies --the microscopes and computers that are his stock in trade today.

He was one of the pioneers in the field.

It was while he was heading an imaging program at the University of Arizona that he began to do research with viruses.

Work with rotaviruses led him to a collaboration with Dr. Mary Estes at Baylor College of Medicine in the Texas Medical Center, who suggested that he move to Houston, where he would have more collaborators and greater access to the technology he needed.

In Houston, he could start a program and build it the way he wanted.

Chiu accepted and began his work here in 1988.

Viruses are natural phenomena, occurring in all animals, including humans.

Most are no more than a few genes swathed in one or more protein coats. No one knows how they came into being or why they exist. In her book A Dancing Matrix : How Science Confronts Emerging Viruses, Robin Marantz Henig proposes that a virus is a "dancing matrix," pieces of errant genetic material that pass from one organism to another, circulating genetic messages from one species to another, much as bees spread pollen from one flower to another.

. . .

This dance, she writes, is a natural way of varying the gene pool, propelling evolution and increasing genetic variability.

The bearer of these genes is infinitesimally tiny, thousands of times smaller than the microscopic cells they invade.

To put the size in perspective, consider that the average person is less than two meters tall. The cells that make up that body are as small as 0.3 microns. (A micron is one millionth of a meter.) And even these cells are thousands of times bigger than the viruses that enter them.

The diameters of the smallest viruses are measured in nanometers -- one-billionth of a meter. To call them living seems to stretch the definition of life itself.

Outside the cell, viruses are fairly inert particles with a few tricks that can gain them entry into cells. And as long as they are outside of these natural hosts, viruses cannot reproduce or metabolize.

But once inside, a virus subverts the cell's genetic machinery, often becoming an instrument of destruction as it overrules the cell's original function, forcing it to function for the virus.

Inside their protein coats, called capsids, some viruses contain a double strand of DNA (the form in which genetic information is usually stored); others a double strand of RNA (the form in which genetic information is usually transmitted). Others have only single strands of either DNA or RNA.

This diversity of genetic materials is one reason medicine has had a difficult time engineering the vaccines and drugs to control the myriad viruses that inhabit the world.

Spikes on the outside of capsids are called capsomeres and contain special areas that are shaped to allow the viruses to attach themselves to the outside of cells -- much as pieces of jigsaw puzzles fit together.

Often the protein coats of viruses mimic the genetic pattern of materials the cell needs -- allowing the virus to sneak past cellular and immune defenses.

When there is a perfect fit between virus and cell, the virus gains entry. Once inside, enzymes divest the virus of its protective protein coat and expose the genetic material (either DNA or RNA), which is the virus' heart and which superimposes the genetic messages it carries over those of the cell itself.

The cell's genetic blueprint is like a computer code. It contains the instructions the cell needs to sustain itself and to carry out specialized functions within the body.

But when the genes of the virus add their own computer program to that of the cell, in many instances, the messages from the virus overtake those of the cell, which ceases to function normally. The cell becomes the site at which the virus takes in or makes materials necessary for its survival. It is also the site of virus reproduction.

Eventually, the cell becomes a virus factory, releasing new viruses into the bloodstream.

Researchers need more specific information to manufacture effective vaccines and drugs. They need to know how the protein coat is assembled, where the antigenic sites attach and how the individual layers of protein interact.

. . .

In the past, scientists used trial and error in their attempts to create vaccines and drugs to fight viruses. Sometimes -- as in the case of polio and small pox -- they were lucky.

In other cases, the virus is more complicated, and attempts have failed --as in HIV.

But now they are trying to define the structure of these particles in an attempt to design drugs that will solve the specific problems each virus presents.

Enter scientists such as Chiu, who use powerful electron microscopes and computers to assemble virus replicas.

On the computer screen, Chiu can rotate virus images, cut them apart and view them from all directions to discover their vulnerabilities and develop biochemical methods of taking advantage of those weaknesses.

The first step in the process is to take pictures of the viruses with an special electron microscope that suspends a frozen virus specimen in a vacuum to eliminate all possible distortion.

When Chiu came to Baylor, his first job was to find a stable, sturdy place without noise or tremors, where he could locate the cryo-electron microscope he uses to make pictures of viruses.

The slightest vibration --from a passing truck, an automobile horn or helicopter --could throw his highly magnified pictures out of whack.

He found that place deep in a cold room of the 1950s-era sub-basement of Baylor's main building, where the structure's utilities -- its electrical conduits, and pipes for hot and cold water -- run above the halls that lead to Chiu's microscope room.

Here, Joanita Jakana works her magic, operating the big cryo-electron microscope that extends into an alcove higher than the 9-foot ceiling. Jakana sits behind a control board of video screens, knobs and dials. She is concentrating on a virus preparation held in the vacuum of the microscope's viewing chamber.

Earlier, the specimen had been carefully prepared to exist in the vacuum within the microscope. Suspended in water while attached to a tiny copper grid, it was placed in the grasp of laboratory tweezers and plunged into liquid nitrogen.

The trick is to freeze the entire specimen before the water can form ice crystals. The water prevents the microscope's vacuum from drying out the sample.

If the freezing is done quickly enough and to a low enough temperature, the water becomes vitreous -- a smooth, glass-like medium against which the virus particles clearly stand out. Ice crystals would deform and destroy the viruses. But as long as Jakana can maintain her specimens at -182 degrees Celsius, she will see a true representation under the microscope.

Jakana begins scanning at low magnification as she searches for the perfect spot for her pictures. If she were to use higher magnification, the bombarding electrons would damage the proteins in the virus. When she finds the right place, she increases the magnifying capability 30,000 to 40,000 times.

. . .

On this day she is looking at a virus that infects bacteria --a collaborative project with Jonathan King, a researcher at the Massachusetts Institute of Technology.

"He wants to know what his virus looks like," she says.

Jakana can give him tiny black-and-white photos that show one dimension of the virus. But taking several pictures of the virus in different orientations, measuring them in several ways and running those numbers through a computer results in a three-dimensional picture that pops up on a video display screen.

From these pictures, the MIT researcher can study how the structure of the virus -- called bacteriophage 22 --varies at stages of its life.

The information may seem inconsequential, given the threats of HIV or Ebola, but what is learned about this virus may provide important information about how all viruses live and replicate.

Chiu's laboratory is one of only a few in the world that has the technology and has attracted the expertise to make the pursuit of such research possible.

The National Institutes of Health designated his program a National Center for Research Resources. But Chiu and his colleagues do not limit their activities to the United States. They collaborate with people all over the world.

One of the most active collaborations is with Frazer Rixon, a scientist with the Virology Unit of the Medical Research Council in Glasgow, Scotland.

Chiu met Rixon while both were on a sabbatical in Cambridge, England.

"It was an opportunity to learn," Chiu said. "We would eat, drink and talk. I dived into that culture."

He and Rixon began talking about the Scot's current enthusiasm -- herpes simplex virus type 1, the virus that causes cold sores.

The two began a collaboration, looking into the structure of that complicated virus using the cryo-electron microscope and computers to assemble a three-dimensional image.

Today, Rixon and one of Chiu's students, Hong Zhou, who recently received his doctorate from Baylor and has gone on to post-doctoral studies at the University of Houston, communicate by the Internet as each works in his own way to uncover the secrets of the herpes virus.

Rixon takes the images that Zhou produces on the computer and develops specially engineered forms of the virus that will provide information about how it infects the body and how it works to subvert the cells it invades.

The images also give clues about particular structural weaknesses the viruses demonstrate.

Structural data show that the capsid -- the outer protein layer -- is formed of three shapes and two proteins.

. . .

Two shapes, one resembling a child's flower drawing with six petals and the other a flower with five petals, make up most of the capsid. The hexon (or six-sided shape) and penton (or five-sided shape) are made of the same protein, Rixon said during a recent Houston visit to meet with his collaborators.

Their work identified the second protein in the shape of a small six-sided cap that sits atop the hexon.

"We think that the hexon shape makes the walls of the herpes virus capsid," Rixon said. "The penton shape makes the top. But the smaller protein appears to lock on the hexon to reinforce those walls and make it stronger. It's just a theory now. But we would not know any of this without this technology."

Building on this kind of knowledge, Rixon eventually might identify a way of destabilizing the herpes structure or inserting a protein that prevents the virus from completing its life cycle.

Mary Estes and B.V.V. Prasad, both of Baylor, hope to do the same with rotavirus.

Prasad strives for greater and greater detail in his pictures of the rotavirus, because he knows they will provide the key to designing drugs and vaccines for its prevention.

"Good structural information provides you with a good, rational explanation of how the virus works," he said. "From that, we can proceed to rational drug design."

Rational drug design is a holy grail for structural biologists. It means taking what one knows about how an organism is made and how it lives and applying that information to making a drug.

With viruses, it might mean making a drug that attaches to the virus at each point where the virus has a structure that allows it to enter the cell. If that structure, called the antigenic site, is the key and the place it fits on the cell is a lock, then a blocking drug could render the key useless.

In the Estes lab, scientists are working with the genes that make up the shell of the rotavirus. Using a special preparation of insect cells, she inserts the rotavirus genes into the cellular DNA. The genes then tell the cell to create the proteins that make up the virus shell. The proteins then assemble themselves into a replica of the rotavirus shell -- one that lacks the essential genetic material needed for replication.

From her studies, Estes, a professor of molecular biology, hopes to develop a vaccine against rotavirus.

She and Prasad, an associate professor of biochemistry and molecular virology, also hope to make drugs that will stop rotavirus and Norwalk virus, another organism that causes diarrheal disease.

Chiu recently visited Japan, where he saw a microscope that could bring even more viral and molecular structures into focus.

The $3 million instrument is too expensive for U.S. scientists who are watching research funds dwindle, Chiu says, "but I think we should have something like this in the United States."

And if there is to be one in the United States, he adds, why not in Houston?

As yet, no "rational drug" designed through structural biology has made it to pharmacy shelves, and Chiu thinks his field will need more refining and better technology like the Japanese microscope before that can become a reality.

Chiu's work is still in its infancy, and many scientists are unaware of its value, but he has strong supporters.

"From structural biology, you can extract information that will be important at the bedside," says Dr. Salih J. Wakil, chairman of the biochemistry department at Baylor. "Molecular medicine is the future. It is important that we know how these organisms are organized," he said.

"When we know these intricate structures, we can fight AIDS and cancer as well as viruses."

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