Decoding A Virus

University molecular biology professor Thomas Shenk and visiting research fellow Wade Bresnahan were separating viral DNA fragments on a gel when they noticed something odd.

When DNA strands are separated on a gel, they normally separate into distinct bands — but Shenk and Bresnahan saw something else, something they had never noticed before.

"When we were analyzing the DNA gel, we saw a smear at the bottom. I knew that's where RNA is," said Shenk, who is the chair of the molecular biology department. And because the virus they were studying — the human cytomegalovirus, also known as HCMV — was supposed to contain only DNA, they were suspicious.

The two virologists ran further tests to make sure their samples were not contaminated. Then they developed a sophisticated procedure that could be used to identify the messages that the RNA carried.

When they had identified the RNA strands, Shenk and Bresnahan — a postdoctoral fellow in Shenk's lab — had shattered a long-held definition of a virus. "Viruses had always been defined as having either RNA or DNA," said Shenk, who specializes in HCMV research, with a focus on the mechanisms by which it replicates and infects cells. What they found was a virus that had both RNA and DNA, something never before observed.

"It was completely novel," said Bresnahan who earned a bachelor's degree from St. Cloud State in Minnesota in 1992 and received his graduate degree from the medical branch of the University of Texas in 1997.

Human cytomegalovirus infections are widespread, especially among young people, senior citizens and those with compromised immune systems — including transplant recipients, cancer patients and AIDS patients. HCMV is a giant among human viruses, containing more than 200 genes — more than any other known human virus.

Genes are composed of DNA, a code that contains the instructions for making the proteins that serve different purposes in the cell, from providing structural support to aiding biological reactions.

Since the genes are found in the nucleus of the cell and proteins are synthesized outside the nucleus in the cytoplasm, the cell needs a go-between that can carry the instructions from the DNA to the ribosomes, where proteins are made. RNA, a close cousin of DNA, can act as that go-between. After RNA is transcribed from DNA in the nucleus, it makes its way to the ribosomes in the cytoplasm, where it provides the instructions for ordering amino acids to make new proteins. Aside from aiding protein synthesis, RNA can also serve other roles such as aiding cellular mechanisms.

Protein synthesis can be carried out only with the proper cellular machinery, which viruses lack. Viruses are simply snippets of protein-encased RNA or DNA that wander around the body and the environment, looking for a cell with the necessary machinery to invade so that it can replicate itself.

Ordinarily, when a virus infects a cell, the virus' genetic material travels into the nucleus, where it is inserted into the genome — the cell's DNA. Some proteins are also injected into the cell. DNA viruses incorporate their DNA directly into the genome, whereas RNA viruses must first make the necessary complementary DNA that preserves the message of the original RNA.

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Once the virus has incorporated its genetic material into the genome, it can start making the proteins it needs to make new viruses that can then spread to other cells.

What makes HCMV unique is that it has both RNA and DNA, the scientists explained. HCMV has a strand of DNA that can code for about 210 different proteins — this DNA goes into the nucleus and inserts itself into the genome so that it can make the necessary proteins for replication. The newly discovered RNA apparently already codes for some of the proteins that the DNA codes for, apparently to start making new proteins before the genetic material is even incorporated into the genome.

Shenk — who came to Princeton in 1984, nine years after completing his postdoctoral work at Stanford University — was thrilled by the pair's discovery. "It was very exciting to find out why the virus is doing this, why it is important, the way the virus interacts with us," he said.

As part of their ongoing research on HCMV, the scientists developed a sophisticated procedure called a "gene array" that was specific for HCMV. The gene array they developed is a collection of all the DNA fragments for HCMV — it can be used to screen for RNA or DNA that might be part of HCMV's genome. If an unknown genetic strand matches any of the DNA fragments, it will stick to the part of the array where its complement is located. This allows Shenk and Bresnahan to identify unknown genetic strands and their messages.

"We envisioned many applications for an HCMV gene array, so we developed the array," Shenk said. "We had noticed that RNA was present in purified virus particles, so we decided to analyze this RNA as a first application for the array." Until they developed the HCMV gene array, they knew that the virus had RNA but they could not identify it.

Bresnahan added, "There was no way of knowing where or what type of RNA — viral or cellular — it was. The HCMV gene array allowed us to identify the specific HCMV transcripts that were packaged."

Developing the HCMV gene array took several months, Bresnahan said. "There was a period of several months between knowing that there was RNA and identifying it," he explained. "Once we had the tools, we could go ahead and test it."

Once they developed the gene array, they were able to confirm that the RNA strands are indeed part of the virus and carry the same messages as part of the viral DNA. They then screened the RNA strands with all the different DNA fragments from the genome. An RNA strand that carries the same message as one of the DNA fragments will hybridize with, or bind to, that fragment, thus allowing Shenk and Bresnahan to identify the messages that the RNA strands carry.

They also ran some experiments to determine if any RNA strands assisted in constructing proteins. To test whether the RNA coded for the construction of proteins, they introduced a drug to the infected cell that prevented the virus from using its DNA. Results showed that even without active DNA, the virus was still able to make proteins with at least some of the RNA strands.

Because they only recently identified the RNA strands, they do not yet fully understand why HCMV contains it. "We've identified it, but we still don't know the function of it," Bresnahan said.

He theorizes that the RNA might "establish an environment suitable for viral infection," essentially clearing the way for the DNA to do its work.

Apparently, the RNA strands help the virus infect a cell more quickly because it helps make new proteins while the DNA is still working itself into the cell's genome. "Presumably, these RNAs are packaged so that they can be translated very early within a newly infected cell, even before the viral DNA reaches the nucleus and begins to be transcribed," explained Shenk, who graduated from the University of Detroit in 1969 and earned his Ph.D. from Rutgers University in 1973.

It has been theorized that the inclusion of the RNA strands makes it possible to synthesize a protein that would not otherwise be able to get to certain parts of the cell. If the DNA were to make those proteins, they would be altered beyond recognition by cellular pathways before they reach their destination — the inclusion of RNA strands apparently works around this problem.

The two scientists speculated that there is also a possibility that at least some, but not all, of the RNA strands do not code for proteins. Rather, some of these strands may instead serve to neutralize the host cell's anti-viral defenses and prevent the cell from self-destructing. Often, cells will attempt to self-destruct in the face of a viral infection to prevent the virus from multiplying and infecting other cells.

When asked if other viruses might also have both RNA and DNA, Shenk replied, "I'm going to guess but I don't know. There could be both in other viruses. We haven't looked at the others yet."

Even though Shenk has spent many years working on the virus, he said he has many questions still unanswered. Aside from the function of the RNA strands just discovered, he is still puzzled by why viruses are sometimes active and sometimes not. "How the virus becomes dormant is something we don't understand at all," he explained.

And the pair is still searching for answers about viruses and the way they behave, a difficult and elusive task that will require years of experimentation.