Anatomy of Infection

Understanding retroviral assembly is key to controlling the world's most lethal communicable diseases

by Rhituparna Chatterjee

More than 25 years have passed since the world first woke up to the nightmare of acquired immunodeficiency syndrome and the human immunodeficiency virus that causes it. Over the intervening two-and-a-half decades, according to statistics compiled by the World Health Organization, 25 million people have died. Of those deaths, according to the Centers for Disease Control and Prevention, more than 530,000 were Americans.

Thankfully, these grim statistics are not the full story. Since AIDS was first identified in 1981, important therapeutic breakthroughs have prolonged the lives and reduced the suffering of millions of patients. These treatments, however, are not a cure. Today, an estimated 39.5 million people are living with HIV/AIDS around the globe. Over the next 25 years, according to UN-sponsored research, millions more will contract the disease. As many as 117 million could die.

Scientists such as Marc Johnson, an assistant professor of molecular microbiology and immunology at MU, are determined to avert such a catastrophe. Progress depends, he says, on better understanding how retroviruses, the class of contagion that includes HIV, assemble new viruses.

"What we're working on are ways to better understand and study the process by which viruses, specifically the virus HIV, manufacture new viruses inside infected cells," Johnson says.

Viruses infect healthy cells because they need such cells to reproduce, he explains. "When a healthy cell is compromised, that cell becomes a sort of assembly plant for the production of more virus particles. These particles then leave the cell and seek out new cells to infect, like tiny insidious parasites. So far, much of the research into preventing the spread of HIV has centered on preventing healthy cells from being infected. This is basically trying to stop the virus in a defensive way -- it's only preventing new virus from infecting cells. Comparatively little has been done to see how the virus can be stopped from manufacturing new particles, which would render it incapable of attacking healthy cells in the first place. That's what my research is about."

HIV is a retrovirus, a tiny, RNA-containing organism that infects cells of the human immune system, rendering it susceptible to other infectious diseases. The HIV life cycle goes something like this: The virus enters a human cell and makes DNA copies of its RNA genome using a viral reverse transcriptase enzyme. Such enzymes reverse the typical DNA-to-RNA flow of genetic information; hence the name "retrovirus." The DNA copy of the genome then goes into the nucleus and "hides" inside a host cell's chromosome.

The virus then exploits the host's molecular machinery to make new viral proteins and new viral RNA, which assembles in the cytoplasm of infected cells to form new viruses. These leave the cell, infect new cells, and start the cycle all over again.

Over the last 10 years, scientists have unraveled many of HIV's molecular secrets. They now have a fairly sophisticated understanding of its genes and its proteins, as well as the ways these interact both with each other and with molecules of the host cell. These insights hastened the discovery of the protease inhibitor class of drugs that, when taken in combination with other medicines, can block HIV reproduction and prolong patients' lives.

Nevertheless, Johnson says, there remains a pressing need for new treatments. Not only do the existing drugs come with a bundle of serious side-effects, but HIV's ability to rapidly mutate can quickly make the drugs ineffective. Another problem, he says, is that most drugs attack very late stages of the virus' life cycle, often well after the newly made viruses have begun to attack new cells in the patient.

"If we can have more drugs that target earlier stages of the virus' life cycle, we can prevent new viruses from forming and leaving the infected cells," he says. "This will be one step closer to our ultimate goal of killing off the last infected cells and ridding the body of the infection."

The viral genome is small as genomes go, only about 9,000 bases long. It codes, or provides information for, just nine genes. Here's where things get complicated, Johnson explains. "There are two major components to a virus particle, the Gag protein and the Env protein," he says. "These pieces are assembled in the infected cell and packaged into the virus particle, but we have very little knowledge about how these two proteins actually find each other."

And there are other questions: Do the individual components communicate with each other, or do they interact via the host cell's proteins? Are there specific sites inside the host cell where these events take place? How does the virus fool the host machineries into helping it multiply?

Johnson joined the search for answers during a post-doctoral stint with retrovirus expert Volker Vogt, a professor of biochemistry, molecular and cell biology at Cornell University. An early project involved coming up with ways to create a visual representation of the Gag protein, a process he still uses today. The choice of an imaging project was obvious, Johnson recalls, especially for a self-described visual person. "It's much easier to convince people of what you are seeing if you can actually show a picture of it," he says.

Virologists often use scanning electron microscopes to observe individual viruses and their structures. Unfortunately, this type of microscopy cannot be used to visualize proteins inside living cells. Johnson has thus turned to fluorescence microscopy, which allows him to observe the Gag protein inside a live HIV-infected cell.

The imaging process is anything but straightforward. Johnson must first join the Gag protein with Green Fluorescent Protein, or GFP, from a jellyfish. This "recombined gene" is Gag-GFP, which Johnson can track in the cell using a fluorescent microscope.

Like most research tools, this one isn't perfect. It does not allow him, for example, to observe how the proteins form new viruses, since individual proteins, or a group of proteins, cannot be distinguished from new viruses. To get around the problem, Johnson has come up with an innovative means of combining fluorescent markers and scanning electron microscopy to get a better picture of virus formation.

"You can think of fluorescent microscopy as being like satellite images. They are very useful at showing you the big picture, but are not very high resolution," Johnson says. "To put these images in context, we collect scanning electron microscopy images of the same cell and combine the two kinds of images. The electron microscopy images cannot be captured in real time like the fluorescent images, but we can get about 100 times greater resolution with this technique. This example shows us not only that the Env proteins have gathered in clusters, but that the clusters are at virus particles' assembly sites on the cell's surface, meaning they have been recruited by the Gag protein to the assembly site."

Using this technique, Johnson and his fellow researchers found that not all the Gag-GFP made their way into new viruses, and that young human immunodeficiency viruses took turns leaving the cell, one by one, often through the same spot on the plasma membrane.

Johnson published the finding in the October 2005 issue of the Proceedings of the National Academy of Sciences. At least two different research groups have adopted his technique, one to study viral assembly and the other to look at viral entry into host cells.

It was an important discovery, although Johnson warns that more research is needed before he can prove this to be the general mechanism for HIV to exit from the host cells. Still, Johnson's former boss was impressed.

"That's entirely his own creation," says Volker Vogt of Johnson's imaging combination. "And it is going to go places, I can say that."

After finishing his six-year stint with Vogt last year, Johnson moved to MU to start his own lab. He says, only half-joking, that the decision to move to Columbia was predestined: Johnson was born in Columbia 34 years ago, on the very same floor that today houses his academic department. "Perhaps it's all just imprinting," he laughs. "Just like the salmon do it."

Despite a new laboratory to set up and new team members to train, Johnson is still able to spend time at the lab bench. He is currently designing a large-scale study to screen molecules for their ability to stop HIV from assembling inside the infected cells. At this point, he says, it is still a "pie in the sky idea," but worth a try. Johnson is also expanding his repertoire of viruses, from HIV to Murine leukemia virus, human T-lymphotropic virus and others. "I am a strong believer in studying multiple related viruses at once when it is possible," he says. "We are trying to figure out riddles.  It's like trying to solve the Rosetta Stone. If scholars had focused only on the Greek writing or only on the Egyptian [demotic] writing, then we never would have learned how to read hieroglyphics. It is exactly that way for the viruses."

At MU's Life Sciences Center, Johnson is part of a core group of virologists. One of them, MU associate professor Donald Burke, says that he knew and appreciated Johnson's work well before he joined the MU faculty.

"There are many people trying to understand how viruses do these things," says Burke. "People who are doing it best are applying creative techniques, and Marc's approach is creative. He is one of the small number of people at the front."

Others agree. Last year Johnson was one of a select group of researchers who received the 2006 Beckman Young Investigator Award from the Arnold and Mabel Beckman Foundation. The California-based organization makes grants to promote research in chemistry and the life sciences, particularly research using instruments and materials that may open up new avenues of scientific exploration.

The support was a welcome vote of confidence, Johnson says, one that affirms that his work and the work of his colleagues may one day help tame some of the world's worst viruses. "The exciting thing," Johnson says, "is that not only can we learn more about how HIV behaves but, since many viruses appear to assemble at the cell surface in similar ways, what we're investigating here has relevance to researchers studying influenza, rabies, measles, Ebola, and even the bird flu."