The greatest barrier for a virus is the immune system’s constant adaptation to new infections, demanding that the pathogen constantly evolve to keep up. Some viruses are aided in this competition by genetic material that mutates frequently, generating subtle changes that are sometimes advantageous in the face of new immune defenses. But it has been less clear how viruses whose genetic material is more stable – those that carry DNA, not RNA – have acquired the mutations they need to survive fast enough to compete with the immune system. Now, Howard Hughes Medical Institute scientists have uncovered a genetic tactic that allows one of these DNA viruses to compete in the arms race against its host’s immune system.
In a paper published August 17, 2012, in the journal Cell, HHMI early career scientist Harmit Malik at the Fred Hutchinson Cancer Research Center and Nels Elde, a former postdoctoral researcher in Malik’s lab who is now at the University of Utah School of Medicine, report on one way that double-stranded DNA viruses keep up with the ever-changing defenses of the immune system. In collaboration with Jay Shendure and Adam Geballe at the University of Washington the scientists have found that the virus vaccinia, a member of the poxvirus family, increases the size of its genome when it confronts the immune system, thereby increasing the odds of a random mutation that will improve its survival. Malik expects that other viruses may also use this genomic expansion as a strategy for adaptation.
“We started out with the less fit state of the virus and simply let the virus come up with its solution.”
Harmit S. Malik
Poxviruses are among the most dangerous viruses for the human species. But as parasitic bundles of protein-wrapped DNA, they are lifeless without a host cell. Once viruses infect a cell, they take advantage of that cell’s machinery to replicate their own genetic material and produce the proteins they need to survive. But to do all of this, the virus must overcome the constantly evolving defenses of the host immune system.
One of the immune system’s strongest cellular defenses is protein kinase R, an enzyme that lies dormant until it detects viruses replicating within a cell. Once active, protein kinase R blocks nearly all protein production in the cell to prevent virus replication, stopping only when the virus is eradicated by treatment or the cell dies.
“This is a quick and potent response that almost every cell can mount,” Malik says. So to survive, viruses must find ways to counteract the activity of protein kinase R.
RNA viruses, like polio and influenza, rely on high mutation rates to optimize the chance of finding a mutation that will defeat protein kinase R. But large double-stranded DNA viruses like poxviruses and herpesviruses are much slower to evolve than RNA viruses, leaving scientists wondering how they adapt to compete in the arms race with the immune system.
Scientists have known for many years that the well-studied vaccinia poxvirus uses two proteins to inhibit the protein kinase R response. The first protein, named E3L, prevents protein kinase R from detecting the double-stranded RNA produced in virus replication. The second, K3L, mimics a crucial host protein that signals the cell to stop growth and division, detracting the host from that signal.
Three years ago, Malik’s group discovered that rapid evolution of protein kinase R determined whether the virus was able to prevent protein kinase R’s defensive response. “But as it turned out,” Malik says, “vaccinia’s K3L protein is actually quite ineffective against the human version of protein kinase R, which can adapt to discriminate between the viral K3L imitator and the true protein.”
Now, using close observation of multiple generations of the vaccinia virus and robust methods of genetic sequencing, the scientists have identified an expansion of the K3L gene that enables vaccinia to defeat protein kinase R and compete in the arms race against the immune system.
To pressure the K3L protein to evolve, the researchers deleted for the gene that encodes E3L from the virus, removing its only other method of defense against protein kinase R. “We started out with the less fit state of the virus,” Malik says, “and simply let the virus come up with its solution, rather than direct or influence its evolution in some way.”
The team was surprised at how quickly vaccinia came up with that solution. The first Petri dish of host cells had only a few vaccinia-infected cells; protein kinase R had defeated the virus in almost all of the cells. When the researchers transferred some of those cells to a new dish, a few more became infected. After only six generations, there were ten times as many infected cells. In all three experimental trials, vaccinia had figured out how to defeat protein kinase R to become ten-fold more infectious.
In search of the genetic mutations responsible for vaccinia’s success at infecting the cell, the scientists collaborated with Shendure to sequence the genome of the evolved, more infectious vaccinia virus.
Michael Eikbush, a research technician in the Malik lab, and Jacob Kitzman in the Shendure lab teamed up to analyze the genome data and found an unusual characteristic in the segment that codes for the K3L protein. For all of the samples, there were three to four times as many K3L-coding segments as for any other gene.
“That immediately was the moment where we knew that this was the mechanism—a sort of amplification of the K3L gene,” Malik says.
But that wasn’t all—when examining the genome data from the viruses that had become ten-fold more infectious, Elde and Malik noticed a change in the genome that would alter a single amino acid in the K3L protein. “This exact mutation had actually come up in a study of vaccinia done in yeast cells by Tom Dever’s lab [at the National Institute of Child Health and Human Development] almost 12 years ago,” he says. In those experiments, a single amino acid substitution in the K3L protein had enabled vaccinia to defeat protein kinase R in yeast cells.
“We immediately had the right intellectual context to know what was going on,” Malik says. Vaccinia viruses first expand the region of their DNA that produces K3L proteins, he explains. Although that DNA mutates slowly, by producing more K3L proteins, the virus increases the probability that one of the K3L genes will randomly arrive at the amino acid mutation that lets K3L block protein kinase R’s response. Viruses harboring this specific mutation successfully infect and replicate inside the host cell.
When the researchers examined the number of K3L genes in the viral genome at each generation of cell division, they found that once the virus has the amino acid substitution it needs, the number of copies of the K3L gene shrinks back down. Maintaining a large genome is energy intensive, Malik explains, so the virus might conserve energy by reducing the gene expansion after finding a useful mutation.
The vaccinia gene expansion resulted in such a quick improvement in the strength of the virus that Malik says the next steps will be to study whether there are other adaptation-driven gene expansions that enable viruses to keep up with the arms race against the immune system.