AT FIRST sight, the industrial city of Bradford in the north of England looks an unpromising place to discover new and exotic forms of life, but appearances can be deceptive. Two years ago, French researchers announced that a mysterious beast plucked from one of the city’s cooling towers years previously was something mightily unusual – a giant virus that seemed to break all the rules.
When word started spreading of the discovery, the near universal response was disbelief. Viruses are by their very nature small. They normally consist of a few snippets of DNA or RNA wrapped inside a simple protein shell 100 or so nanometres across – no more than what is needed to invade cells, replicate and get out. And rather than being viewed as independent life forms, each one is seen as a non-living “bag of chemicals”, a glitch in the genetic program of life.
The Bradford bug blew this dogma to bits. Dubbed Mimivirus, it was a mammoth 500 nanometres in diameter – 750 if you count its hairy coat. That is not only several times larger than any other virus, but larger even than many bacteria. What’s more, Mimivirus’s genome contains an astonishing 911 genes arrayed over 1.2 million letters, or nucleotides, of double-stranded DNA. Most viruses don’t even make double figures in genes. About half of Mimivirus’s genes are new to science. Dozens more are involved in processes long believed to be surplus to a virus’s requirements. In another viral first, Mimivirus boasts some genes from the “universal core genome”, the 60 or so genes common to every bacterium, plant and animal that has been sequenced. All told, it’s as genetically sophisticated as some free-living organisms.
Advertisement
Not surprisingly, scientists are scratching their heads over this mysterious discovery. Why would a virus need to be so big? Why does it have so many genes? Does it force us to rethink what a virus is? As if all this weren’t enough, some biologists now believe that the discovery raises some fundamental questions about the origin and evolution of life. Mimivirus may even be distantly related to you and me.
“Mimivirus breaks the boundary that used to be considered sacred – the boundary between viruses and cellular life forms,” says Eugene Koonin, an expert in evolutionary genomics at the National Center for Biotechnology Information in Bethesda, Maryland. “It’s mind-boggling,” adds virologist James Van Etten of the University of Nebraska in Lincoln.
The common view of viruses as non-living bags of chemicals dates back to 1935, when biochemist Wendell Stanley of the Rockefeller Institute in New York analysed tobacco mosaic virus and found it had none of the molecular equipment needed to perform the metabolic functions found in living cells. Hence the view of viruses as little more than rogue fragments of genetic material that blindly invade cells to commandeer their molecular machinery. When most virologists think of viruses, they typically picture HIV, which has only nine genes and less than 10,000 letters of genetic code, or the hepatitis D virus, which gets by with only a single gene.
That’s not to say every virus is a minimalist. Since around 1990, microbiologists have been quietly discovering more and more viruses with genomes between 100,000 and 300,000 nucleotides long. They have also come across genes that were thought to be useless to viruses, such as ones involved in photosynthesis.
“There’s a lot more sophistication in viruses than this image that we typically have of them as simply renegade replicons that kill cells,” says Luis Villarreal, director of the Center for Virus Research at the University of California, Irvine. “They have attained a level of sophistication that is well beyond what most people think.” Still, no one was prepared for what came next.
“Not only is this virus big, but we’ve found evidence that it is becoming bigger”
Unusual suspects
The Mimivirus story actually begins in 1992, when Timothy Rowbotham, a microbiologist who had been tracking the source of a pneumonia outbreak in Bradford, took samples from the cooling tower and discovered amoebas infected with a mysterious parasitic microbe. Thinking it was a bacterium, Rowbotham named the bug Bradford coccus and set about trying to classify it. When he failed, he did what microbiologists often do with unknown microbes – he stored it away in a freezer and left it at that. When Rowbotham retired in 1998, the still unstudied microbe made its way into the lab of Bernard La Scola, a bacteriologist at the Mediterranean University in Marseille, France.
La Scola also assumed Bradford coccus was a bacterium. Under the light microscope it was the size and shape of a bacterium. It tested positive for a chemical staining test called the Gram stain, placing it firmly in a category with bacteria rather than viruses. “In bacteriology when something stains Gram-positive, it is always a bacterium – always,” says La Scola. “With its size and this aspect of Gram-staining, we were sure that it was a bacterium.”
Yet when La Scola set out to analyse part of the microbe’s ribosomal RNA – a molecule associated with the protein-making apparatus that has become a universal barcode for bacterial classification – he couldn’t find it. Blaming his failure on a procedural mistake, he repeated the same experiment more than two dozen times, to no avail.
Eventually La Scola decided to have a look at the source of his frustration using an electron microscope. With this vastly more powerful tool he discovered Bradford coccus looked nothing like any bacterium he had ever seen. Its outer shell was in the form of an icosahedron – a gem-like polyhedron of 20 triangular faces, common to many viruses. Searching through a borrowed set of virology classification guides, he discovered similarities between Bradford coccus and members of a group known as nucleocytoplasmic large DNA viruses (NCLDVs). What startled him was that even the largest of these lookalikes was several times smaller than the beast lurking in his lab. “That’s when we realised we had something interesting,” he says.
However, a bacterium-sized virus was not something microbiologists were ready to swallow. In 2002, La Scola submitted a description of what he was now calling Mimivirus – after its skills as a microbial mimic – to Nature. The editors rejected the paper as inconclusive, but after preliminary sequencing of the Mimivirus genome had revealed several viral-like genes, the journal Science accepted a brief report (vol 299, p 2033). This was followed late in 2004 by the publication of the entire genome, which showed that Mimivirus carries 26 of the 31 genes common to all NCLDVs. Even so, doubts persisted.
“A lot of people thought, well maybe it’s a novel bacterium and they just haven’t realised it yet,” says Elodie Ghedin, a virus expert at the Institute for Genomic Research in Rockville, Maryland. “But it’s not a bacterium. There’s no debate now, absolutely none.”
Besides its colossal size, what is most striking about Mimivirus is its complexity. Among its vast repertoire of genes – the majority of which show all the signs of being fully operational – are many that have functions no one expected to see in a virus. Several are involved in translation, the first step in the process that makes proteins from genes. Mimivirus also has genes that help metabolise large molecules, genes for repairing DNA, and genes to help proteins fold properly. It has genes for building and transporting amino acids, plus three versions of a DNA-untangling enzyme known as topoisomerase. As if all that weren’t enough, it has roughly 450 novel genes whose functions remain unknown. “A parasite like Mimivirus should not have so many genes,” says La Scola. “That’s the paradox.”
Preliminary investigations into the biology of Mimivirus reveal some unusual traits that could explain part of this mystery. Its hardiness at extreme temperatures has prompted speculation that it is adapted to survive long periods outside its host. Its outer coat is also highly complex, comprising two membranes wrapped in a protein shell that is surrounded by a dense thicket of hair-like strands. Some suggest this indicates Mimivirus has evolved to look and taste like a bacterium, a favourite meal of amoebas. If true, it means Mimivirus infects its host by disguising itself as food. Adaptations such as these would probably require the service of at least a few extra genes.
Human connection
Beyond this, however, nothing about Mimivirus appears to require so many genes. Its life cycle isn’t unusual. Nor does it seem equipped for infecting anything but amoebas, as La Scola discovered when he mixed the virus with a wide range of cells, including human ones (though that’s not to say we’re in the clear – see “Attack of the giant virus?”). To get a better understanding of what Mimivirus does with all its genes, La Scola plans to use “lab on a chip” DNA microarrays to identify when different genes are activated.
What Mimivirus does with all its genes, however, is just one puzzle in a much bigger battle over its evolutionary significance.
In evolutionary biology, viruses have traditionally been placed outside the mainstream “tree of life”, the unifying pedigree that links all living organisms (èƵ, 3 September 2005, p 26). One reason is that, being unable to reproduce without a host, they must have appeared after the first cells. Another is that viral evolution is less about standard “descent with modification” than about stealing genes from the viral hosts, a process known as horizontal gene transfer.
However, scientists led by Jean-Michel Claverie, a molecular evolutionist at the Mediterranean University who teamed up with La Scola in 2001, favour a more controversial version of events. According to Claverie, Mimivirus isn’t a ragbag collection of stolen genes jerry-built from the bottom up, but the descendant of a fully-fledged life form that was once far more complex – and possibly even free-living. Such an ancestor may have existed before cells appeared, while nature was still experimenting with simpler designs. Only later would it become a parasite.
Claverie says he reached this conclusion while picking through Mimivirus’s genes one at a time. For one thing, he says, how do you explain those 450 or so unknown genes? If the virus is a collection of stolen genes, why do so many of them bear no resemblance to anything in the vast catalogue of known genes? Sure, it is possible that accumulated mutations could have masked their origins over the course of evolution, and that the Mimivirus genes that are clearly recognisable were simply acquired more recently. Yet if the genes were picked up randomly over time, one would expect to see a range of variation, from unrecognisable to obviously similar. Claverie saw no such pattern.
At the same time, the deeper into the genome he went, the more he began to feel like an archaeologist unearthing an ancient ruin. This was particularly so when he came to Mimivirus’s translation genes, which bore a surprising likeness to the versions found in all cellular life. Here was something that looked more like the crumbling remains of a formerly intact piece of architecture, not something assembled by the fickle winds of chance.
Of course, if Mimivirus is the descendent of a free-living life form, traces of this ancestry might be lingering in its genes. To find out, Claverie turned to the seven genes that Mimivirus shares with the universal core genome – those common to all life sequenced so far. Using models designed to determine relatedness between organisms by comparing the accumulation of mutations in shared genes, he compared Mimivirus with representatives from all three established domains of life – bacteria, archaea and eukaryotes. As he suspected, Mimivirus popped out on a branch near the base of the tree of life, before the evolution of the eukaryotic cells it now infects.
The analysis has raised eyebrows among scientists trying to understand the evolution of cells. For one thing, viruses are not supposed to belong on the tree of life. It also boosts a controversial idea about one of the biggest mysteries in evolution – the origin of cell nuclei, including those in our own cells.
Some biologists argue that, much as cells’ mitochondria are derived from parasitic bacteria, the nucleus was originally a DNA virus that became permanently incorporated into a bacterium-like cell. The trouble was that nuclei seemed too complex to be derived from viruses – until now. “Mimivirus is very much like what I’d hoped would be discovered,” says Philip Bell, a molecular biologist at Macquarie University in Sydney, Australia, and a leading proponent of the virus-to-nucleus idea. “It’s almost a missing link, going from a relatively simple virus to something you could imagine as being complex enough to be a nucleus.”
Evolutionary puzzle
Meanwhile, Claverie has uncovered evidence that horizontal gene transfer has played only a limited role in Mimivirus evolution. In a gene-by-gene comparison with Entamoeba histolytica, the only amoeba that has been sequenced to date, he found only five Mimivirus genes that were more closely related to the amoeba than to anything else. More recently a comparison between Mimivirus and its host amoeba, for which roughly 10,000 genes are known, also failed to reveal any unusual similarity.
Finally Claverie discovered that roughly half of Mimivirus’s genes are controlled by the same molecular switch, a degree of uniformity unheard of in nature. “If you believe Mimivirus is getting its genes from everywhere, it seems difficult to understand how you could put the same signal in front of each of these new captured genes,” he says. “This seems to indicate that the genome has been together for a very long time.”
For some researchers, Claverie’s ideas are right on the mark. In his own analyses of DNA viruses, Van Etten has found no evidence of large-scale horizontal gene transfer. Instead he has discovered that many viral proteins are simpler versions of equivalents found in cells, suggesting that the virus’s are prototypes.
Others remain doubtful. David Moreira, an evolutionary microbiologist at Paris-Sud University in Orsay, France, believes viruses accumulate mutations much faster than other organisms. This means that when they are compared with other organisms, viruses appear more ancient than they actually are, a trap called long-branch attraction.
In his own analysis Moreira looked at Mimivirus’s seven universal core genes one at a time, in each case comparing it with 46 other species. None of the results confirmed Claverie’s data. Two genes even turned out to be closely related to amoebas – for Moreira a clear indication of horizontal gene transfer.
Koonin, meanwhile, says the scarcity of amoeba-like genes isn’t enough to rule out gene transfer. For one thing, Mimivirus has most likely shared its host with many other parasitic bacteria and viruses. It has probably also had many different hosts in the past. This, he says, explains why some Mimivirus genes closely resemble those found in eukaryotes, while others are more bacteria-like.
As for the single switch controlling so many genes, Koonin points out that many bacteria insert their own activation system in front of newly transferred genes, and it is possible that Mimivirus does something similar. “The Mimivirus,” he says, “is not a primitive or degraded form of cellular life.”
Claverie isn’t backing down, however. He argues that there is limited evidence for long-branch attraction. “It’s just an easy way out for people who don’t want to recognise that viruses might be very ancient organisms.”
Riding on the outcome of this debate is whether Mimivirus deserves recognition as a “proper” life form – and possibly even special status as a new domain of life, on a par with bacteria, archaea and eukaryotes.
Koonin thinks it doesn’t. He points out that Mimivirus is still completely dependent on its host for basic metabolic processes such as protein synthesis and energy production. And it lacks protein-building ribosomes, one of the fundamental features of life. “So it’s still a virus,” he says. “And a virus is a virus is a virus. There’s no objective reason to set Mimivirus aside from the rest of the viral world.”
Claverie agrees that by this definition the Mimivirus is still a virus, but argues that a term broad enough to include everything from the minuscule hepatitis D to the giant Mimivirus is useless. He has found evidence that Mimivirus and a few other large DNA viruses are under fundamentally different evolutionary pressures from other viruses. He has demonstrated that there is a big jump in genome size between the largest viruses and the rest of the viral pack, suggesting the former are being selected for greater size and complexity, while the latter are under pressure to remain lean. Claverie has also found evidence that Mimivirus has undergone at least two episodes of genome duplication in the past. “Not only is this virus big,” he says, “but we’ve found evidence that it is becoming bigger.” He is now proposing that large DNA viruses be placed in a separate category called “giruses”.
“My guess is they’re everywhere, and who knows, maybe we’ll find a much larger virus still”
Even more controversially, Claverie thinks giruses should be declared the fourth domain of life. To build his case he is focusing on those Mimivirus genes that are unlike any others in nature. If the proteins made by these genes are as unusual as their gene sequences suggest, then it would be strong evidence that Mimivirus is the relic of a life form from the deep past. “They may be genes that are more than just undiscovered,” says Claverie. “They may be from a new type of organism, a new domain of life.”
As for the largely philosophical question of whether Mimivirus is alive, Claverie says there should no longer be any doubt. “Everybody doing virology thinks these things are alive,” he says. “How much DNA do you need to be considered a living being?”
Regardless of how all these debates unfold, one thing is clear: we will never view viruses the same way. The discovery of Mimivirus has sparked a microbiological gold rush to see what other surprises lie hidden beyond this new frontier. Late in 2005, microbiologist Curtis Suttle of the University of British Columbia in Vancouver, Canada, began sequencing a giant virus collected more than a decade ago from the Gulf of Mexico. It was recently found to contain Mimivirus-like genes, and Suttle estimates its genome at 730,000 letters long. “It’s not as big as Mimivirus,” he says. “But it would be an easy silver medallist.”
Claverie, meanwhile, has been comparing Mimivirus genes against a large database of microbial genes extracted only recently from the Sargasso Sea by a team of explorers led by genome entrepreneur Craig Venter. The prevalence of more than 100 Mimivirus-like gene sequences in the new database suggests Mimivirus is no one-off.
“There are probably more members of this family out there that are not exactly like Mimivirus but closely related,” says Ghedin, who collaborated with Claverie. “My guess is they’re everywhere, and who knows, maybe we’ll find a much larger virus still.”
Even if Mimivirus remains one of a kind, chances are it will go down as the discovery that thrust viruses into biology’s mainstream. It may even be proof that, deep down in our cell nuclei, we’re all descended from viruses. Not bad for an obscure bug from Bradford.
Attack of the giant virus?
It resists high temperatures, strong alkali and bombardment by sound waves. It is stable for long periods of time. Its tough protein coat withstands treatments that would rip other viruses to shreds. Surely the last thing anyone wants to hear is that the Mimivirus could threaten humans.
Unfortunately there is evidence this may be the case. In a study published a year ago in the journal Emerging Infectious Diseases, researchers at the University of Alberta in Edmonton, Canada, examined 376 pneumonia patients and found that roughly 10 per cent of them had antibodies to Mimivirus. A previous study in France had uncovered similar results, and other tests showed that mice inoculated with Mimivirus developed a pneumonia-like disease.
Although there is still no direct evidence that Mimivirus can cause pneumonia in humans, researchers are taking the possibility seriously, not least because a large number of pneumonia cases are caused by unidentified agents. What’s more, many of the known causes of the disease are bacteria that, like Mimivirus, normally live inside amoebas.
The only puzzle is that lab strains of Mimivirus have not shown an ability to infect human cells. Whether this is merely a quirk of being reared in the lab is one of the questions researchers are now trying to answer.
