Pushing viruses over the error threshold

14 May 2009

The capacity of RNA viruses to produce prodigious numbers of mutations is a powerful advantage. But remember that selection and survival must balance genetic fidelity and mutation rate. Many mutations are not compatible with viral replication. Consequently, if mutation rates are high, at some point accumulating base changes lead to lethal mutagenesis – the population is driven to extinction. The error threshold is a mathematical measurement of the genetic information that must be maintained to ensure survival of the population. The results of experiments clearly show that RNA viruses evolve close to their error threshold.

The first clues that viruses exist on the edge came from studies in which cells infected with the RNA viruses poliovirus and vesicular stomatitis virus were treated with base analogs that act as mutagens. These compounds, such as 5-azacytidine, are incorporated into the growing RNA chain during replication, leading to incorporation of incorrect bases. Treatment of virus infected cells with such drugs dramatically inhibits the production of new infectious virus particles, but stimulates mutagenesis of the viral RNA by only 2- or 3-fold. When the same experiment is done with cells infected with a DNA virus, the mutation frequency increases by several orders of magnitude.

A direct demonstration of error catastrophe was achieved by treating poliovirus-infected cells with the mutagen ribavirin. Treatment with a concentration of ribavirin that causes a 9.7-fold increase in mutagenesis lead to a 99.3% loss in poliovirus infectivity. A graph of the results shows that poliovirus exists at the edge of viability.

poliovirus-error-catastrophe

The line on the graph shows the infectivity of poliovirus RNA as a percentage of untreated viral RNA. The introduction of 2 mutations per genome reduces infectivity to about 30% of wild type RNA, while infectivity is nearly eliminated with 7 mutations per genome.

Ribavirin pushes poliovirus beyond the error threshold because it causes the RNA polymerase to make more errors than it already does. What would be the property of a poliovirus mutant resistant to this drug? A poliovirus mutant resistant to ribavirin was selected by passing the virus in the presence of increasing concentrations of the drug. Resistance to ribavirin is caused by a single amino acid change, G64S, in the viral RNA-dependent RNA polymerase. RNA synthesis of ribavirin-resistant poliovirus is characterized by fewer errors, in the absence of drug, compared with the parental virus. Therefore the mechanism of resistance to the mutagen ribavirin is achieved through an amino acid changes that makes a viral RNA polymerase with greater fidelity.

Now that we have an RNA polymerase that makes fewer errors, we can use it to limit viral diversity and test the theory that viral populations, not individual mutants, are the target of selection.

Crotty, S. (2001). RNA virus error catastrophe: Direct molecular test by using ribavirin Proceedings of the National Academy of Sciences, 98 (12), 6895-6900 DOI: 10.1073/pnas.111085598

Pfeiffer, J. (2003). A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity Proceedings of the National Academy of Sciences, 100 (12), 7289-7294 DOI: 10.1073/pnas.1232294100

  • Mauricio Carrillo-Tripp

    Where did this G64S mutant come from? was it just a product of the natural process you've been talking about?

    “Now that we have an RNA polymerase that makes fewer errors, we can use it to limit viral diversity and test the theory that viral populations, not individual mutants, are the target of selection.” Wow, has this been done?

  • http://www.virology.ws profvrr

    The G64S mutant was selected by growing virus in the presence of ribavirin. The drug selects for pre-existing mutants in the quasispecies. So yes, it's a product of the natural process I've been writing about. Tomorrow I'll write about how this mutant was used to test the theory. Yes, it's been done.

  • http://www.facebook.com/people/Corey-Philipp/799897312 Corey Philipp

    Does the history of viral mutagens mirror those of the animal and bacterial worlds. At first we didn't have the wonderful compounds to speed up the mutation rate and we had to have poor graduate students look for mutation at the normal rate? I know that in Drosophila there was a time when you would do an F1, F2, etc crosses and continue to look for the deviation from the wild type until you found your mutant. Then mutagens like UV light, x-rays, drugs, etc were used to greatly speed these processes up. Likewise, do virologists use high energy photons to mutate viruses or does that not work so well? It seems like you could blast a plaque, once you have one, with x-rays and get viral mutants. Of course if you blast a viral infected cell culture with x-rays you might get some other results because of the damage to the host cell genome.

    Also, can ribavirin be used as an antiviral and are there other drugs that could be used as an antiviral that would modify rna dependent polymerase activity? It seems like there could be nonspecific interference with the host cell rna polymerase if you were to administer ribavirin to humans. I guess the trick would be to find drugs that would cause rna modifications to only those rna’s made by rna depdent polymerase. That may not be possible since one rna looks like the other.

  • http://www.zonegrippeaviaire.com/showthread.php?t=2560 gsgs

    is there also a threshold for synonymous mutations ?

  • Dunbar

    Your last comment is puzzling to me. The unit of selection is individual mutants, at the very least.
    To Corey, ribavirin is used clinically (http://en.wikipedia.org/wiki/Ribavirin). To gsgs, I don't see why there wouldn't be a synonymous mutation threshold given that the hosts have codon biases.

  • http://www.virology.ws profvrr

    Great question. Yes, there is indeed a threshold for synonymous
    mutations. Two recent papers have shown that for poliovirus. Check out
    “Modulation of poliovirus replicative fitness in HeLa cells by
    deoptimization of synonymous codon usage in the capsid region” at
    http://bit.ly/15K00K.

  • http://www.virology.ws profvrr

    Great question. Yes, there is indeed a threshold for synonymous
    mutations. Two recent papers have shown that for poliovirus. Check out
    “Modulation of poliovirus replicative fitness in HeLa cells by
    deoptimization of synonymous codon usage in the capsid region” at
    http://bit.ly/15K00K.

  • http://www.virology.ws profvrr

    In some systems it is necessary to accelerate the mutation rate,
    particularly for DNA based organisms with error correction. But for
    RNA viruses, all the mutants you ever need is in a tube of virus. All
    you need to do is devise a selection scheme.

    Ribavirin is used clinically to treat many viral infections, but there
    are side effects – because the drug is not specific for viral
    polymerases, as you point out. Many other base analogs are available,
    particularly for treatment of AIDS. Acyclovir, for treatment of herpes
    simplex infections, is very specific because the drug is activated
    only in cells infected with the virus.

  • http://www.virology.ws profvrr

    Individual viruses emerge after selection, but they are not the target
    of selection. The target for selection is the entire population.

  • trine

    Hi Vincent,
    Are there substances found naturally that act like ribavirin, pushing up the number of mutations per genome?
    Thanks! T

  • Pingback: Increased fidelity reduces viral fitness()

  • trine

    Vincent,
    So today in the NYTimes, the story about the assistant principle who died of swine flu said he had been given “the experimental drug” ribavirin. This must be the same stuff you are talking about here, right? So the thinking about how it would work is that it pushes the virus over the error threshold once an infection has started?

    From the NYTimes: “Mr. Wiener, 55, had been “overwhelmed” by the illness, despite beginning a course of treatment with an experimental drug, Ribavirin, after he failed to respond to other antiviral drugs, according to Ole Pedersen, a spokesman for Flushing Hospital Medical Center, where Mr. Wiener had been a patient since Wednesday.”

  • http://www.virology.ws profvrr

    Yes, ribavirin is the same drug that we have been talking about. It is
    not licensed for use in treating influenza, hence it's called
    'experimental'. It is licensed for use in treating RS and HCV in the
    US. Note that it is 'over the counter' in Mexico where it is used for
    treating flu. But it has at least five different mechanisms of action,
    not just pushing viruses over the error threshold. It could work that
    way with influenza, but it hasn't been looked at.

  • http://www.virology.ws profvrr

    Yes, ribavirin is the same drug that we have been talking about. It is
    not licensed for use in treating influenza, hence it's called
    'experimental'. It is licensed for use in treating RS and HCV in the
    US. Note that it is 'over the counter' in Mexico where it is used for
    treating flu. But it has at least five different mechanisms of action,
    not just pushing viruses over the error threshold. It could work that
    way with influenza, but it hasn't been looked at.