Sunday, 16 February 2014

Viral polymerases and pathogenesis
Read an introduction to viral evolution and you'll pretty quickly come across a sentence equating to 'viruses mutate and evolve fast and this is because their polymerases lack proofreading ability'. Sure, but that's a big generalisation. Large DNA viruses, for example, tend to have polymerases that are much less error-prone (again a generalisation). Nevertheless, in the world of RNA viruses it is a good rule of thumb that the polymerases tend to be error-prone when copying their genetic code. Superficially, such apparent laxity would appear to be detrimental to virus survival. However, if faithful replication of a nucleic acid was important, then the replication would indeed be faithful, but virus evolution occurs at a population level as opposed to the individual.

The viral sequences in GenBank are consensus sequences, in essence a read-out which represents the most commonly occurring base at each particular position in the genome. In a sense the concept of the consensus can be somewhat misleading: if RNA viruses have a mutation rate that is sufficiently great that it cannot copy a genome without making an error, then it's conceivable that the consensus per se does not, in reality, actually exist. Instead, a virus is a population of sequences evolving together, exploring sequence space with a great level of diversity.
Virologists tend to roll out the term quasispecies to describe this, but there remains some debate as to the validity of this theory, certainly in relation to Eigen's initial proposal.

Survival of the flattest: when mutation rates are low, all viruses accumulate together; if the peak is steep and narrow, then a low mutation error results in all of the viruses being exceptionally fit, (A outcompetes B). If mutation rates are high, then fitness is spread, in which case a spread of highly fit viruses is preferable to an extremely fit virus surrounded by viruses of low fitness (B outcompetes A, from Wilke 2005). 
Having such diversity means that the virus can tackle things that get in the way of progress, most obviously an immune response; if the response impacts upon the fitness of one sequence, then there's another that is capable of preserving the virus' existence. In order to generate such diversity, a virus must necessarily make mistakes and thus viruses tend to live quite close to the maximum permitted error rate for a particular lifestyle. Too much error, and the virus suffers error catastrophe and becomes extinct, as there are insufficient fit sequences. Too little error though, and sufficient diversity becomes hard to generate.

A paper came out recently describing a variant of Chikungunya virus (CHIKV) with altered levels of fidelity. A CHIKV mutant had previously been isolated by growing the virus in the presence of the antiviral ribavirin, which resulted in a virus with increased fidelity. This virus had a single mutation in the viral polymerase. The authors systematically changed this amino acid and looked at the effect it had. Of the 19 options attempted, 12 viruses were viable.

Figure 1 Mutagenizing position 483 variants allows isolation of mutator variants.
Mutator variants: A. the impact of different amino acids on the susceptibility to ribavirin treatment. B. mutation frequencies of selected variants compared to wild-type virus; G and W result in increased rates of mutation. C. average diversity of confirmed fidelity variants at each point across the genome; anti-mutator variant with a Y results in lower diversity across the genome. D. due to the greater diversity, mutator strains are better able to escape virus neutralisation by antibody (CHK-102).  
If these viruses have altered fidelity, then they should have different sensitivities to ribavirin, and indeed some viruses were less sensitive to ribavirin (antimutator strains) whereas some were more sensitive to ribavirin (putative mutator strains). When they checked for sequence diversity, they found a matching trend that sensitivity to ribavirin correlated with diversity, i.e. mutator viruses that make mistakes are susceptible to the treatment. A more highly mutated genome would in theory result in more non-viable/unfit genomes, and indeed they found that the progeny virus population of mutator strains was less infectious, even though their replication in mammalian cells was largely unaffected. When they injected some of the viruses into mice, there was a correlation between the frequency of mutation and viral loads. In line with the in vitro data, the mouse model revealed a decrease in pathogenicity associated with the increased error rates of mutator strains.

Whilst mutator strains replicated fine in mammalian cells, they replicated poorly in mosquito cells. As might be expected under such pressure, in vivo infections of mosquitoes, as well as passage in insect cells, resulted in a reversion of the mutator strains to a more wild-type level of fidelity. It would be interesting to see whether these viruses could be transmitted to subsequent hosts via mosquito bites.

Mosquito infections: Aedes  albopictus (A-C) or Aedes aegypti (D-F) mosquitoes were fed blood containing wild-type (WT) or mutator versions of CHIKV. Virus levels of all viruses was equivalent in the 

Another way in which they confirmed the impact of the changes associated with the polymerase fidelity and its impact upon virus phenotype was to specifically alter the equivalent position in another alphavirus: Sindbis virus (SINV). When they tested the mutated version of SINV, they found the same outcome, notably relatively little impact upon replication levels in mammalian cells, attenuation of pathogenicity in mouse models, lowered titres in insect cell cultures, and reversion to 'wild-type' in mosquito infections, thus confirming the importance of the mutator phenotype.

Mutator (and antimutator) viruses such as these may offer ways in which we can study further how viruses live close to the error threshold - perhaps with the prospect of adding evidence for or against the 'quasispecies' concept.
But replication competent virus but with attenuated phenotype? This rings the vaccine bell. At least potentially. There's more to do, and the authors highlight this, in particular with regard to how the viruses under selective pressure in vivo will behave and evolve in different hosts. But they're certainly an intriguing prospect.

Rozen-Gagnon, K., Stapleford, K.A., Mongelli, V., Blanc, H., Failloux, A-B., Saleh, M-C., Vignuzzi, M. (2014). Alphavirus Mutator Variants Present Host-Specific Defects and Attenuation in Mammalian and Insect Models PLOS Pathogens DOI: 10.1371/journal.ppat.1003877

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