Describing a viral quasispecies

QuasispeciesVirus populations do not consist of a single member with a defined nucleic acid sequence, but are dynamic distributions of nonidentical but related members called a quasispecies (illustrated at left). While next-generation sequencing methods have the capability of describing a quasispecies, the errors associated with this technology have limited progress in our understanding of the genetic structure of virus populations. A new method called CirSeq reduces next-generation sequencing errors to allow an accurate description of viral quasispecies.

The key to eliminating sequencing errors is a clever approach based on the conversion of viral RNAs to circular molecules. When copied with reverse transcriptase, tandemly repeated cDNAs are produced (illustrated below). Mutations in the original viral RNA will be shared by all repeats derived from a circle, but not errors produced during copying or sequencing. The latter can be computationally subtracted, reducing sequencing error to a point that is much lower than the estimated mutation rate of an RNA virus.CirSeq

CirSeq was used to characterize poliovirus populations produced by seven serial passages in HeLa cells. The calculated mutation frequency, 2 X 10-4 mutations per nucleotide, was substantially lower compared with estimates determined by conventional sequence analysis. Over 200,000 sequence reads per nucleotide position were used to detect >16,500 variants per population per passage. This number represents ~74% of all possible alleles. Many mutations were detected at nearly all positions in the viral RNA. Most mutations occur at a frequency between 1 in 1000 to 1 in 100,000. The conclusion is that the virus population produced in HeLa cells consists mainly of genomes with the consensus sequence, and small amounts of many variant genomes. These variants are only those that give rise to viable viruses; lethal mutations are not observed.

CirSeq was also used to calculate the mutation rate of poliovirus. The rates vary according to type: transitions occurred at a rate of 2.5 X 10-5 to 2.6 X 10-4 substitutions per site, while transversions were observed at a rate of 1.2 X 10-6 to 1.5 X 10-5 substitutions per site. Nucleotide-specific differences in mutation rate were also observed: C to U and G to A transitions were 10 times more frequent than U to C and A to G. These rates are consistent with previously determined values using other methods.

This method can also be used to determine the fitness of each base at every position in the genome, according to changes observed during the seven passages in HeLa cells. This analysis allows determination of which bases are neutral, and which are selected, and when combined with analysis of protein structure, can provide new insights into viral functions.

By enabling a sequencing approach that gives an accurate description of virus populations at a single-nucleotide level, CirSeq can be used to provide an unprecedented view of how virus populations change during evolution.

Increased fidelity reduces viral fitness

pvrtgWe have spent over a week discussing the effects of polymerase error rates on viruses. RNA viruses have the highest error rates in nature, a property that is believed to benefit the viral population. For example, selective pressure from the immune system or antiviral drugs may lead to changes that are beneficial for the population. In fact, it has been hypothesized that high error rates are required for survival of RNA viruses in complex environments. The isolation of a poliovirus mutant with an RNA polymerase that makes fewer errors during replication made it possible to test this hypothesis.

Infection of an animal host poses perhaps the greatest challenges to viral propagation. Transmission, entry, host defenses, tissue diversity and anatomical restrictions all are serious obstacles to the ability of a virus to replicate, disseminate, and successfully spread to other hosts. Therefore the effect of viral diversity is most stringently tested in infection of an animal.

In these experiments, mice were inoculated with the poliovirus mutant containing the G64S amino acid change in the viral RNA polymerase that causes enhanced fidelity. Infection of mice with poliovirus typically leads to symptoms of poliomyelitis that are similar to those in humans. Compared with the wild-type parental virus, the G64S mutant was less pathogenic: it caused significantly less paralysis and lethality. This effect could be a consequence of restricting the viral quasispecies, or a replication defect in mice caused by the G64S mutation. To distinguish between these possibilities, the G64S mutant was propagated in cells in the presence of a mutagen, a procedure which expanded the number of viral mutants. This treatment – basically expanding the quasispecies – lead to a significant increase in lethality of the G64S virus, to nearly the same extent as wild type virus.

Why would a less complex quasispecies lead to reduced pathogenicity? Viral growth and spread in an animal likely requires a diverse viral population, comprising many mutants, which can replicate efficiently in the many different cell types in an animal. Support for this idea comes from a competition experiment in which the poliovirus G64S mutant was mixed with wild type virus and inoculated into the leg muscle of a mouse. Several days later the mice were sacrificed and the virus that had reached the brain was characterized.  The results showed that both wild type and the G64S virus could replicate in muscle, but the mutant virus spread to the brain less frequently.

These results show that mutations do benefit viral populations, especially in complex environments such as an animal. The ability to produce a quasispecies may allow virus populations to respond to the different environments encountered during spread between hosts, within organs and tissues, and in response to the pressure of the host immune response.

We’ll shortly return to influenza virus replication, but I hope you have been able to follow what to many must be a somewhat arcane discussion. From the silence I suspect that I might have lost some of you – it might help to go back over some of the posts. I’ll try to put up an index of some sort to make it easier to find articles. The blog format isn’t great when it comes to finding older material – once posts scroll off the bottom of the page, they don’t receive further notice.

Pfeiffer, J., & Kirkegaard, K. (2005). Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice PLoS Pathogens, 1 (2) DOI: 10.1371/journal.ppat.0010011

Vignuzzi, M., Stone, J., Arnold, J., Cameron, C., & Andino, R. (2005). Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population Nature, 439 (7074), 344-348 DOI: 10.1038/nature04388