Good viruses visiting bad neighborhoods

Marco VignuzziWhat would happen to an RNA virus if its genome were placed in a bad neighborhood? The answer is that fitness plummets.

RNA virus populations are not composed of a single defined nucleic acid sequence, but are dynamic distributions of many nonidentical but related members. In the past I have referred to these populations as quasispecies but that is no longer the preferred term: mutant swarms or heterogeneous virus populations should be used instead.

The term for all possible combinations of a viral genome sequence is sequence space; for a 10,000 nucleotide genome this would be theoretically 410,000 different genomes – a huge number, more than the atoms in the universe. Any RNA virus population occupies only a fraction of this sequence space, in part because many mutations are deleterious. Studies have shown that viral genomes occupy specific parts of sequence space, called neighborhoods, and movement to different neighborhoods is important for viability. If the viral genome is placed in a bad neighborhood – one that is detrimental for virus fitness – the ability to explore sequence space is restricted.

An example of the effect of changing viral sequence space is shown by a study in which hundreds of synonymous mutations (they did not change the amino acid sequence) were introduced in the capsid region of poliovirus (link to paper). Such rewiring, which placed the virus in a different sequence space, reduced viral fitness and attenuated pathogenicity in a mouse model. In other words, the viral genome was placed in a bad neighborhood, from where it could not move to other neighborhoods needed for optimal replication and pathogenesis. While the genome rewiring did not affect the protein sequence, it might have had deleterious effects on RNA structures or codon or dinucleotide frequency. For example, introduction of codon pairs that are under-represented in the human genome can produce less fit viruses.

A recent study avoids these potential issues by introducing changes in the viral genome that do not affect protein coding, RNA structures or codon or dinucleotide frequency, yet place the viral genome in a different sequence space (link to paper). All 117 serine/leucine codons in the capsid region of Coxsackievirus B3 were changed so that a single nucleotide mutation would lead to a stop codon, terminating protein synthesis and virus replication (this virus is called 1-to-Stop). The serine codons were changed to UUA or UUG; one mutation changes these to the terminators UAA, UGA, or UAG. Another virus was made in which two mutations were needed to produce a stop codon (NoStop virus).

1-to-Stop viruses replicated normally, but when mutagenized, they had significantly lower fitness than wild type or NoStop viruses. Extensive passage of the virus in cells, which would be expected to cause accumulation of mutations, had the same effect on fitness. When a high fidelity RNA polymerase was introduced into 1-to-Stop virus, it replicated like wild type virus. Similar results were obtained with an influenza virus when one of its 8 genome segments was rewired to produce 1-to-Stop and NoStop counterparts.

The 1-to-Stop Coxsackieviruses were attenuated in a mouse model of infection. Furthermore, mice infected with 1-to-Stop virus were protected against replication and disease after challenge with wild type virus. These observations suggest that rewiring viral RNA genomes could be used to design vaccines.

These findings show that recoding a viral genome places it an different sequence space than wild type virus, in which single mutations can lead to inactivation of viral replication. This new neighborhood is unfavorable (‘bad’) because the virus cannot readily move to other neighborhoods to accommodate the effects of mutation.

For more discussion of viral sequence space and rewiring viral genomes, listen to the podcast This Week in Evolution #24: our guest is Marco Vignuzzi (pictured), senior author on the second paper discussed here.

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