Why do rna viruses evolve so quickly

Double-strand RNA-dependent adenosine deaminases ADARs are another type of host enzymes that edit viral genomes by deaminating adenosines in long double-stranded RNA and converting them to inosines. The latter base-pair with guanosines, resulting in A-to-G base substitutions [ 67 ]. ADAR-driven hyper-mutation was first demonstrated in measles virus [ 69 ] and has since been suggested for a variety of RNA viruses including human parainfluenza virus [ 70 ], respiratory syncytial virus [ 71 ], lymphocytic choriomeningitis virus [ 72 ], Rift Valley fever virus [ 73 ], and noroviruses [ 74 ].

Uracil can be found in DNA abnormally due to spontaneous or enzymatically induced cytidine deamination, leading to G-to-A mutations. Failure to incorporate UNG produces a fourfold increase of the HIV-1 mutation rate in actively dividing cells, and of fold in macrophages [ 75 , 76 ].

Variations in the concentration and balance of dNTPs among cell types may also influence viral mutation rates [ 77 ]. Although analysis of HIV-1 mutations in various cell lines revealed no obvious mutation rate differences, it nevertheless showed differences in the type of mutations produced [ 78 ].

In contrast to cells, viruses can adopt a variety of replication modes. Under this theoretical model, there is only one round of copying per cell. In practice, this means that each infecting genome is used to synthesize a single reverse-complementary intermediate which in turn is used as template for synthesizing all progeny genomes.

This contrasts with semi-conservative replication, in which each strand is copied once to produce progeny molecules that are, in turn, used as templates in the next round of copying.

Since under semi-conservative replication the number of strands doubles in each cycle, the virus necessarily has to undergo multiple replication cycles within each cell to produce enough progeny. Under stamping machine replication the mutation frequency observed after one cell infection equals the mutation rate, but under semi-conservative replication this frequency is also determined by the number of replication cycles, as mutants become amplified.

This means that a given viral polymerase will produce more mutations per cell if replication is semi-conservative than if replication is stamping machine-like. These two models are indeed two extremes of a continuum of possible replication modes. For instance, a virus can produce multiple progeny molecules per round of copying which then undergo a second replication cycle in the same cell to end up producing hundreds or thousands of progeny molecules. Viral replication modes and mutation accumulation.

As opposed to cells, which use only semi-conservative replication, viruses can adopt a variety of replication modes. In the stamping machine model, a single template strand is used to synthesize all progeny genomes within a given cell. Under this model, the mutation frequency after one cell infection cycle will equal the mutation rate except if mutations occur during the first round of copying from genome to anti-genome , in which case they will be present in all of the viral progeny.

Under semi-conservative replication, multiple rounds of copying are required to produce enough progeny, thus allowing for the intra-cellular accumulation of mutations.

Longer cell infection cycles late burst can allow for the production of more progeny viruses. Under semi-conservative replication, this will require more rounds of copying but, if replication follows the stamping machine model, the number of rounds of copying will not change more progeny genomes will be produced from the same template.

Hence under this model, a late-burst virus variant will undergo fewer total rounds of copying at the population scale than early-burst variants and will tend to accumulate fewer mutations. It has been suggested that the stamping machine model has been selectively favored in RNA viruses because it compensates for the extremely high error rate of their polymerases [ 79 — 81 ].

However, empirically-informed modeling of the poliovirus replication cycle indicated multiple rounds of copying per cell [ 85 ]. Similarly, single-cell analysis of the genetic diversity produced by vesicular stomatitis virus revealed that some mutations are amplified within cells, implying that multiple rounds of copying take place per cell [ 86 ]. However, it remains unknown whether a given virus can modify its replication mode in response to specific selective pressures in order to promote or down-regulate mutational output.

To a large extent, the replication mode of most viruses should be dictated by the molecular mechanisms of replication and, hence, should be subjected to strong functional constraints. In contrast, semi-conservative replication is probably the only mechanistically feasible replication model for viruses with large DNA genomes.

Changes in lysis time can be thought of as another mechanism for regulating the production of mutations in viral populations. Lysis is a tightly regulated process and, in theory, viral fitness is maximized for some intermediate lysis time [ 89 — 91 ].

If lysis occurs before this optimum, the infected cell will release a small amount viral progeny and hence few cells will be infected in the next infection cycle, retarding population growth. Yet if lysis occurs after the optimum, a large amount of progeny will be produced per cell but cell-to-cell transmission will be delayed. However, the optimum can also vary according to mutation rate.

Interestingly, 5-fluorouracil selected for an amino acid replacement in the N-terminal region of the phage lysis protein V2A. This change conferred partial resistance to the drug, but also delayed lysis [ 93 ]. In turn, delayed lysis was concomitant with an increase in the viral yield per cell, since progeny virions had more time to accumulate intracellularly.

Therefore, at the population level, growth of the V2A variant occurred through longer infection cycles with increased per-cell productivity. However, because the virus replicates following a stamping machine model, each infection cycle should involve only one round of copying regardless of lysis time. As a result, population growth required fewer total rounds of copying in the delayed lysis variants than in the wild-type, meaning that mutations had fewer opportunities to accumulate Fig.

Therefore, delayed lysis increased the ability of the phage to tolerate mutagenesis. The fidelity of a given polymerase varies according to certain template properties. It is well known that misalignments at homopolymeric runs can cause frameshift mutations and base substitutions [ 94 ]. Sequence context may influence the fidelity of HIV-1 RT by modulating enzyme binding and dissociation [ 95 ].

Also, RNA secondary structures have been shown to promote template switching, a process that does not lead to new mutations but produces recombinant viruses [ 96 — 98 ]. Shuttle vectors are systems in which most or all sequences except essential cis-acting elements such as the Rev-responsive element or long terminal repeats have been removed from the viral genome. Shuttle vectors allow propagating HIV-1 in the absence of selection because all required functions are provided in trans by helper plasmids that are freshly provided in each infection cycle [ ] Fig.

The shuttle vector simply carries forward sequences of interest, which can be reporter genes for selecting and visualizing transduced cells, or transgenes for engineering purposes. However, the vector also accepts HIV-1 sequences. These will have no role in the infection cycle, as they are not expressed. Because selection is absent, such HIV-1 sequences cloned in a shuttle vector can be used for interrogating the viral mutation rate in cognate templates, which is helpful for testing the effects of sequence context or RNA structure on mutation rate.

Cell culture systems for the accumulation of mutations in the absence of selection. A resistance gene RES, red is inserted to allow for the selection of cells containing the vector. Any short sequence of interest SEQ, blue , including HIV sequences, can be cloned in the shuttle vector and propagated in the absence of selection. The shuttle vector DNA is co-transfected with helper plasmids encoding the Gag capsid and Pol RT, integrase proteins as well as a viral glycoprotein suited for transducing a given cell line here vesicular stomatitis G protein, VSV-G, which has a broad tropism.

Pseudotyped viruses are produced, used for transduction, and cells carrying the retroviral shuttle vector are selected with the appropriate antibiotic. The infection cycle can be restarted at any time by transfecting the two helper plasmids. Two cistrons are separated by an internal ribosome entry site IRES.

The right cistron encodes HCV non-structural NS proteins required for replication, but lacks the envelope proteins and hence does not support viral budding. The left replicon carries a resistance gene to select cells carrying the replicon.

Reporters such as luciferase can be also cloned in this cistron. Replicon RNA is obtained by in vitro transcription and transfected into Huh7 hepatoma cells. Cells are selected using the appropriate antibiotic and passaged before confluence to allow vigorous replication of the viral RNA. Using this system, we recently characterized the distribution of mutations along the HIV-1 envelope, integrase, vif , and vpr genes [ 99 ].

We found that a 1 kb region encompassing the V1—V5 loops of the gp envelope protein accumulated approximately three times fewer mutations than other regions of the HIV-1 genome.

This coldspot mapped to the outermost domains of gp, which are preferred targets of circulating antibodies and show extensive glycosylation. Examination of this region revealed two differential properties. To more directly test the effect of RNA structure on HIV-1 RT fidelity, we used in vitro polymerization assays with two different templates: a random sequence and RNA from potato spindle tuber viroid, which shows a marked, stem-like secondary structure [ ].

Using a conceptually similar approach, we recently characterized the accumulation of mutations along the HCV genome under weak or no selection using a bicistronic replicon by cloning HCV sequences at a site commonly used for inserting reporter genes Fig.

This revealed extreme mutation rate variations across individual nucleotide sites of the viral genome, with differences of orders of magnitude even between adjacent sites [ ]. In that system, we found little or no effect of RNA structure on mutation rate, but a more significant effect of base identity, such that A and U bases were more prone to mutation than G and C.

The finding that HIV-1 has a reduced mutation rate in the genome region encoding the outermost domains of the gp envelope protein reveals an uncoupling between mutation rate and genetic diversity, as these domains are the most variable regions of the HIV-1 genome, mainly as a consequence of immune pressure [ ].

This indicates that HIV-1 has not evolved the ability to target mutation to regions wherein they are more likely to be needed for adaptation. Similarly, strong selection at the protein level may have favored amino acid replacements within this region even at the cost of disrupting pre-existing RNA secondary structures and, as a consequence, these RNA structural changes would have modified replication fidelity [ 99 ]. In HCV, we found no significant differences in mutation rate across genes [ ], as opposed to genetic variation, which concentrates in specific genomes regions including external domains of the E2 envelope protein [ ].

This again supports the view that RNA viruses cannot target mutations to specific genomes regions to improve their adaptability. This contrasts with bacteria and DNA viruses, in which mechanisms of error-prone replication have evolved at specific loci involved in host-pathogen interactions [ — ]. A well-characterized system of mutation targeting, called diversity-generating retro-elements DGRs , is found in large DNA bacteriophages [ ]. DGRs are typically located in genes involved in host attachment, a step of the infection cycle that is subject to rapid changes depending on host species availability.

The cDNA is then transferred to the VR, producing a large number of variants of the mtd gene capable of interacting with new host ligands [ ]. DGRs have also been described in plasmids, bacterial and archaeal chromosomes, and archaeal viruses [ — ].

It therefore appears that at least some prokaryotic DNA viruses have evolved the ability to target mutations to specific regions, as opposed to RNA viruses. Diversity-generating retro-elements have not been described in eukaryotic viruses, but these viruses can use other mechanisms of mutational targeting that involve recombination. The inverted terminal repeats of vaccinia virus contain 10— base repeated sequence motifs known to experience frequent unequal crossover events and rapid changes in copy number [ , ].

Recombination has been shown to promote the rapid production of genetic diversity in other genome regions of the vaccinia virus involved in immune escape and the colonization of novel hosts.

Protein kinase R PKR is a central effector of innate antiviral immunity that induces translational shutoff, modifies protein phosphorylation status, alters mRNA stability, and induces apoptosis [ ]. Poxvirus proteins K3L and E3L block PKR and have evolved as antagonists of innate immune responses in a host-specific manner [ , ].

Experimental deletion of E3L renders vaccinia virus more susceptible to host antiviral responses, imposing a strong selection pressure in the other PKR suppressor K3L to increase its function [ ].

Serial transfers of E3L-deleted vaccinia virus led to an elevated K3L copy number, a recombination-driven process that allowed the virus to overexpress this gene. This gain-of-function mutation had a direct fitness benefit, but also increased the number of available targets for the appearance of subsequent selectively advantageous point mutations in K3L. Remarkably, upon selection of these mutants K3L copy numbers were again reduced.

Hence, recombination led to an evolutionary process characterized by expansion and contraction of a specific genome region. These so-called genomic accordions have been posited to mediate adaptive duplications in other poxviruses such as myxoma virus [ ]. Interesting interplays between recombination and mutation rates have also been recently found in RNA viruses.

These two processes are primarily controlled by the viral polymerase since, in RNA viruses, recombination takes place when the viral polymerase switches between different template genomes present in the same cell [ ]. The estimated recombination rates of different riboviruses and retroviruses correlate positively with estimated mutation rates [ ].

High mutation rates confer viruses the ability to rapidly produce advantageous mutations, but also inflate the genetic load of the population. In turn, frequent recombination allows beneficial mutations to unlink from deleterious genetic backgrounds, as well different beneficial mutations to be combined into the same genome. As such, recombination is expected to enhance adaptation when a large number of alleles coexist in the same population, a scenario that typically takes place at high mutation rates [ ].

Experimental evidence supporting the joint effects of recombination and mutation rates in viral adaptability has been recently obtained using poliovirus polymerase mutants that individually alter replication fidelity or recombination rate [ ]. In another recent work, a low-fidelity variant of Sindbis virus was found to exhibit increased recombination [ ].

This variant showed low fitness and a greater tendency to accumulate defective interfering particles i. Therefore, it appears that high mutation and recombination rates enhance viral adaptability, but only up to a certain point, beyond which both processes contribute to the accumulation of deleterious alleles in the population. Some of these processes underlie large-scale patterns of variation among viruses, such as differences between RNA and DNA viruses, between viruses with small and large genomes, and between single-strand and double-strand viruses, but important mechanistic aspects behind these differences still remain uncharacterized.

Furthermore, mutation rates are not static and can evolve in response to selective pressures, as exemplified by fidelity variants selected under mutagenic conditions in a variety of viruses. In addition to polymerase fidelity, other mutation rate-determinants such as access to DNA repair may have also changed in response to selective pressures during viral evolution. In RNA viruses, both low- and high-fidelity polymerase variants tend to have a negative impact in viral fitness in complex environments, suggesting that RNA virus mutation rates have been evolutionarily optimized.

It appears that large DNA viruses have adopted a different and more elaborate strategy consisting of targeting mutations to specific genome regions subject to rapidly varying selective pressures, such as genes encoding attachment proteins or inhibitors of innate immunity responses. Mutation targeting mechanisms such as DGRs and recombination-driven gene copy amplification are probably not accessible to small DNA viruses with compact genomes. Furthermore, mutation rate evolution in small DNA viruses is further constrained by the fact they do not encode autonomous replication systems.

Therefore, small DNA viruses should rely on repair avoidance and on use of host-encoded error-prone DNA polymerases to elevate their mutation rates and achieve faster adaptation.

Elucidating the mutational mechanisms of small DNA viruses is a current challenge in virus molecular biology and evolution. Other exciting unresolved questions include unveiling the interplays between mutation and recombination, the roles played by viral accessory proteins in determining mutation rates, the effects of host-encoding enzymes on viral diversity and evolution, whether mutation accumulation can be evolutionary adjusted by modifying viral replication modes, and how template sequences regulate viral mutation rates.

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Do vaccines for viruses need to be updated when variants arise? Is that true with all viruses? Some people are trying to develop a universal influenza vaccine, to try to target the antibody generation toward a part of the molecule that cannot change without making the molecule not work anymore. But I think the evidence is that the variants are not escaping the vaccine dramatically.

And of course, that also depends how quickly we can get this vaccination round to complete—how many different variants are going to emerge by the time most people are vaccinated. The coronavirus definitely is not going to be like that. But if it does become endemic and it circulates in the population all the time, then there is a chance that a slightly different virus might emerge and we may need a booster.

Another question is, as people become immune thanks to vaccinations, is that going to be a strong pressure on the virus? Variants may emerge because people are immune to the old virus. Taylor McNeil can be reached at taylor. Skip to main content. Of course, a lower mutation rate comes with the tradeoff that it will also limit the smaller fraction of beneficial mutations—alleles that are beneficial in the current environment and that will help an organism leave more descendants over time.

It would also limit the accumulation of neutral or nearly neutral variation in populations that might be beneficial if circumstances change, alleles that could be beneficial in a new environment or after climactic change [ 5 ]. The mutation rate of all cellular life is under selection, and cells have evolved many ways of tweaking their mutation rates—largely to lower the mutation rate inherent in a fast-moving, processive polymerase replicating their large genomes. These involve proofreading components of the polymerases themselves and a variety of other proteins and systems to check for errors in DNA and to repair common kinds of DNA damage [ 6 ].

Some DNA viruses with larger genomes also have DNA repair proteins, and the very largest RNA viruses have some ability to proofread and correct replication errors [ 7 ]. Mutant viruses and cells with lowered mutation rates can be isolated by exposing cells or viruses to mutagens, but just as there are proteins and alleles that decrease mutation rates, there are mutations to break those proteins and other alleles that increase mutation rates, which are beneficial in some environments [ 8 ].

RNA viruses are perhaps the most intriguing biological entities in which to study mutation rates. They encode their replication machinery, and thus their mutation rates can be optimized for their fitness in comparison to small DNA viruses that use the polymerases of their host cells.

Their ability to rapidly change their genome underlies their ability to emerge in novel hosts, escape vaccine-induced immunity, and evolve to circumvent disease resistance engineered or bred into our crops [ 10 , 11 ]. The exogenous mutagen causes enough additional mutations, which are often deleterious, so that the progeny RNA viruses are of lower fitness, eventually leading to ecological collapse of the population [ 14 ]. Another way in which researchers have seen the constraints imposed by the high mutation rate of RNA viruses is in their limited genome size—the mutation rates per nucleotide are too high to increase their genome size without having a higher per-genome accumulation of mutations [ 9 , 15 ].

Researchers have suggested that RNA virus mutation rates have evolved to be just under the threshold for lethal mutagenesis sometimes referred to as error threshold [ 16 ] but that selection for genetic diversity and other consequences of a high mutation rate push RNA viruses to near their catastrophic limits.

It has been hard to assess this assumption and verify that RNA viruses have their optimal mutation rates due to natural selection on mutation rate. The 3D:G64S strains not only have a lower mutation rate than wild-type polio but also are less fit in several ways: in one-step growth curves, in cell culture passaging, and in mice, in which they have reduced virulence the 3D:G64S strains more slowly invade the central nervous system.

They are more fit than wild-type poliovirus only in the presence of mutagens, in which their lower mutation rate reduces the inherent number of mutations in each progeny genome, so more exogenous mutations can be tolerated. The 3D:G64S strain also has measurably less genetic diversity during infections, which has suggested a link between population diversity and virulence as well as the adaptability that is conferred by having more standing genetic variation and being able to more rapidly create more variation.

However, these conclusions are largely correlational and theoretical, as it has been difficult to conduct experiments to definitively prove that it is indeed the reduced mutation rate of 3D:G64S and not other effects of this mutation causing the reduced virulence and fitness observed in experiments.

In this issue of PLOS Biology , Fitzsimmons and colleagues show that reduced replication speed explains more of the effects of the 3D:G64S than its reduced mutation rate per se [ 19 ]. If a task is critical to do without any errors at all, it will likely need to be done more slowly with more care and attention. Fitzsimmons and colleagues demonstrate that a compensatory mutation in 3D:G64S can restore replication speed but not affect the lower mutation rate of 3D:G64S, and this increases viral fitness 2C:VL.

This key experiment teased apart two highly correlated traits to reveal that replication rate affects fitness more than mutation rate. The process of entering the mouse central nervous system is a severe bottleneck and is dominated by drift compared to selection—both the wild type and 3D:G64S polioviruses have similar diversities in the mouse central nervous system [ 19 ].

Deep sequencing of cell culture-passaged wild type and 3D:G64S populations revealed that both lacked genetic diversity at a meaningful level SNPS at 0. Finally, the wild type and 3D:G64S increased fitness by identical amounts after passaging in cell culture, refuting that the lower mutation rate of the 3D:G64S strain reduces adaptability.

Altogether, this new work suggests that the 3D:G64S strain has a lower fitness because of slower replication, not its reduced mutation rate. RNA viruses like poliovirus likely have higher mutation rates than what would be optimal for the organism because higher mutation rates are, in part, a byproduct of selection for faster genomic replication.