Mutation in rna viruses

These warnings will probably not halt the question as to whether mutations will arise in SARS-CoV-2, enabling it to spread more efficiently between humans or generate a higher case fatality rate. Large deletions in the open reading frame 8 ORF8 region and mutations in the spike S protein were discovered during the early stages of the outbreak and eventually dominated the epidemic, suggesting that these were adaptations to humans 12 , Based on this observation, some hypothesized that virus genetic changes in part drove the SARS epidemic, but this claim is unsubstantiated Will adaptation precipitate more deaths?

It is time to reshape our conception of mutations. Mutations are not indicative of outlandish and devastating new viral characteristics. Instead, they can inform our understanding of emerging outbreaks. Any claims over the consequences of mutation demand careful experimental and epidemiological evidence.

Mutation is an inevitable consequence of being a virus. The pattern and time course of mutations in a virus genome are key for estimating phylogenetic trees, which, in turn, depict the course epidemics in effectively real time The developing field of genomic epidemiology is currently being employed in the mitigation and control of the SARS-CoV-2 outbreak.

The rapid and open access deposition of virus genomes, most of which differ by mutation, is enabling precise investigations into patterns of spread. To this end, websites like Virological.

Rather than fearing mutation, perhaps it is now time to embrace it. Begley, S. DNA sleuths read the coronavirus genome, tracing its origins and looking for dangerous mutations. Gulland, A. Coronavirus: researchers warned to be on alert over mutations that could speed up disease spread. Yuan, L. Science , — Grubaugh, N. Trends Microbiol. Holmes, E. Bull, J. Evolution 48 , — Wain, L. Tsetsarkin, K. PLoS Pathog. Article Google Scholar.

The observation that most highly variable and rapidly evolving DNA viruses have small genomes including double-strand viruses indirectly supports an effect of genome size [ 3 ]. Candidate mechanisms that might account for mutation rate differences between large and small DNA viruses may involve virus—DDR interactions.

Whereas many viruses appear to evade DDR, others seem to use it for their own benefit [ 25 , 26 ]. Polyomaviruses, papillomaviruses and parvoviruses induce and depend on DDR signaling pathways for efficient replication [ 30 — 32 ]. These viruses share the property of having small, circular DNA genomes which do not encode a polymerase.

As such, they depend directly on the cellular replication machinery, as opposed to larger DNA viruses. It is possible that some small viruses promote the DDR to prolong the S cell-cycle phase, which offers a more favorable environment for replication. By adopting circular genomes, these viruses would also avoid the formation of genome concatemers, a typical effect of DDR in linear viral genomes such as, for instance, adenoviruses [ 33 ].

The DDR comprises error-prone DNA polymerases for re-synthesis of excised strands [ 34 ], and involvement of these polymerases in viral replication may lead to higher mutation rates.

Intrinsic polymerase fidelity i. Polymerase variants with altered fidelity have been artificially selected in a number of RNA viruses by subjecting laboratory populations to mutagenic treatments [ 35 ].

For instance, serial passaging of poliovirus in the presence of the base analog ribavirin led to the selection of a polymerase variant G64S with threefold increased fidelity [ 36 ]. This same mutation also confers increased fidelity in the related human enterovirus 71 [ 37 ], and other amino acid replacements such as LF have also been shown to modify the replication fidelity of this virus [ 38 ].

Passaging of coxsackievirus B3 also a member of the enterovirus genus in the picornavirus family in the presence of ribavirin or 5-azacytidine selected for another fidelity variant in the viral polymerase AV [ 39 ]. Outside picornaviruses, fidelity variants have been more recently obtained by serial mutagen treatment in chikungunya virus [ 40 ], influenza A virus [ 41 ], and West Nile virus [ 42 ].

Several antivirals and notably many antiretroviral drugs are base analogs. Resistance to these treatments is well documented in the HIV-1 RT and some of these variants modify replication fidelity, as determined in vitro or in cell cultures [ 13 ]. Intrinsic fidelity can be determined by residues located inside or outside the catalytic domain [ 43 , 44 ].

For instance, reorientation of the triphosphate moiety of the incoming nucleotide is a fidelity checkpoint in poliovirus polymerase [ 45 ]. Interestingly, recent work has shown that replication fidelity can also be determined by proteins of the replication complex other than the viral polymerase. Serial passages of chikungunya virus in the presence of nucleoside analogs favored the appearance of substitution GD in the RNA helicase nsP2 [ 40 ].

This variant increased replication fidelity through mechanisms linked to reduced helicase activity, increased replication kinetics, and resistance to low nucleotide concentrations [ 46 ].

Fidelity variants demonstrate the ability of RNA viruses to evolutionarily adjust mutation rates in response to selection acting on mutation rate or other traits. DNA virus mutation rates also respond to selection, as shown in earlier work with bacteriophage T4 in which a series of polymerase variants were identified following chemical mutagenesis [ 47 ].

T4 polymerase variants showing strongly increased fidelity have been described as opposed to more modest effects in RNA viruses and tend to map to the central palm and the carboxyl-terminal thumb subdomain of the viral polymerase.

Mutator phenotypes have also been described in T4. This phenotype can be conferred by changes in replication factors such as single stranded DNA-binding proteins or helicase proteins [ 48 ]. Similar results were obtained with herpes simplex virus type 1 HSV-1 , for which mutations in the conserved regions of the polymerase domain were found to modify replication fidelity.

However, this variant rapidly evolved a compensatory substitution LF that restored DNA replication fidelity in this genetic background [ 49 , 50 ]. Since RNA virus polymerases typically lack this activity, no such mutators can be produced, except for coronaviruses [ 51 ]. Furthermore, the genetic diversity of RNA viruses is probably closer to an upper tolerability limit beyond which the population genetic load increases to levels incompatible with virus survival [ 3 , 52 ]. Therefore, both biochemical and population-genetic factors limit the appearance of strong mutators in RNA viruses.

Whereas post-replicative repair probably plays a role in determining DNA virus mutation rates as discussed above , RNA virus mutation rates are strongly influenced by other host-encoded factors.

Apolipoprotein B mRNA-editing catalytic polypeptide-like enzymes APOBEC are a family of cellular cytidine deaminases that function as an innate cellular defense against retroviruses [ 53 ].

There are seven APOBEC3 paralogs in the human genome A—D and F—H which have been shown to also edit retroelements and other viruses, including hepatitis B virus [ 59 ], papillomaviruses [ 60 ], and herpesviruses [ 61 ].

Editing is strongly dependent on sequence-context. DNA editing hotspots have been identified and depend both on sequence context and DNA secondary structure [ 62 ]. In many cases, hyper-mutation leads to loss of infectivity and hence effectively exerts its antiviral action.

However, APOBECs can also produce moderately mutated, viable viruses, thus raising the question whether these deaminases may contribute to viral diversity and evolution, immune escape, and drug resistance [ 64 — 66 ]. 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 [ ].

How viruses mutate largely has to do with how they make copies of themselves and their genetic material, says Marta Gaglia , an associate professor of molecular biology and microbiology at the School of Medicine. DNA is almost always double stranded, and each of the sugars has a base attached: adenine A , cytosine C , guanine G , and thymine T.

The A, C, G, and T pair up with each other; that makes it very stable as a molecule. RNA is a similar molecule. Viruses can have all sorts of different genomes: double-stranded, single-stranded DNA, single-stranded or double-stranded RNA genome—it just depends on the virus.

DNA and RNA have slightly different chemistry and the proteins that make them are slightly different. That has some implications for the mutation rates and for the kind of molecule that the viruses must encode to be able to survive. If viruses have double-stranded DNA genomes, they kind of work the same way as DNA does normally in us, and they can use all the enzymes of the cell they have invaded. One base will pair and bind with the other base. But sometimes mutations occur if a wrong pairing happens.

If A by mistake ends up pairing with, say, C, then that will be a mutation, because it will change the coding. Our DNA-synthesizing machinery tends to have an error correction mechanism. We set out to search for shared evolutionary characteristics that may aid in gaining a broader understanding of RNA virus evolution, and constructed a phylogeny-based data set spanning thousands of sequences from diverse single-stranded RNA viruses of animals.

Strikingly, we found that the vast majority of these viruses have a skewed nucleotide composition, manifested as adenine rich A-rich coding sequences.