Historical Perspective on the Discovery of the Quasispecies Concept

Annual Review of Virology Historical Perspective on the Discovery of the Quasispecies Concept Esteban Domingo,1,2 Carlos García-Crespo,1 and Celia Perales1,2,3 1Department of Interactions with the Environment, Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain; email: [email protected] 2Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, 28029 Madrid, Spain 3Department of Clinical Microbiology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain Annu. Rev. Virol. 2021. 8:51–72 The Annual Review of Virology is online at virology.annualreviews.org https://doi.org/10.1146/annurev-virology-091919105900 Copyright © 2021 by Annual Reviews. This work is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See credit lines of images or other third-party material in this article for license information Keywords mutation, population dynamics, adaptation, mutation rate, sequence space, residue conservation, antiviral strategies, lethal mutagenesis Abstract Viral quasispecies are dynamic distributions of nonidentical but closely related mutant and recombinant viral genomes subjected to a continuous process of genetic variation, competition, and selection that may act as a unit of selection. The quasispecies concept owes its theoretical origins to a model for the origin of life as a collection of mutant RNA replicators. Independently, experimental evidence for the quasispecies concept was obtained from sampling of bacteriophage clones, which revealed that the viral populations consisted of many mutant genomes whose frequency varied with time of replication. Similar findings were made in animal and plant RNA viruses. Quasispecies became a theoretical framework to understand viral population dynamics and adaptability. The evidence came at a time when mutations were considered rare events in genetics, a perception that was to change dramatically in subsequent decades. Indeed, viral quasispecies was the conceptual forefront of a remarkable degree of biological diversity, now evident for cell populations and organisms, not only for viruses. Quasispecies dynamics unveiled complexities in the behavior of viral populations, 51 Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. with consequences for disease mechanisms and control strategies. This review addresses the origin of the quasispecies concept, its major implications on both viral evolution and antiviral strategies, and current and future prospects. 1. INTRODUCTION In virology, quasispecies is defined as the set of mutant genomes that comprise viral populations. Mutant ensembles are subjected to episodes of competition, selection, and random drift; they can act collectively as a unit of selection. The concept applies to all RNA viruses for which genomic sequences within infected cells and organisms have been compared. It does not refer to differences among consensus sequences of independent viral isolates, which recapitulate influences on later stages of virus evolution. The quasispecies population structure was identified during the 1970s and early 1980s, prior to the era of routine sequencing, molecular cloning, and PCR, due to a patient screening for mutations by RNA fingerprinting of biological clones of viruses. Years later, viral genetic heterogeneity was observed by use of rapid sequencing of biological and molecular clones. In the past decade, the extreme complexity of RNA viral populations and their change in mutant composition with time have been fully confirmed by deep sequencing methodologies. Although the rate of genetic change is 104-to10 7-fold larger for RNA viruses than for their host organisms (1), host environments also present nonuniform challenges to viruses. The biosphere diversity in which viruses are installed has consequences for the understanding of virus-host interactions because variant virus forms often meet with unique intracellular environments. The present-day scenario is in sharp contrast with concepts of mutational stasis that prevailed in genetics well into the twentieth century. Because the origin of quasispecies lies in replication with frequent production of mutant genomes,it is also valid for DNA viruses whose replication is catalyzed by low-fidelity polymerases or that use RNA as a replicative intermediate. Following the discovery of quasispecies,some authors attributed viral genome heterogeneity to sequencing artifacts and even questioned the value of the concept for viral genetics. However, the sequencing of genomes extracted from biological clones (not dependent on in vitro amplification of viral RNA) and quantification controls of the mutations identified in molecular clones rendered it unlikely that high mutation rates and frequencies were overestimates. The levels of genetic heterogeneity inferred from deep sequencing data are reasonably close to those calculated from the initial sequence comparisons, although recent sequencing platforms allow an unprecedented penetration into the repertoire of minority mutations present in viral populations. Mutant distributions offer an interpretation of adaptation based on the dynamic replacement of genome subpopulations within replicating ensembles. This is not a minor facet because responding to harsh selective constraints or to subtle environmental changes is what viruses have to do most of the time, including when they spread within organisms to cause disease. Interestingly, quasispecies dynamics has provided a molecular interpretation of why some vaccine and antiviral therapeutics fail and has suggested new approaches for viral disease prevention and treatment. Among the latter is lethal mutagenesis—virus extinction by an externally induced excess of mutations—which has acquired new impetus with the need to respond to the coronavirus disease 2019 (COVID-19) emergence (Sections 9 and 10). The quasispecies concept had two independent origins, one from theoretical biophysics and the other from experimental virology; both took place during the 1970s. Here we succinctly describe them from a historical perspective, with references to present-day virology. At the time of their formulation, however, some connections between theory and empiric observations were 52 Domingo •García-Crespo •Perales Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. not obvious. John Holland and colleagues (2) were the first to bring to light the biological significance of high mutation rates of RNA viruses in the context of a DNA-based biosphere, with medical implications of the collision between the contrasting DNA and RNA worlds. Following the account of the origin of quasispecies, we recapitulate its major implications for virology as we perceive them half a century later. 2. ORIGIN OF QUASISPECIES THEORY Manfred Eigen and Peter Schuster in Göttingen, Germany, developed quasispecies theory to explain self-organization and evolution of primitive RNA (or RNA-like) molecules (replicons) that might have populated Earth at the onset of life. They approached self-organization of replicating molecules by linking principles of Darwinian evolution and information theory (3, 4). Darwinian evolution was explicitly investigated in the experiments of Sol Spiegelman and colleagues (5), who showed that replicating RNA molecules evolved in the test tube. Information theory had previously highlighted the requirement for the biological meaning imprinted in a molecule to be faithfully transmitted to the following generations. The major departure from this traditional view was to describe mathematically a system involving multiplication of molecules with a regular production of error copies as inherent to the replicative process (3, 4) (Figure 1). With equation 1 in Figure 1, replication and mutation became firmly linked. The error copies of a master sequence (defined as the one with the highest replicative fitness in relation to its error copies) was referred to as a comet tail by Eigen and was later known as mutant spectrum, distribution, cloud, or swarm in virology. There are two impediments to mutant distributions attaining a population equilibrium implied by the initial formulation of quasispecies theory: (a) the random nature of mutations and (b)the statistical fluctuations in genome frequencies during the replicative process, which continue upon successive infectious cycles. Mutations are random because they arise from quantum mechanical uncertainties regarding the electronic distribution of some atoms in template and nucleotide substrate residues; altered electronic distributions may modify base pairing or base stacking interactions that exert an influence on template copying. When and where a mutation will arise during RNA virus replication is largely unpredictable; the certainty is that mutations will occur at an average rate of about one mutation per 10,000 nucleotides copied (Section 3). Statistical fluctuations may affect the variant genome production, competition, and selection, mainly through viral population migrations entailing reductions in population size, notably bottlenecks of different intensity (Figure 1). The random nature of mutations (even accounting for differences in mutation type preferences among polymerases or biases at some template sites) concurrent with modifications of virus population size are key ingredients of the unpredictable course of virus evolution. Remarkably, however, features reminiscent of determinism—consisting of similar behavior in parallel evolutionary lineages—are occasionally recorded under some experimental conditions, and they beg for an interpretation at the molecular level. In viral dynamics, a population equilibrium (meaning a steady mutant distribution that is constant in time) is beyond reach under usual replication conditions, even after extensive virus multiplication in cell culture in the absence of external influences. Populations can at most be portrayed as consisting of sequential, very short equilibrium steps (Section 7). A prediction of quasispecies theory is that there is a limit to the information content that can be stably inherited for a given average template copying fidelity of the replication machinery. This concept is mathematically formulated as an error threshold relationship that includes as one of its terms the average polymerase error rate above which the information cannot be propagated www.annualreviews.org •Discovery of the Quasispecies Concept 53 Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. a Experimental evolution Cytolytic, persistent, clonal infections Adaptation to alternative host ... Selective pressures Plaque-to-plaque transfers Virus sample ... ... b dxi dt= (Ai Qi – Di)xi +W ik xk – Φi Σk = 1, k ≠ i n lnσ0 1 – q lnσ0 ν lnσ0 p ν < νmax = and p < pmax = = Concentration of i, k Accuracy of replication of i Degradation of i Synthesis of i from k Replication of i xi , xk Wik xk Ai Qi Di Flux of i from the replication ensemble Amount of information Superiority of the master Average copying fidelity Average error rate σ0 q p Φi ν Equation 1 Equation 2 Figure 1 Basic equations of quasispecies theory and meaning of quasispecies for viral populations. (a) Equations describe replication (variation of the concentration of genome i as a function of time) with production of error copies (equation 1) and the maximum accepted amount of genetic information that can be stably maintained for a given superiority of the master sequence and average copying fidelity (equation 2). Terms are explained in the box. Derivation of the equations and extensions to nonequilibrium conditions can be found in Reference 8. (b) Organisms (in this case a pig) infected by RNA viruses (or some rapidly mutating DNA viruses) include complex and dynamic mixtures of variant genomes (represented by horizontal lines with colored symbols to depict mutations). A virus sample is a minimal representation of the variants present in the infected organism, and it may be used for experimental evolution studies to test the effect of selective pressures or bottlenecks, realized by plaque-to-plaque transfers (left panels). Quasispecies may facilitate transmission to a new host, in this case humans, where they will form new, diverse, and compartmentalized mutant spectra (right). Figure adapted with permission from References 33 and 98. (equation 2 of Figure 1). For viral genomes the information content can be equated with the number of nonredundant protein-coding regions and regulatory elements they harbor. Two transformations can be distinguished: (a) the loss of superiority of the master sequence while drift of components of the mutant spectrum is still allowed and (b) the complete disappearance of the population due to transgression of an extinction threshold (6, 7). Maintenance of viral identity is compatible with transmission of variant versions of a virus, sometimes exhibiting 54 Domingo •García-Crespo •Perales Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. different phenotypes. The application of the concept embodied in equation 2 is the basis of lethal mutagenesis as an antiviral strategy (Section 9). The generally nondeterministic nature of virus evolution does not invalidate quasispecies as a theoretical framework to approach viral dynamics. If the initial theory was not a sufficient stimulus for experimental inquiries, extensions of quasispecies and its associated error threshold relationship have been formulated for entities that exhibit a finite population size and that confront variable fitness landscapes (treated in several chapters of Reference 8). The quasispecies display of primitive replicons was an important component of the catalytic hypercycle, which was put forward as a mechanism of self-organization in the process of life construction; the hypercycle combines the coding capacity of nucleic acids with the catalytic potential of proteins in sustainment of primitive life forms (4, 9). Before we describe the experimental origin of quasispecies, it is pertinent to mention that Eigen asserted on several occasions that the value of a theoretical model lies in its capacity to evoke experimental testing (3, 10, 11). The experiments that quasispecies theory has inspired in virology have been (and continue to be) highly revealing of the nature of viral populations and of how they adjust to changing environments. 3. EXPERIMENTAL ORIGIN OF QUASISPECIES The first evidence of RNA virus quasispecies was obtained in the laboratory of Charles Weissmann in Zürich, Switzerland, also in the 1970s, independent of the theoretical development of the concept. The discovery was preceded by many years of studies on the mechanism of Escherichia coli bacteriophage Qβreplication, purification of its RNA replicase, and the elaboration of a cell-free system for sustained phage QβRNA amplification that even today stands as the most efficient in vitro RNA replication system (12–14). A stepwise in vitro QβRNA synthesis procedure using purified replicase allowed the templatedirected introduction of a mutagenic nucleotide analog into preselected positions of the full-size complementary RNA product. In the second round of copying, the analog directed the incorporation of either the correct or the incorrect nucleotide, generating a transition mutation at the preselected site of some progeny genomes. Curiously, the analog N4-hydroxy-CTP used at the time (15, 16) is the same pyrimidine analog produced intracellularly by prodrug NHC (β-D-N4-hydroxycytidine-5-isopropyl ester) used half a century later for lethal mutagenesis of coronaviruses (17, 18) (Section 10). Relevant tools were provided by Walter Fiers and colleagues (19) in Ghent, Belgium, with the initial RNase-based RNA sequencing procedures and a T1-oligonucleotide fingerprinting method that was applied to the identification of mutations in P32-labeled viral RNA (20). Synthesis of QβRNAs carrying a mutation at preselected genomic sites was the birth of sitedirected mutagenesis and reverse genetics (21), amply used today in virology and cell biology. A mutant QβRNA synthesized by this procedure that included a transition at the 3extracistronic region was viable (produced infectious progeny that maintained the engineered mutation). At the time, the result came as a surprise because the high identity of the 3extracistronic region among related bacteriophages (that was revealed by the very first results of RNA sequencing) had been taken as evidence that extracistronic mutations would be lethal. The naïve distinction between viability and lethality, without consideration of an intermediate behavior, reflects how our perception of the effect of mutations in viral genomes has changed since then. Upon replication in E. coli cells, the extracistronic mutant reverted to the wild-type sequence, indicating that the mutant displayed a selective disadvantage relative to its wild-type counterpart. A rate of 10−4reversion events per nucleotide copied was calculated by combining reversion and wild-type mutant competition experiments (22). The value obtained was several orders of magnitude higher than the www.annualreviews.org •Discovery of the Quasispecies Concept 55 Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. mutation rates calculated by John Drake (23) for some DNA viruses and microorganisms. In the following decades, high mutation rates for RNA viruses and the difference with cells and complex (large genome size) DNA viruses were corroborated (24–26). Confirmation of the specificity of the site-directed mutagenesis procedure required many infections of E. coli spheroplasts with the designed Qβmutant RNAs and analyses of progeny RNA. In the course of such experiments, it became obvious that phage populations (either the progeny of site-directed mutants or standard, nonmutagenized clonal populations) were composed of pools of mutants present at different frequencies. “The genome of Qβphage cannot be described as a defined unique structure, but rather as a weighted average of a large number of different individual sequences” (27, p. 735). Weissmann presented these results to Eigen and his colleagues at a Max Planck Winter Seminar in Klosters, Switzerland, in January 1978, and the connection between quasispecies theory and experimental observations was made. One of the attendees recalled that Eigen stood up and said, “Quasispecies in reality!” The subject was discussed at several Winter Seminars in following years with participation of Christof Biebricher, whose work with colleagues on in vitro QβRNA replication was an essential link between theory and experimentally determined kinetic parameters (28). The calculation of a mutation rate and the conclusion that phage Qβpopulations consisted of mutant spectra provided a quantitative interpretation of many observations with RNA viruses that had been reported in preceding decades, such as frequent reversion of temperature-sensitive mutants and high frequency of plaque morphology bacteriophage variants or lesion-type mutants in plant viruses, among others (additional examples and references are collected in Reference 29). Quasispecies research continued on several theoretical and experimental fronts in addition to virology. These domains include evolutionary optimization and the origin of life, replicator networks, extensions to cellular biology such as the dynamics of bacterial populations and cancer cells, chromosomal instability, conformation heterogeneity of prions, or generalization of the error threshold concept (different areas of quasispecies research have been summarized in Reference 8). Thus, from both theoretical and experimental perspectives, the quasispecies concept has been instrumental for the investigation of dynamic systems, whether they are viruses, cells, or molecules. 4. OVERVIEW OF QUASISPECIES IMPACT FOR VIROLOGY The introduction of quasispecies in virology led to some controversy because some experts argued that the concept was unnecessary, that it provided a misleading view of virus evolution, and that some virologists referred to the term quasispecies inappropriately (see, for example, the exchange of letters in References 30, 31). Additional concerns were expressed on the theory and the experimental evidence of mutant spectra. It was argued that a deterministic mathematical formulation such as that used for the initial quasispecies theory (implying infinite steady-state mutant distributions in equilibrium) could not represent viral populations. From the experimental side, it was suggested that mutation frequencies might have been overestimated due to errors introduced during viral RNA template amplification procedures (discussed in Reference 1). In our view, four developments in the past two decades have largely resolved the above concerns and have reinforced quasispecies theory as a framework to understand key events in virus-host interaction, viral pathogenesis, and the very early stages of virus diversification and evolution. The first developments are extensions of quasispecies theory to finite populations in variable fitness landscapes—i.e., extensions to mathematically describe mutant spectra under nonequilibrium conditions (1, 8) (see also Section 2). The second developments are observations that suggest that mutant swarms can act as units of selection. They include suppressive effects of mutant spectra on high fitness genomes, 56 Domingo •García-Crespo •Perales Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. interference, cooperation, or complementation among components of a mutant spectrum, and quasispecies memory. These interactions extend mutant spectrum composition and behavior beyond the standard mutation-selection balance (for reviews of mutant spectra acting as units of selection, see References 1, 8, 32, 33). Third is that confirmation through control experiments that the number of mutations introduced during reverse transcription–PCR amplification procedures prior to molecular cloning and Sanger sequencing and by ultra-deep sequencing did not inflate in a significant manner the number of mutations genuinely present in the RNA template molecules of the virus samples. No significant discrepancies have been observed in quantifications of mutant spectrum complexities using these different methods, or those resulting from sequencing biological viral clones, without cloning of individual molecules amplified in vitro. Fourth is that the large proportion of low-frequency variants and their dynamic change in frequency (e.g., by comparing sequential samples from a virus replicating in cell culture or in a live host organism) currently being revealed by ultra-deep sequencing portray a level of complexity not contemplated in the concept of genetic polymorphism of population genetics, at least as classically formulated (1). Therefore, although alternative procedures are available to understand short-term and longterm viral evolution, quasispecies theory stands as a solid framework to interpret virus-host interactions, as well as virus diversification in individual infected hosts, prior to transmission and subsequent interhost evolutionary events. Three major domains of quasispecies influence, and some of their ramifications, are schematically represented in Figure 2. They are conceptual departures such as a new definition of wild type, the role of mutant spectra in virus adaptability and behavior, and the need to consider viral dynamics when planning antiviral interventions. Central to the ramifications is that These strategies may hinder selection of escape mutants and vaccination-promoted virus evolution High connectivity of sequence space Exploration guided by fitness gradient Mutations may permit genome segmentation Reinforced by recombination and reassortment QUASISPECIES IMPACT FOR VIROLOGY Adaptability explained by mutant spectrum dynamics Influence on antiviral interventions; lethal mutagenesis Wild-type virus defined as a set of genomes Genome ensemble may dictate behavior Consensus sequences insufficient to interpret host-virus interactions Capacity of sequence space exploration Potential for salient evolutionary transitions Virus extinction by crossing an error threshold Advantage of multi-epitopic vaccines and combination therapies Fate of individual genomes dependent on mutant spectrum Intra-mutant spectrum interactions: cooperation, complementation, interference Relevant transitions (i.e., extinction by lethal mutagenesis) do not alter the consensus Individuals may differ phenotypically from the ensemble * * * * * * * * * * * * New prospects of sequential treatments and synergistic lethal mutagenesis * * * Figure 2 Scheme of quasispecies implications for viruses, and their connections. Major departures are a new meaning of wild type, a more precise understanding of adaptation at the molecular level, and the necessity to open new avenues for the control of viral disease. Each major departure has ramifications written succinctly below the arrows. It puts into focus the main points discussed in this article. See text for evidence and references. www.annualreviews.org •Discovery of the Quasispecies Concept 57 Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. complex mutant spectra—fueled by high mutation rates—are dynamically renewed in response to environmental perturbations and stochastic (chance) effects. The perturbations comprise subtle modifications of environmental parameters (intracellular ionic conditions or metabolite concentrations, temperature, etc.), components of the immune response, and external introductions (antiviral agents, vaccination). Despite our consideration of viral genomes in terms of consensus sequences (a largely unavoidable simplification), a deeper understanding of the nature of viral populations and virus-host interactions is gained from the composition of mutant spectra. The latter cannot be regarded as mere mutant aggregates acting independently of one another; internal interactions of cooperation or interference can be established in what has been called the social behavior of viral populations. The first evidence was provided by the suppressive potential of a vesicular stomatitis virus (VSV) population (32), and additional cases have been reported (reviewed in References 33, 34). The presence in mutant spectra of a molecular memory of those genomes that were dominant in previous phases of the same evolutionary lineage is yet another consequence of the weight of the mutant ensemble, with medical implications (35). High mutation rates and the quantity of tolerated mutations recorded under various biological circumstances render the presence of mutant spectra the norm; even viruses within a plaque on a cell monolayer are not genetically homogeneous (36, 37). DNA viruses whose replicative machinery displays higher fidelity or includes proofreading-repair activities, and even more those whose progeny DNA can benefit from postreplicative repair pathways, are expected to have a low net mutational input. Their quasispecies features would be displayed under different time frames and population size parameters (38), as also documented for cellular organisms (several chapters of Reference 8). 5. MUTANT SPECTRA AS PHENOTYPIC RESERVOIRS Mutant clouds equip viral populations with multiple related minority genomes present at low frequency. Those that are better suited to respond to a selective constraint than the dominant ones will increase in frequency once the constraint is in place. Clouds participate in a permanent game of challenges and responses. Basal constraints are those inherent to the cellular environments in which viruses replicate, and they act on both viral protein and RNA (extracistronic but also within open reading frames); RNA structures may be an important part of the viral phenotype (recent example in Reference 39). In differentiated hosts, clouds vary among organs, tissues, and probably even individual cells (40). The long-term historical adaptation of viruses to the cell types they infect has shaped the core genome organization and encoded proteins to provide replicability and an acceptable longterm survival probability. The central viral genome organization—which determines virus identity and assignment to one of the groups defined by the International Committee on Taxonomy of Viruses—should be flexible enough to face host alterations, including nutritional influences (41). The core genome concept has parallels with that of the pan-genome of bacteria, wherein all members of a bacterial species share a core element while individuals differ in some nonidentical features of their genetic information (42). Because a large proportion of mutations that occur during viral genome replication results in fitness decrease, mutant spectra tend to be populated mainly by genomes with a limited number of mutations relative to either the nonmutated class (when such a class can be specified) or genomes with the lowest number of mutations in that population (27, 43). Heavily mutated genomes, whose origin has sometimes been traced to apolipoprotein B messenger RNA editing complex,and adenosine deaminase acting on double-stranded RNA activities tend to be rare.They may survive in specific environments—such as the human brain for hypermutated measles virus 58 Domingo •García-Crespo •Perales Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. RNA derivatives responsible for some neurological syndromes—or due to positive epistatic interactions among mutations or,more generally, tolerance to constellations of accumulated mutations. Mutations that bring about a phenotypic change have been identified as minority components of mutant spectra of populations that exhibited a different phenotype. Phenotypes embodied in minority genomes cannot be disregarded on the grounds that their becoming prominent is unlikely. A population bottleneck (either intrahost or interhost) entails a certain probability that one of the hitherto hidden traits may be expressed by the chance promotion of the genome encoding that trait to dominance. The biological effect of a single mutation can be recognized following site-directed mutagenesis of a viral genome (Section 3), but its presence in mutant spectra may not have been directly demonstrated. Its fitness cost may mean it is present at frequencies below detectability. Detectability of a mutation may also depend on the accompanying mutations in the same genome and other genomes. Fortunately, advances in deep sequencing, with increasingly lower cutoff values for mutant detection and new methods for sequencing entire viral genomes (44), should contribute to clarifying if a residue responsible for one phenotype is present at low frequency in the mutant spectrum of the virus exhibiting a different (or the opposite) phenotype. Still another category of mutations comprises those whose presence in a mutant spectrum is inferred from the experimental design that identifies them. It includes mutants that are resistant to inhibitors, antibodies, or cytotoxic T cells (CTLs) and that are selected from a viral population that collectively displays sensitivity to the constraint. In a study with human immunodeficiency virus type 1 (HIV-1), the magnitude and genetic barrier for CTL escape were comparable to those measured for escape to antiretroviral therapy (45). Escape mutations may incur a fitness cost, but high mutation rates and large population sizes facilitate finding mutational pathways for fitness recovery. The frequency of monoclonal antibody-resistant mutants (MARMs), determined for several RNA viruses, falls in the range of 10−3to 10−7, independent of the number of circulating serotypes of the virus in nature (reviewed in Reference 1). There is no reason to suspect that the mutation rate at the genomic residues that lead to the MARM phenotype differs on average from the mutation rate at other sites for which we lack a quantification marker; the corresponding mutant frequencies may, however, be modified following subsequent replication rounds. These considerations predict the presence in mutant spectra of individual viral genomes with deviant phenotypes, as increasingly reported in the literature. Selection for an alternative viral phenotype may take place indirectly on infected cells. In parainfluenza virus 5 (PIV5), some amino acid substitutions in protein P—which is part of the virus replication complex—may repress viral transcription and replication, thus allowing subsets of infected cells that express low levels of viral proteins to escape killing by the cellular immune response (particularly by CTLs); such selective survival of P protein mutants mediates PIV5 persistence (46). Both lytic and persistent variants were detected in the mutant spectrum of a dog PIV5 isolate (that had been minimally passaged in cell culture), suggesting the coexistence of lyticand persistent-prone genomes in PIV5 quasispecies. In general, the presence in mutant spectra of minority genomes exhibiting decreased cytopathology may favor their selection as part of the multiple mechanisms involved in viral persistence (47). Thus, CTL escape can be mediated by amino acid substitutions that impede recognition of the relevant epitopes or indirectly through limited expression of viral proteins at the surface of infected cells. Quasispecies components may also modulate the immune response. Natural VSV isolates include clones that differ from the majority of the population in capacity to respond to or to induce interferon (IFN), a fact that established IFN induction as a quasispecies marker (48). The IFN response may be affected by amino acid substitutions in viral proteins such as Sendai virus protein C (49) or PIV5 protein V (50). Avian influenza virus subpopulations with enhanced www.annualreviews.org •Discovery of the Quasispecies Concept 59 Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. 3. Mutant spectra are reservoirs of phenotypically relevant variants that may affect the interaction with the host, disease processes, and response to treatments. Mutant genomes may act collectively as units of selection. 4. A consensus sequence is a simplified representation of a viral population. It may not be present in the virus population it intends to represent. Studies with hepatitis C virus suggest that residue conservation according to alignments of consensus sequences or sequences compiled in data banks does not fit conservation of the same residues in mutant spectra. 5. Lethal mutagenesis is an antiviral design inspired in the error catastrophe concept of quasispecies theory. It consists of the extinction of a virus by introducing in the replicating viral genome a number of mutations incompatible with maintenance of infectivity. There is increasing evidence of its effectiveness in cell culture and in vivo. FUTURE ISSUES 1. Clarification is needed of the extent of operation and biological implications of quasispecies dynamics for complex DNA viruses and cellular populations, in particular tumor cells. 2. Application of ultra-deep sequencing techniques and new bioinformatics approaches to achieve deeper penetration into the composition of mutant spectra is needed, in particular the use of whole-genome sequencing to identify mutation linkage and to interpret fitness effects of epistasis in dominant and minority genomes. 3. Implementation of expanded data banks for viral genomes that incorporate mutant spectrum sequences for a stricter criterion of residue conservation should help in the design of universal ligands and vaccines. 4. It will be necessary to distinguish viable and defective genomes in the mutant spectra characterized by deep sequencing and the role of defective genomes in the interactions of the viable virus subset with its host. 5. Advances in lethal mutagenesis designs should center on finding (or designing) new mutagenic agents that increase the error rate of the viral (but not of the cellular) polymerases and that can act synergistically to avoid selection of escape viral mutants. DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. ACKNOWLEDGMENTS We are indebted to many students and colleagues for work on the quasispecies concept. The research at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO), SAF2017-87846-R and BFU2017-91384-EXP from Ministerio 66 Domingo •García-Crespo •Perales Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. de Ciencia, Innovación y Universidades (MCIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER), and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121 and CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. LITERATURE CITED 1. Domingo E. 2020. VirusasPopulations. Amsterdam: Elsevier. 2nd ed. 2. Holland JJ, Spindler K, Horodyski F, Grabau E, Nichol S, VandePol S. 1982. Rapid evolution of RNA genomes. 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Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. VI08_TOC ARjats.cls August 13, 2021 12:27 Annual Review of Virology Volume 8, 2021 Contents History I Am Here: It Took a Global Village Mavis Agbandje-McKenna pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp1 Reflections on a Career in Plant Virology: A Chip Floating on a Stream Andrew O. Jackson pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp23 Historical Perspective on the Discovery of the Quasispecies Concept Esteban Domingo, Carlos García-Crespo, and Celia Perales pppppppppppppppppppppppppppppppp51 Ecology and Evolution Understanding the Complex Phage-Host Interactions in Biofilm Communities Diana P. Pires, Luís D.R. Melo, and Joana Azeredo pppppppppppppppppppppppppppppppppppppppp73 Bacteriophage-Bacteria Interactions in the Gut: From Invertebrates to Mammals Joshua M. Kirsch, Robert S. Brzozowski, Dominick Faith, June L. Round, Patrick R. Secor, and Breck A. Duerkop ppppppppppppppppppppppppppppppppppppppppppppppppppp95 Interaction of Viruses with the Insect Intestine Enhao Ma, Yibin Zhu, Ziwen Liu, Taiyun Wei, Penghua Wang, and Gong Cheng ppppp115 Integrating Viral Metagenomics into an Ecological Framework Pacifica Sommers, Anushila Chatterjee, Arvind Varsani, and Gareth Trubl ppppppppppppp133 Quantitative Temporal Viromics Alice Fletcher-Etherington and Michael P. Weekes pppppppppppppppppppppppppppppppppppppppp159 The Social Life of Viruses Rafael Sanjuán pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp183 Genome Replication, Regulation of Gene Expression, and Biosynthesis The Ends Dictate the Means: Promoter Switching in Herpesvirus Gene Expression Andrew E. Hale and Nathaniel J. Moorman pppppppppppppppppppppppppppppppppppppppppppppp201 Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. VI08_TOC ARjats.cls August 13, 2021 12:27 Assembly and Egress Virus Structures and Dynamics by Magic-Angle Spinning NMR Gal Porat-Dahlerbruch, Amir Goldbourt, and Tatyana Polenova pppppppppppppppppppppppp219 Virus Cell Biology Adenosine Deaminases Acting on RNA (ADARs) and Viral Infections Christian K. Pfaller, Cyril X. George, and Charles E. Samuel pppppppppppppppppppppppppppp239 Targeted Restriction of Viral Gene Expression and Replication by the ZAP Antiviral System Mattia Ficarelli, Stuart J.D. Neil, and Chad M. Swanson ppppppppppppppppppppppppppppppp265 Pathogenesis Bacteriophage ICP1: A Persistent Predator of Vibrio cholerae Caroline M. Boyd, Angus Angermeyer, Stephanie G. Hays, Zachary K. Barth, Kishen M. Patel, and Kimberley D. Seed pppppppppppppppppppppppppppppppppppppppppppppppp285 Advances in Viroid-Host Interactions Beatriz Navarro, Ricardo Flores, and Francesco Di Serio ppppppppppppppppppppppppppppppppp305 New Insights into Chikungunya Virus Infection and Pathogenesis Vasiliya Kril, Olivier Aïqui-Reboul-Paviet, Laurence Briant, and Ali Amara ppppppppppp327 Conquering the Host: Determinants of Pathogenesis Learned from Murine Gammaherpesvirus 68 Yiping Wang, Scott A. Tibbetts, and Laurie T. Krug pppppppppppppppppppppppppppppppppppppp349 Viruses and Metabolism: The Effects of Viral Infections and Viral Insulins on Host Metabolism Khyati Girdhar, Amaya Powis, Amol Raisingani, Martina Chrudinová, Ruixu Huang, Tu Tran, Kaan Sevgi, Yusuf Dogus Dogru, and Emrah Altindis ppppp373 Sex Differences in Respiratory Viral Pathogenesis and Treatments Rebecca L. Ursin and Sabra L. Klein ppppppppppppppppppppppppppppppppppppppppppppppppppppppp393 Immunity Bacteriophages and the Immune System Medeea Popescu, Jonas D. Van Belleghem, Arya Khosravi, and Paul L. Bollyky ppppppppp415 Vaccines Prospects for the Global Elimination of Hepatitis B Timothy M. Block, Kyong-Mi Chang, and Ju-Tao Guo ppppppppppppppppppppppppppppppppppp437 Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use. VI08_TOC ARjats.cls August 13, 2021 12:27 Antivirals Small-Molecule Inhibition of Viral Fusion Glycoproteins Han-Yuan Liu and Priscilla L. Yang ppppppppppppppppppppppppppppppppppppppppppppppppppppppp459 The Current Status of Latency Reversing Agents for HIV-1 Remission Anthony Rodari, Gilles Darcis, and Carine M. Van Lint ppppppppppppppppppppppppppppppppp491 Viral Vectors and Therapeutics FAST Proteins: Development and Use of Reverse Genetics Systems for Reoviridae Viruses Yuta Kanai and Takeshi Kobayashi ppppppppppppppppppppppppppppppppppppppppppppppppppppppppp515 Education Virology in the Classroom: Current Approaches and Challenges to Undergraduateand Graduate-Level Virology Education David B. Kushner and Andrew Pekosz ppppppppppppppppppppppppppppppppppppppppppppppppppppp537 Errata An online log of corrections to Annual Review of Virology articles may be found at http://www.annualreviews.org/errata/virology Annu. Rev. Virol. 2021.8:51-72. Downloaded from www.annualreviews.org Access provided by Centro de Biologia Molecular on 11/17/21. See copyright for approved use.