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Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, 28049-Cantoblanco, Madrid, Spain
ABSTRACT
INTRODUCTION
MUTATION RATES, MUTANT FREQUENCIES AND THE QUASISPECIES STRUCTURE
OF RNA GENOMES
QUASISPECIES AS RESERVOIRS OF
VIRAL VARIANTS
THE INFLUENCE OF VIRUS POPULATION SIZE ON VIRAL EVOLUTION: A LINK WITH NUTRITIONAL STATUS
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Mutation rates during RNA virus replication are several orders of magnitude larger than those operating during replication of cellular DNA. This results in the continuous generation of mutant genomes and in their rating in competition with other variants present and arising in the population. The dynamic mutant distributions that constitute RNA virus populations are termed quasispecies. This concept has facilitated links between population genetics and virology and has a number of important implications for viral pathogenesis and the control of viral disease. One of them is that the mutant spectra in RNA viruses constitute large reservoirs of genetic and phenotypic variants with potentially altered biological properties. Individual mutants kept in a low proportion under a set of environmental conditions may become dominant following an environmental change. Relevant to this review are possible links between the alteration of quasispecies distributions and nutritional deficiencies and oxidative stress in cells. In addition to being a possible mechanism of viral pathogenesis, oxidative stress, and other environmental modifications resulting from nutritional imbalances, may promote population disequilibrium in replicating viruses. In particular, the increased mutagenesis mediated by oxidative DNA damage could also affect replicating RNA and integrated provirus, extending the mutant repertoire of viruses. Also, the impairment of humoral and cellular immune functions may delay or prevent viral clearance, leading to an expanded representation of viral mutants in the infected organism. Thus, nutritional deficiencies are a potential source of viral mutants with altered biological properties.
KEY WORDS: virus · mutant · quasispecies · virulence · viral diseaseINTRODUCTION
Viruses with RNA as genetic material include important pathogens of humans and animals. Diseases such as influenza, poliomyelitis and several forms of hepatitis and encephalitis are caused by RNA viruses. So are many emergent viral diseases such as AIDS, new forms of respiratory disease caused by hantaviruses, hemorrhagic fevers associated with new arenaviruses, and many others (Morse 1993
, Novella et al. 1995). More than 70% of the known viruses either have RNA as genetic material or replicate via an RNA intermediate. Such ubiquity must originate, at least in part, from the genetic flexibility of RNA viruses. Mutation, homologous and nonhomologous recombination, and segment reassortment (in viruses with a segmented genome, such as influenza virus) contribute to genetic variation and to evolution of RNA viruses (reviews in Domingo et al. 1988, Gibbs et al. 1995, Holland 1992, Morse 1994, and references therein). This review summarizes some biological implications of the high mutation rates operating during RNA virus replication and retrotranscription. Possible links between nutritional status and RNA virus evolution are discussed.
MUTATION RATES, MUTANT FREQUENCIES AND THE QUASISPECIES STRUCTURE
OF RNA GENOMES
3 to 105 substitutions per nucleotide copies (s.nt1), which means that the input of mutations per genomic residue is at least 103-fold larger than the input during cellular DNA replication. The biochemical basis for such a difference is the absence of efficient proofreading and postreplicative repair activities associated with RNA replicases and reverse transcriptases (Friedberg et al. 1995, Steinhauer et al. 1992). Mutation rates may vary depending on the particular viral replicase and reverse transcriptase or on the sequence context of the template nucleotide copied (Richetti and Buc 1990); they may also vary depending on environmental changes (relative concentrations of nucleotide substrates, intracellular ionic composition, presence of mutagens, etc.). There is evidence that RNA viruses replicate near the error threshold, the minimal fidelity compatible with maintaining their genetic information (Eigen and Biebricher 1988, Holland et al. 1990).
QUASISPECIES AS RESERVOIRS OF
VIRAL VARIANTS , Wimmer et al. 1993). Because not all mutations are equally tolerated, mutation rates are not necessarily an accurate predictor of mutant frequencies. The latter are influenced not only by the rate at which mutations arise (a biochemical event) but also by their effect on fitness (overall replication ability). Mutant frequencies may vary when individual genomic positions are compared, but they are generally in the range of 103-105 s.nt1 (reviewed in Domingo and Holland 1994, Holland 1992). This range of mutant frequencies implies that most individual genomes in a virus population will differ in one or more nucleotides from the average or consensus sequence of the population. This type of genetic organization is termed the quasispecies structure of RNA viruses, and it has remarkable biological implications. Quasispecies as a theoretical concept was first developed by Eigen and his colleagues (Eigen and Schuster 1979) and shown to be an adequate descriptor of RNA viruses at the molecular level (reviews in Domingo et al. 1988, Holland 1992; historical account in Domingo et al. 1995).
Fig. 1.
Mutant spectra of HIV-1 genomes in four patients (137, 49, V75-5, and D17/+20). The regions encoding amino acids 41-108 (left boxes) and 181-219 (right boxes) of reverse transcriptase (RT) are shown. For each population, mutations relative to the consensus (top lines) are indicated. The relative abundance of each clone is given as the percentage of the total number of clones analyzed. Arrows indicate presence of deletions. Samples 137, 49 and V75-5 correspond to patients not treated with RT inhibitors. D17/+20 corresponds to a patient subjected to treatment with 3
-azido-3-deoxythymidine (AZT). Note the effect of selection on codon 67, related to AZT resistance. For further details see Nájera et al. (1995). [Reprinted from Nájera et al. (1995) with permission from American Society of Microbiology.]
[View Larger Version of this Image (28K GIF file)]
, Wain-Hobson 1994). Because the genomic complexity of RNA viruses is in the range of 3000-30,000 nucleotides, all single mutants, most double mutants, and decreasing proportions of triple and quadruple mutants will be potentially present in many viral populations. This signals an important difference between quasispecies and genetic polymorphism as applied to cellular organisms. Because of the much higher genomic complexity of cellular DNA, only a very tiny minority of all possible single mutants, and even lower proportions of multiple mutants, will be found in cellular organisms. Furthermore, because variant viruses are continuously being generated, quasispecies are enormous and dynamic mutant distributions with remarkable adaptability (Eigen and Biebricher 1988). Not only are independent isolates of the same virus genetically distinct, but each infected individual contains a spectrum of mutants. Although the human immunodeficiency virus type 1 (HIV-1) constitutes one of the most dramatic examples of quasispecies diversification within infected individuals (Fig. 1) (Coffin 1995, Wain-Hobson 1994), similar observations have been made upon sequence analysis of many virus populations (Domingo et al. 1988, Domingo and Holland 1994, Holland 1992). A direct example of the survival value of quasispecies was provided by the presence in HIV-1 populations of mutations related to resistance to inhibitors of the viral reverse transcriptase and protease even in individuals not treated with the relevant inhibitors (Borman et al. 1996, Lech et al. 1996, Nájera et al. 1995). Another example is the presence of mutant viruses resistant to neutralization by monoclonal antibodies at frequencies that generally are in the range of 103-106. The limited protection afforded by antiviral synthetic vaccines, in particular peptide antigens, originates (at least in part) from the quasispecies structure, which may provide variants capable of escaping an immune response elicited by another variant (Domingo and Holland 1992). Additional examples of the biological relevance of viral quasispecies can be found in recent reviews (Domingo et al. 1988, Gibbs et al. 1995, Holland 1992, Morse 1994).
THE INFLUENCE OF VIRUS POPULATION SIZE ON VIRAL EVOLUTION: A LINK WITH NUTRITIONAL STATUS
, Duarte et al. 1992). Such transfers represent a severe form of bottleneck since the virus population is periodically reduced to one. In contrast, large population passages, in which competitive rating among variants is allowed, result in fitness gains (Novella et al. 1995). More modest variations in population size may also have a significant influence in the type of variants that become dominant in evolving quasispecies. A recent example has been provided by model studies with foot-and-mouth disease virus (FMDV) in cell culture. Parallel passage of either 2 × 105 or 2 × 108 infectious units (I.U.) in triplicate series documented the dominance of an unusual antigenic variant in each of the three passage series involving 2 × 108 I.U. and in none of the passage series involving 2 × 105 I.U. (Sevilla and Domingo 1996). A simple prediction using the Poisson distribution shows that, obviously, the total number of single and multiple mutants potentially present in viral populations increases with the population size (Domingo et al. 1985, Domingo and Holland 1992).
). A second mechanism stems from the impairment of immune response as a result of nutritional imbalances. There is evidence that nutritional deficiencies can alter the following processes related to immune function: cell activation, proliferation and movement, development of lymphoid tissue, synthesis of antibodies and immunoreactive factors (such as cytokines), phagocytosis, and others (Bendich and Chandra 1990, Frei 1994, Sherman 1992). Both the humoral and cellular arms of the immune system seem to be affected because their response is based on rapid cell proliferation that may be impaired by oxidative damage, in particular by lipid peroxidation of cell membranes. Paradoxically, reactive oxygen species are involved in normal immune reactions, and damage to the immune system may originate from imbalances in the relative levels of oxidants and antioxidant molecular species. This is suggested by the enhancement of immune function when adequate levels of vitamin E and selenium, two of the main natural antioxidants, are restored (Frei 1994).
, Beck et al. 1995) have recently described the rapid evolution in selenium-deficient mice of an apathogenic (amyocarditic) coxsackievirus B3 into a pathogenic form that caused heart disease. Six nucleotide substitutions distinguished the virulent virus from the avirulent parent. These authors attributed the generation of the mutant form to higher virus titers in the hearts of selenium-deficient mice due to decreased antigen-specific T cell responses, or to an elevated rate of oxidative damage to the RNA genome (Beck 1997, Beck et al. 1995). As expected, once generated, the variant virus remained virulent even in animals with adequate selenium levels. This example illustrates how nutritional imbalances may modify the course of viral evolution and contribute to the emergence of mutant viruses with altered pathogenic potential.
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
A Principle of Natural Self-Organization. Springer, Berlin, Germany.
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