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Viruses 2012, 4(11), 2786-2805; doi:10.3390/v4112786
Published: 6 November 2012
Abstract: Lymphocytic choriomeningitis virus (LCMV) has contributed to unveil some of the molecular mechanisms of lethal mutagenesis, or loss of virus infectivity due to increased mutation rates. Here we review these developments, and provide additional evidence that ribavirin displays a dual mutagenic and inhibitory activity on LCMV that can be relevant to treatment designs. Using 5-fluorouracil as mutagenic agent and ribavirin either as inhibitor or mutagen, we document an advantage of a sequential inhibitor-mutagen administration over the corresponding combination treatment to achieve a low LCMV load in cell culture. This advantage is accentuated in the concentration range in which ribavirin acts mainly as an inhibitor, rather than as mutagen. This observation reinforces previous theoretical and experimental studies in supporting a sequential inhibitor-mutagen administration as a possible antiviral design. Given recent progress in the development of new inhibitors of arenavirus replication, our results suggest new options of ribavirin-based anti-arenavirus treatments.
1. Introduction: complexity of LCMV populations
Michael B.A. Oldstone wrote that “LCMV has proven to be a Rosetta stone for uncovering numerous phenomena in virology and immunology”. The several crucial achievements were listed by Oldstone in an introduction to an outstanding compilation of review articles on arenavirus biology . Yet, arenaviruses pose important challenges regarding the mechanism of replication and regulation of gene expression of a segmented genome, as well as disease mechanisms, prevention and treatment. Persistent arenavirus infections are highly prevalent among rodents, and when some of the viruses infect humans they may cause severe disease, including hemorrhagic fevers [2,3,4]. The prototypic arenavirus LCMV is increasingly regarded as a neglected human pathogen [5,6,7,8]. Unfortunately, the number of preventive or therapeutic possibilities for arenavirus-associated disease is very restricted. Despite promising developments [9,10,11,12], no licensed vaccines are generally available, and current therapy is essentially limited to an off-label use of the antiviral agent ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) (Rib) [13,14]. This is why arenavirus-related diseases are an interesting target to explore new antiviral designs.
Lack of effective vaccines and antiviral treatments is a general difficulty for diseases associated with highly variable RNA viruses, due to quasispecies dynamics . The root of the problem is that mutations occur at such a high rate during RNA genome replication that even what is generally termed the “wild type” virus is, at any time, an average of a multitude of sequences. Although our attention is often fixed on a consensus sequence or on specific mutants identified in mutant spectra, in reality we deal with mutant clouds of rather indeterminate composition despite transient dominance of some specific mutant residues. Analyses of mutant spectra by ultra-deep sequencing have confirmed the extreme complexity of viral populations in nature, in support of previous evidence obtained by molecular cloning and sequencing ([16,17], among other examples ). LCMV participates of high mutation rates and quasispecies dynamics, with a direct implication in viral persistence and disease ([18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]; reviews in [33,34]). The range of mutation frequencies calculated with genomes sampled from mutant spectra of LCMV populations passaged in cell culture in absence of mutagenic agents is in the range of 1.0 × 10-4 to 7.5 × 10-4 substitutions per nucleotide (s × nt-1), and mutation frequencies reached 3.6 × 10-3 and 1.1 × 10-3 s × nt-1 when the virus was passaged in the presence of 5-fluorouracil (FU) or Rib, respectively [35,36,37,38,39,40]. Mutation frequencies are in agreement with values of Shannon entropy, a measure of the proportion of different sequences in a genome distribution. Both parameters are used to quantify the complexity of mutant spectra, which is influenced by the number of passages from a clonal origin (i.e. starting with a plaque-purified virus), the multiplicity of infection (MOI), and the viral gene analyzed. Several studies indicate that the complexities of mutant spectra of LCMV are comparable to those determined for other RNA viruses .
2. Contributions of LCMV to lethal mutagenesis
Mutations often entail a fitness cost, and this applies also to drug-escape mutants. The almost systematic selection of drug-resistant viral mutants can be explained by the existence of multiple pathways to resistance, some with limited fitness cost (under a genomic sequence context or a given environment), and to the selection of compensatory (fitness-enhancing) mutations in viral genomes that continue expressing the resistance phenotype . Under this scenario, it has been amply recognized that new antiviral strategies must be investigated to avoid or minimize the selection of drug or multi-drug-escape mutants, and the consequent treatment failure. Strategies already implemented or under development are combination therapies, splitting of treatment between an induction and a maintenance regimen, use of drugs that target cellular functions, and combination of immunotherapy and chemotherapy. As reviewed in , none of these strategies is free of the problem of selection of escape mutants.
An additional strategy now under investigation is lethal mutagenesis, also termed virus entry into error catastrophe, in recognition of its conceptual origins. It consists in achieving large reductions of viral load and ideally virus extinction by increasing the mutation rate of the virus above a critical error rate that sets a limit for virus viability. Current research involves the use of mutagenic base or nucleoside analogues which are converted intracellularly in the nucleoside-triphosphate derivatives which are incorporated into viral RNA during elongation. They do not act as chain terminators, a mechanism that results mainly in inhibition, but they allow continued chain elongation, a mechanism that results in mutagenesis. The latter is a result of the ambiguous pairing with the corresponding complementary purine or pyrimidine nucleotides, when the analogue is either a substrate or a template residue . Lethal mutagenesis was inspired in the concept of crossing an error threshold, or transition into error catastrophe, established as an important corollary of quasispecies theory [42,43]. Crossing the error threshold implies loss of genetic information. It was reasoned that drugs that increase the mutation rate of a virus above a viability threshold may highly diminish the chances of virus escape. Mutagenesis can provide an advantage over conventional non-mutagenic inhibitors because mutagenized genomes may suppress the most infective subpopulations of genomes coexisting in the same quasispecies, as discussed in the next section. A number of base and nucleoside analogues are currently under development as potential new antiviral mutagenic agents [44,45,46,47,48,49,50].
At present it is not known whether in the context of a clinical application of lethal mutagenesis , mutagen-resistant mutants will be as readily selected as inhibitor-resistant mutants. Rib-resistant mutants have been isolated in cell culture [52,53,54,55,56,57,58,59,60,61,62,63,64,65], and in patients subjected to Rib monotherapy . Rib resistance can have multiple mechanisms, as evidenced by the fact that resistance mutations have been mapped in several viral genes. When resistance is associated with amino acid substitutions in the polymerase, it can be due to restoration of polymerase activity or to alterations of nucleotide recognition. The latter can occur through at least three mechanisms: average increase of template-copying fidelity, specific decrease of Rib-triphosphate incorporation (without a significant effect on general fidelity), or modulation of the transition types produced in the presence of Rib (without alteration of the average mutation frequency among the components of the mutant spectrum) [55,57,58,59,61].
LCMV has had an important contribution regarding the feasibility of a lethal mutagenesis-based approach in vivo , and in the proposal of the lethal defection model of virus extinction (participation of defective viruses in decrease of replicative competence of a viral quasispecies ), two of the highlights of lethal mutagenesis research (Table 1). In addition, early experiments evidenced that LCMV could be efficiently extinguished by enhanced mutagenesis  (comment by Eigen ). The transition towards extinction did not involve any modification of the consensus genomic sequence, and entailed a 102- to 103-fold decrease in specific infectivity of LCMV . These were revealing observations to interpret the molecular events associated with virus extinction.
|Table 1. Some highlights in letahal mutagenesis research|
|Observation and implications||References|
|• J.J. Holland and colleagues explore for the first time quasispecies error catastrophe with real viruses and show that poliovirus and vesicular stomatitis virus have very limited tolerance to increased mutagenesis.|||
|This study was the birth of experimental studies on the application of the concept of error catastrophe developed by M. Eigen, P. Schuster and colleagues.|
|• L.A. Loeb, J. Mullins and colleagues show that a mutagenic pyrimidine analogue impairs HIV-1 replication in cell culture. They coin the term “lethal mutagenesis”.|||
|This study suggested the use of mutagenic agents as antiretroviral drugs.|
|• E. Domingo, P. Lowenstein and colleagues show that lymphocytic choriomeningitis virus and foot-and-mouth disease virus can be extinguished by mutagenic agents, and that low viral load and low viral fitness favor extinction.||[35,71]|
|These experiments suggested that modification of virus population parameters, specifically a decrease in fitness and low viral load, may help in producing virus extinction.|
|• S. Crotty, R. Andino, C.E. Cameron and colleagues demonstrate that the antiviral ribonucleoside analogue ribavirin is mutagenic for poliovirus.|||
|This important discovery implied that ribavirin might be exerting some of its antiviral clinical activity as a mutagen. This is still a debated issue, but there is evidence that ribavirin is mutagenic for a number of RNA viruses including LCMV.|
|• 5-Fluorouracil impeded the establishment of a persistent LCMV infection in mice.|||
|This experiment constitutes a proof of principle of the feasibility of a lethal mutagenesis-based antiviral approach in vivo.|
|• Experimental and theoretical evidence for the lethal defection model of virus extinction.|||
|These results introduced the concept that a mutagenic agent may not only “kill” virus but that, more subtly, the agent may be generating interfering genomes that participate in the impairment of viral replication and eventual extinction. The results suggested also the possibility of guiding internal interactions within mutant spectra to achieve extinction through modest mutagenic intensities.|
|• When a mutagen participates in therapy, a sequential inhibitor-mutagen administration might have an advantage over the corresponding combination treatment.||[73,74,75]|
|These studies illustrate that the interactions among drugs must be considered with regard to efficacy, in particular in the case of antiviral inhibitors used with virus-specific mutagenic agents. The general advantage of a combination therapy need not apply when a mutagen is involved in therapy.|
|• 5-Azacytidine can induce lethal mutagenesis of HIV-1|||
|This result suggests that some nucleotide analogues can be incorporated both into RNA and DNA during the retroviral life cycle. It shows also that there is room for classic antiviral and anti-cancer agents to find an application in lethal mutagenesis.|
|• First clinical trial involving administration of a pyrimidine analogue to AIDS patients. The resident HIV-1 was mutagenized although no virus extinction was achieved.|||
|In addition to representing the first clinical trial based on lethal mutagenesis, this result opens the possibility of improved efficacy in vivo using either combination or sequential administration of inhibitors and mutagens, in conjunction with provirus mobilization from carrier cells, a point under active investigation.|
5. Concluding remarks
Quasispecies did not exist as a scientific concept until 1971, when Manfred Eigen published his pioneer treatise . Since its completion by Eigen and Schuster , the theory has been extended to finite populations of replicons under non-equilibrium conditions, and several of its ground-breaking predictions (adaptability, limitations for sustained maintenance of information, ensembles of mutants as units of selection, etc.) have been applied and confirmed with complex biological entities such as the RNA viruses, with their highly compact genomes. Despite many unknowns in viral dynamics, the new insight provided by quasispecies is now permeating several aspects of RNA virus biology, including medical aspects such as the design of antiviral therapies. Treatment of arenavirus infections may also benefit from these developments.
Conflict of Interest
The authors declare no conflict of interest.
We thank several colleagues for important contributions and discussions on lethal mutagenesis and LCMV, in particular John Holland who initiated this line of experimental research. Work in Madrid supported by grant BFU2011-23604 to E.D. and RyC-2010-06516 and AGL-2011-25025 to V.M. and by Fundación R. Areces. The Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) is funded by Instituto de Salud Carlos III. H.M. was supported by a predoctoral contract from Instituto de Salud Carlos III (FI08-00775). A.G.-P. acknowledges grant P09-CV1-5428 from Junta de Andalucía.
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