Mutagenesis-Induced, Large Fitness Variations with an Invariant Arenavirus Consensus Genomic Nucleotide Sequence

Abstract

Enhanced mutagenesis may result in RNA virus extinction, but the molecular events underlying this process are not well understood. Here we show that 5-fluorouracil (FU)-induced mutagenesis of the arenavirus lymphocytic choriomeningitis virus (LCMV) resulted in preextinction populations whose consensus genomic nucleotide sequence remained unaltered. Furthermore, fitness recovery passages in the absence of FU, or alternate virus passages in the presence and absence of FU, led to profound differences in the capacity of LCMV to produce progeny, without modification of the consensus genomic sequence. Molecular genetic analysis failed to produce evidence of hypermutated LCMV genomes. The results suggest that low-level mutagenesis to enrich the viral population with defector, interfering genomes harboring limited numbers of mutations may mediate the loss of infectivity that accompanies viral extinction.Mutagenic agents administered alone or in combination with antiviral inhibitors can drive RNA virus populations to extinction (4, 18, 20, 21, 41, 42, 46, 48, 50, 51, 53, 65, 67, 72, 77, 79; reviewed in references 6, 27, 28, 31, and 39). Loss of infectivity has been interpreted as an irreversible transition that occurs when template copying fidelity falls below a critical value termed the error threshold. Such a transition has been termed virus entry into error catastrophe, or lethal mutagenesis (50), and its existence has been supported by several theoretical studies (5,31,60,83). In agreement with this concept, analysis of the mutant spectra of virus populations on their way to extinction has shown 2-to 17-fold increases in mutation frequencies, calculated among components of the mutant spectra, as well as increases in Shannon entropy (a measure of the different types of sequences present in a mutant spectrum) to nearly maximal values (that is, each component of the mutant spectrum showed a unique sequence) (reviewed in references 27 and 28).The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) (12,63,64) showed extreme sensitivity to 5-fluorouracil (FU)-induced mutagenesis (41, 72) compared with other RNA viruses subjected to comparable doses of the mutagen (79). The extreme sensitivity of LCMV to FU offered an opportunity to analyze the capacity of LCMV to regain infectivity following FU mutagenesis as well as the modification of genomic nucleotide sequence variations as the virus moves toward or away from the error threshold and to explore the possible dominance of hypermutated genomes in the transition to extinction. The results reveal a remarkable capacity of LCMV populations to modify their fitness level while maintaining an invariant consensus sequence. Multiple molecular clones were analyzed to define a sequence at nucleotides 470 to 550 within the intergenic region of genomic segment L. A number of standard and mutated oligonucleotide primers have failed to produce evidence of hypermutated viral sequences in the L polymerase gene. The results suggest that limited mutagenesis may be sufficient to ...

Lethal Mutagenesis of Foot-and-Mouth Disease Virus Involves Shifts in Sequence Space

“…The present study has revealed that such a melting is mediated by an early movement in sequence space toward highly mutated genomes that precedes a decrease in viral load and the subsequent extinction. An RNA close to what could classify as hypermutated genome was detected in a previous analysis of lymphocytic choriomeningitis virus (LCMV) subjected to 5-fluorouracil mutagenesis (33). However, a shift in sequence space was not noticed using previous approaches based on standard biological or molecular cloning and sequencing.…”

“…Movements in sequence space may be a relevant ingredient in the transition toward error catastrophe. An invariant consensus sequence may conceal lethal defection by an early shift in regions of sequence space prone to produce defector genomes (31,33,34).…”

Foot-and-Mouth Disease Virus Mutant with Decreased Sensitivity to Ribavirin: Implications for Error Catastrophe

“…Viral extinction through error catastrophe or lethal mutagenesis has been supported by many experimental results and by theoretical studies when the latter are based on realistic assumptions such as the coexistence of genomes with different fitness levels in the mutant spectrum (10,11,17,18,28,29,43,54,71; see reviews in references 2 and 27). The transition toward viral extinction occurs with a decrease of specific infectivity, an increase in the complexity of the mutant spectrum, and an invariant consensus sequence of the viral genome (18,19,42,46,55,56,68), a set of properties that distinguish extinction associated with error catastrophe from extinction due solely to the inhibition of viral replication. Furthermore, direct experimental evidence indicates that mutagenesis is required to produce viral extinction (55,57).…”

Ribavirin Can Be Mutagenic for Arenaviruses

“…This virus was used for all cell culture infections described in the present study. Procedures to grow BHK-21 and Vero cells and to infect cells with LCMV have been previously described (39)(40)(41)64). Briefly, for single-step infections, BHK-21 cells were infected with LCMV at a multiplicity of infection (MOI) of 10 PFU/cell in the presence or absence of ribavirin, mycophenolic acid (MPA), or guanosine or combinations of these drugs at the concentrations indicated for each experiment.…”

“…Because of the need to explore new strategies for the control of arenavirus-associated disease, we took LCMV as a model system to investigate lethal mutagenesis or virus extinction through an increase in the mutation rate (for reviews, see references 6, 24, 32, and 38). The analogue 5-fluorouracil (FU) is an effective mutagenic agent for LCMV and can lead to viral extinction through a decrease in the specific infectivity of the mutagenized virus (39)(40)(41). Ribavirin has also been used to extinguish RNA viruses by lethal mutagenesis (38).…”