Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes

McKnight, Kevin L.; Simpson, Dennis A.; Lin, Seh Ching; Knott, Travis A.; Polo, John M.; Pence, David F.; B., Johannsen, Diana; Heidner, Hans W.; Davis, Nancy; Johnston, Robert E.

doi:10.1128/jvi.70.3.1981-1989.1996

Cited by 78 publications

(50 citation statements)

References 61 publications

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“…5) indicates that there might also be other mutations responsible for the adaptation to Vero E6 cells. This explanation is in line with the results observed for other RNA viruses suggesting that an altered phenotype of a given mutation may vary substantially depending on the particular genetic background in which it arises (30).…”

Section: Discussionsupporting

confidence: 89%

Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment

Lundkvist

Cheng

Sjölander

et al. 1997

J Virol

107

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This paper reports the establishment of a model for hantavirus host adaptation. Wild-type (wt) (bank vole-passaged) and Vero E6 cell-cultured variants of Puumala virus strain Kazan were analyzed for their virologic and genetic properties. The wt variant was well adapted for reproduction in bank voles but not in cell culture, while the Vero E6 strains replicated to much higher efficiency in cell culture but did not reproducibly infect bank voles. Comparison of the consensus sequences of the respective viral genomes revealed no differences in the coding region of the S gene. However, the noncoding regions of the S gene were found to be different at positions 26 and 1577. In one additional and independent adaptation experiment, all analyzed cDNA clones from the Vero E6-adapted variant were found to carry substitutions at position 1580 of the S segment, just 3 nucleotides downstream of the mutation observed in the first adaptation. No differences were found in the consensus sequences of the entire M segments from the wt and the Vero E6-adapted variants. The results indicated different impacts of the S and the M genomic segments for the adaptation process and selective advantages for the variants that carried altered noncoding sequences of the S segment. We conclude that the isolation in cell culture resulted in a phenotypically and genotypically altered hantavirus.

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Section: Discussionsupporting

confidence: 89%

Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment

Lundkvist

Cheng

Sjölander

et al. 1997

J Virol

107

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“…Mammalian cell lines Vero and BHK-21 were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% newborn calf serum (NCS; Gibco) and 1% penicillinstreptomycin (P-S; Sigma) at 37°C with 5% CO 2 . Sindbis viruses were generated from the pTR339 infectious clone and were linearized with XhoI (28). Linearized products were then purified by phenol-chloroform extraction and subsequently used for in vitro transcription of viral RNAs using an SP6 mMESSAGE mMACHINE kit (Ambion).…”

Section: Viruses and Cellsmentioning

confidence: 99%

Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles

Poirier

Mounce

Rozen-Gagnon

et al. 2016

J Virol

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Low-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. Additionally, for many RNA viruses, replication to high titers results in the production of defective interfering particles (DIs) that also attenuate infection. We hypothesized that fidelity, recombination, and DI production are tightly linked. We show that a Sindbis virus mutator replicating at a high multiplicity of infection manifests an earlier and greater accumulation of DIs than its wild-type counterpart. The isolated DIs interfere with the replication of full-length virus in a dose-dependent manner. Importantly, the ability of the mutator virus to overproduce DIs could be linked to an increased recombination frequency. These data confirm that RNA-dependent RNA polymerase fidelity and recombination are inversely correlated for this mutator. Our findings suggest that defective interference resulting from higher recombination rates may be more detrimental to RNA virus mutators than the increase in mutational burden.IMPORTANCE Replication, adaptation, and evolution of RNA viruses rely in large part on their low-fidelity RNA-dependent RNA polymerase. Viruses artificially modified in their polymerases to decrease fidelity (mutator viruses) are attenuated in vivo, demonstrating the important role of fidelity in viral fitness. However, attenuation was attributed solely to the modification of the viral mutation rate and the accumulation of detrimental point mutations. In this work, we described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction. Because DIs are known for their inhibitory effect on viral replication, our work suggests that fidelity variants may be attenuated in vivo via several mechanisms. This has important implications in the development of fidelity variants as live attenuated vaccine strains.

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“…All newly generated DNA plasmids were Sanger sequenced in full (GATC Biotech) to confirm mutagenesis of position 483 and to ensure no second-site mutations were introduced. Select SINV mutants were constructed in the same fashion from the pTR339 wildtype infectious clone [60]. CHIKV and SINV expression plasmids were linearized with NotI or XhoI respectively, purified by phenol-chloroform extraction and ethanol precipitation, and subsequently used for in vitro transcription of viral RNAs using the SP6 mMESSAGE mMACHINE kit (Ambion).…”

Section: Cells and Virusesmentioning

confidence: 99%

Alphavirus Mutator Variants Present Host-Specific Defects and Attenuation in Mammalian and Insect Models

et al. 2014

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Arboviruses cycle through both vertebrates and invertebrates, which requires them to adapt to disparate hosts while maintaining genetic integrity during genome replication. To study the genetic mechanisms and determinants of these processes, we use chikungunya virus (CHIKV), a re-emerging human pathogen transmitted by the Aedes mosquito. We previously isolated a high fidelity (or antimutator) polymerase variant, C483Y, which had decreased fitness in both mammalian and mosquito hosts, suggesting this residue may be a key molecular determinant. To further investigate effects of position 483 on RNA-dependent RNA-polymerase (RdRp) fidelity, we substituted every amino acid at this position. We isolated novel mutators with decreased replication fidelity and higher mutation frequencies, allowing us to examine the fitness of error-prone arbovirus variants. Although CHIKV mutators displayed no major replication defects in mammalian cell culture, they had reduced specific infectivity and were attenuated in vivo. Unexpectedly, mutator phenotypes were suppressed in mosquito cells and the variants exhibited significant defects in RNA synthesis. Consequently, these replication defects resulted in strong selection for reversion during infection of mosquitoes. Since residue 483 is conserved among alphaviruses, we examined the analogous mutations in Sindbis virus (SINV), which also reduced polymerase fidelity and generated replication defects in mosquito cells. However, replication defects were mosquito cell-specific and were not observed in Drosophila S2 cells, allowing us to evaluate the potential attenuation of mutators in insect models where pressure for reversion was absent. Indeed, the SINV mutator variant was attenuated in fruit flies. These findings confirm that residue 483 is a determinant regulating alphavirus polymerase fidelity and demonstrate proof of principle that arboviruses can be attenuated in mammalian and insect hosts by reducing fidelity.

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Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes

Cited by 78 publications

References 61 publications

Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment

Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment

Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles

Alphavirus Mutator Variants Present Host-Specific Defects and Attenuation in Mammalian and Insect Models

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