Intragenic suppression of a deletion mutation of the nonstructural gene of an influenza A virus

Treanor, John J.; Buja, R; Murphy, Brian R.

doi:10.1128/jvi.65.8.4204-4210.1991

Cited by 15 publications

(7 citation statements)

References 29 publications

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“…The possibility of deleting large parts of the protein should reduce the probability of repairing the deletion or generating second-site suppressor mutations. It should, however, be considered that even deletion mutants might phenotypically revert by acquiring intragenic or extragenic suppressor mutations (31,32).…”

Section: Discussionmentioning

confidence: 99%

Transfectant Influenza A Viruses with Long Deletions in the NS1 Protein Grow Efficiently in Vero Cells

Egorov

Brandt

Sereinig³

et al. 1998

J Virol

189

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We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (ts) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40°C selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutantswere similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.

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Section: Discussionmentioning

confidence: 99%

Transfectant Influenza A Viruses with Long Deletions in the NS1 Protein Grow Efficiently in Vero Cells

Egorov

Brandt

Sereinig³

et al. 1998

J Virol

189

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“…e Results previously reported (25) we evaluated the stability of the ts phenotype rather than the att phenotype itself because the cumulative experience with the transfectant viruses indicated that the ts mutations themselves specified attenuation and therefore are an appropriate surrogate marker for the att phenotype. The ts or att phenotype of influenza A viruses can be modified by reversion of mutant sequences as well as by the development of intragenic or extragenic suppressor mutations (21,26,27,29). Since diverse genetic mechanisms can act to alter the ts and att phenotypes, it is not surprising to observe that viruses with single ts mutations, or even those with two ts mutations on different genes, readily undergo genetic modification resulting in the loss of the ts or att phenotype following replication in vivo (13,24,26,27).…”

Section: Discussionmentioning

confidence: 99%

Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine

Subbarao

Park

Lawson

et al. 1995

J Virol

Self Cite

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We have previously described a strategy for the recovery of a synthetic influenza A virus wild-type (wt) PB2 gene (derived from influenza A/Ann Arbor/6/60 [AA] virus) into an infectious virus. It was possible to introduce an attenuating temperature-sensitive (ts) mutation at amino acid residue 265 of the AA wt PB2 gene and to rescue this mutant gene into infectious virus. Application of this new technology to influenza A virus vaccine development requires that multiple attenuating mutations be introduced to achieve a satisfactorily attenuated virus that retains the attenuation (att) phenotype following replication in vivo. In this report, we demonstrate that putative ts mutations at amino acids 112, 556, and 658 each indeed specify the ts and att phenotypes. Each of these mutations was introduced into a cDNA copy of the AA mutant mt265 PB2 gene to produce three double-mutant PB2 genes, each of which was rescued into an infectious virus. In general, the double-mutant PB2 transfectant viruses were more ts and attenuated in the lower respiratory tracts of hamsters than the single-mutant transfectant viruses, and the ts phenotype of two of three double-mutant PB2 transfectant viruses was stable even after prolonged replication in the upper respiratory tracts of immunocompromised mice. Two triple-mutant PB2 transfectant viruses with three predicted amino acid substitutions resulting from five nucleotide substitutions in the cDNA were then generated. The triple-mutant PB2 transfectant viruses were more ts and more attenuated than the double-mutant PB2 transfectant viruses. These results indicate that sequential introduction of additional ts mutations into the PB2 gene can yield mutants that exhibit a stepwise increase in temperature sensitivity and attenuation compared with the preceding mutant(s) in the series. Furthermore, the level of temperature sensitivity of the transfectant viruses correlated significantly with the level of attenuation of these viruses in hamsters. Although the triple-mutant PB2 transfectant viruses were attenuated in hamsters, intranasal administration of these viruses elicited a vigorous serum hemagglutinationinhibiting antibody response, and this was associated with resistance of the lower respiratory tract to subsequent wt virus challenge. These observations suggest the feasibility of using PB2 reverse genetics to generate a live influenza A virus vaccine donor strain that contains three attenuating mutations in one gene. It is predicted that reassortant viruses derived from such a donor virus would have the properties of attenuation, genetic stability, immunogenicity, and protective efficacy against challenge with wt virus.

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“…An interesting example of regaining fitness after an extended in-frame deletion in the coding region of the genome was documented for a spontaneous mutant of influenza A virus (504). This mutant was missing 36 nt in the RNA segment coding for the NS1 protein and exhibited ts and small-plaque phenotypes.…”

Section: Negative-strand and Double-stranded Rna Virusesmentioning

confidence: 99%

Emergency Services of Viral RNAs: Repair and Remodeling

Agol

Gmyl

2018

Microbiol Mol Biol Rev

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Reproduction of RNA viruses is typically error-prone due to the infidelity of their replicative machinery and the usual lack of proofreading mechanisms. The error rates may be close to those that kill the virus. Consequently, populations of RNA viruses are represented by heterogeneous sets of genomes with various levels of fitness. This is especially consequential when viruses encounter various bottlenecks and new infections are initiated by a single or few deviating genomes. Nevertheless, RNA viruses are able to maintain their identity by conservation of major functional elements. This conservatism stems from genetic robustness or mutational tolerance, which is largely due to the functional degeneracy of many protein and RNA elements as well as to negative selection. Another relevant mechanism is the capacity to restore fitness after genetic damages, also based on replicative infidelity. Conversely, error-prone replication is a major tool that ensures viral evolvability. The potential for changes in debilitated genomes is much higher in small populations, because in the absence of stronger competitors low-fit genomes have a choice of various trajectories to wander along fitness landscapes. Thus, low-fit populations are inherently unstable, and it may be said that to run ahead it is useful to stumble. In this report, focusing on picornaviruses and also considering data from other RNA viruses, we review the biological relevance and mechanisms of various alterations of viral RNA genomes as well as pathways and mechanisms of rehabilitation after loss of fitness. The relationships among mutational robustness, resilience, and evolvability of viral RNA genomes are discussed.

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Intragenic suppression of a deletion mutation of the nonstructural gene of an influenza A virus

Cited by 15 publications

References 29 publications

Transfectant Influenza A Viruses with Long Deletions in the NS1 Protein Grow Efficiently in Vero Cells

Transfectant Influenza A Viruses with Long Deletions in the NS1 Protein Grow Efficiently in Vero Cells

Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine

Emergency Services of Viral RNAs: Repair and Remodeling

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