Involvement of the N-terminal portion of influenza virus RNA polymerase subunit PB1 in nucleotide recognition

Bình, Nguyễn Trọng; Wakai, Chitose; Kawaguchi, Akiko; Nagata, Kyosuke

doi:10.1016/j.bbrc.2013.12.071

Cited by 15 publications

(17 citation statements)

References 45 publications

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“…Ribavirin and 5-azacytidine exhibited comparable activity in this assay, with 2 to 3 log 10 reductions in influenza virus titer at 10 M. 5-Fluorouracil was less potent, with similar reductions at 80 M. The A/Panama/2007/1999 (H3N2) strain appeared to be less sensitive to both ribavirin and 5-fluorouracil than the other three strains. This strain is not known to be inherently resistant to these drugs and does not contain either PB1 D27N or PB1 V43I, two mutations that are known to confer ribavirin resistance (32,45). The reduced sensitivity could be due to the reduced replicative capacity of A/Panama/2007/1999 in MDCK cells, where its average titer of 3.2 ϫ 10 5 TCID 50 /ml in the absence of drug was at least 1.5 log 10 lower than those of the other strains.…”

Section: Anti-influenza Virus Effects Of Nucleoside Analogsmentioning

confidence: 99%

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Effective Lethal Mutagenesis of Influenza Virus by Three Nucleoside Analogs

Pauly

Lauring

2015

J Virol

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Lethal mutagenesis is a broad-spectrum antiviral strategy that exploits the high mutation rate and low mutational tolerance of many RNA viruses. This approach uses mutagenic drugs to increase viral mutation rates and burden viral populations with mutations that reduce the number of infectious progeny. We investigated the effectiveness of lethal mutagenesis as a strategy against influenza virus using three nucleoside analogs, ribavirin, 5-azacytidine, and 5-fluorouracil. All three drugs were active against a panel of seasonal H3N2 and laboratory-adapted H1N1 strains. We found that each drug increased the frequency of mutations in influenza virus populations and decreased the virus' specific infectivity, indicating a mutagenic mode of action. We were able to drive viral populations to extinction by passaging influenza virus in the presence of each drug, indicating that complete lethal mutagenesis of influenza virus populations can be achieved when a sufficient mutational burden is applied. Population-wide resistance to these mutagenic agents did not arise after serial passage of influenza virus populations in sublethal concentrations of drug. Sequencing of these drug-passaged viral populations revealed genome-wide accumulation of mutations at low frequency. The replicative capacity of drug-passaged populations was reduced at higher multiplicities of infection, suggesting the presence of defective interfering particles and a possible barrier to the evolution of resistance. Together, our data suggest that lethal mutagenesis may be a particularly effective therapeutic approach with a high genetic barrier to resistance for influenza virus. IMPORTANCEInfluenza virus is an RNA virus that causes significant morbidity and mortality during annual epidemics. Novel therapies for RNA viruses are needed due to the ease with which these viruses evolve resistance to existing therapeutics. Lethal mutagenesis is a broad-spectrum strategy that exploits the high mutation rate and the low mutational tolerance of most RNA viruses. It is thought to possess a higher barrier to resistance than conventional antiviral strategies. We investigated the effectiveness of lethal mutagenesis against influenza virus using three different drugs. We showed that influenza virus was sensitive to lethal mutagenesis by demonstrating that all three drugs induced mutations and led to an increase in the generation of defective viral particles. We also found that it may be difficult for resistance to these drugs to arise at a population-wide level. Our data suggest that lethal mutagenesis may be an attractive anti-influenza strategy that warrants further investigation. I nfluenza virus is a single-stranded, negative-sense RNA virus with a genome consisting of 8 segments (1). Like other RNA viruses, influenza virus replicates with extremely low fidelity. Its RNA-dependent RNA polymerase (RdRp) complex, which includes the viral proteins PB1, PB2, PA, and NP (2, 3), has a mutation rate of approximately 2.3 ϫ 10 Ϫ5 substitutions per nucleotide per cell ...

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Section: Anti-influenza Virus Effects Of Nucleoside Analogsmentioning

confidence: 99%

“…Within host cells, it alters GTP pool concentrations by inhibiting IMP dehydrogenase (IMPDH) (29,30). Other modes of action may include direct inhibition of the influenza virus RdRp (31,32) and interference with capping of viral RNA (33). Some data suggest that ribavirin affects inflammatory and T-cell responses in vivo (34)(35)(36).…”

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confidence: 99%

Effective Lethal Mutagenesis of Influenza Virus by Three Nucleoside Analogs

Pauly

Lauring

2015

J Virol

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“…In some cases, however, the precise mechanisms of resistance remain unclear and fidelity may or may not be involved. In the Orthomyxoviridae, an influenza A virus variant D27N, in the PB1 catalytic subunit of the viral replicase, was isolated that displays higher activity than wild type virus in the presence of RBV, as well as of an inhibitor of purine biosynthesis, methotrexate [7]. The authors suggest that this residue is involved in nucleotide recognition, but whether resistance results from increased fidelity or from increased processivity remains to be determined.…”

Section: Ribavirin and Its Isolated Resistant Variantsmentioning

confidence: 99%

“…In recent years, these studies have focused on whether RBV is directly inhibiting the RdRp, mutagenizing the genome, inhibiting IMPDH, or a combination of the three.Inhibition of RNA synthesis is observed for the influenza virus polymerase in cells treated with either RBV or methotrexate, an inhibitor of purine synthesis that decreases intracellular concentrations of purines. In this case, loss of polymerase activity at low concentrations of nucleotide is considered to be the culprit [7]. For hepatitis E virus (HEV) replication in vitro, RBV has the same antiviral effect as two other IMPDH inhibitors, mycophenolic acid (MPA) and 5-ethynyl-1-b-D-ribofuranosylimidazole-4-carboxamide (EICAR) that can be neutralized by the addition of guanosine.…”

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confidence: 99%

Ribavirin: a drug active against many viruses with multiple effects on virus replication and propagation. Molecular basis of ribavirin resistance

Beaucourt

Vignuzzi

2014

Current Opinion in Virology

112

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Ribavirin has proven to be effective against several viruses in the clinical setting and a multitude of viruses in vitro. With up to five different proposed mechanisms of action, recent advances have begun to discern the hierarchy of antiviral effects at play depending on the virus and the host conditions under scrutiny. Studies reveal that for many viruses, antiviral mechanisms may differ depending on cell type in vitro and in vivo. Further analyses are thus required to accurately identify mechanisms to more optimally determine clinical treatments. In recent years, a growing number of ribavirin resistant and sensitive variants have been identified. These variants not only inform on the specific mechanisms by which ribavirin enfeebles the virus, but also can themselves be tools to identify new antiviral compounds.

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“…This helix ␣1 domain contains a crucial, highly conserved PB1 basic residue, Arg-45, that is involved in NTP binding and nucleotidyltransferase reactions. Our group reported that a single amino acid mutation in the PB1 helix ␣1 domain induced changes in nucleotide recognition and the viral RNA synthesis reaction (40,41). To examine the structural effects of V43I substitution, we built a homology model using the PB1 polymerase of bat/Guatemala/060/2010 (H17N10), with which PR8-PB1 shares 79% identity, as a template (38) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Generation of a Genetically Stable High-Fidelity Influenza Vaccine Strain

Naito

Mori

Ushirogawa

et al. 2017

J Virol

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Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production.IMPORTANCE Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines.

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Involvement of the N-terminal portion of influenza virus RNA polymerase subunit PB1 in nucleotide recognition

Cited by 15 publications

References 45 publications

Effective Lethal Mutagenesis of Influenza Virus by Three Nucleoside Analogs

Effective Lethal Mutagenesis of Influenza Virus by Three Nucleoside Analogs

Ribavirin: a drug active against many viruses with multiple effects on virus replication and propagation. Molecular basis of ribavirin resistance

Generation of a Genetically Stable High-Fidelity Influenza Vaccine Strain

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