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Generation and characterization of influenza A viruses with altered polymerase fidelity.

Cheung PP, Watson SJ, Choy KT, Fun Sia S, Wong DD, Poon LL, Kellam P, Guan Y, Malik Peiris JS, Yen HL - Nat Commun (2014)

Bottom Line: We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses.However, a decrease in viral population diversity at day 3 post inoculation is associated with a tenfold reduced lethality and neurotropism in mice.Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Li Ka Shing Faculty of Medicine, Centre of Influenza Research, School of Public Health, The University of Hong Kong, No. 21 Sassoon Road, Pokfulam, Hong Kong SAR, China.

ABSTRACT
Genetic diversity of influenza A viruses (IAV) acquired through the error-prone RNA-dependent RNA polymerase (RdRP) or through genetic reassortment enables perpetuation of IAV in humans through epidemics or pandemics. Here, to assess the biological significance of genetic diversity acquired through RdRP, we characterize an IAV fidelity variant derived from passaging a seasonal H3N2 virus in the presence of ribavirin, a purine analogue that increases guanosine-to-adenosine mutations. We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses. The H5N1 PB1-V43I-recombinant virus replicates to comparable titres as the wild-type virus in vitro or in the mouse lungs. However, a decrease in viral population diversity at day 3 post inoculation is associated with a tenfold reduced lethality and neurotropism in mice. Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis.

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Effects of PB1-V43I mutation on guanosine selectivity and mutational frequencies(a) Effect of increasing concentrations of guanosine on VN04 wild-type or PB1-V43I polymerase complex using mini-genome assay. P-values are based on Student’s t-test. The error bars denote standard deviation calculated for samples performed in triplicates. The experiment was repeated twice independently. (b) Firefly luciferase mRNA expression (copy numbers) under increasing concentration of guanosine. P-values are based on Student’s t-test. (c) Guanosine and ribavirin competition assay to determine the effect of guaosine in reversing the inhibitory effects of ribavirin on wild-type versus PB1-V43I polymerases. P-values are based on Student’s t-test. The mean ± SD relative polymerase activities under different concentrations of ribavirin and gunaosine were determined from triplicate samples. The experiment was repeated twice independently. (d) Mutation frequencies of recombinant Wuhan95 or VN04 wild-type and PB1-V43I viruses in the HA gene were determined by clonal sequencing. Data was expressed as the percentage of clones with 0 or ≥1 mutations in the HA gene. One of the three independently repeated experiments is shown. Fisher’s exact test was performed to determine the P-values.
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Figure 5: Effects of PB1-V43I mutation on guanosine selectivity and mutational frequencies(a) Effect of increasing concentrations of guanosine on VN04 wild-type or PB1-V43I polymerase complex using mini-genome assay. P-values are based on Student’s t-test. The error bars denote standard deviation calculated for samples performed in triplicates. The experiment was repeated twice independently. (b) Firefly luciferase mRNA expression (copy numbers) under increasing concentration of guanosine. P-values are based on Student’s t-test. (c) Guanosine and ribavirin competition assay to determine the effect of guaosine in reversing the inhibitory effects of ribavirin on wild-type versus PB1-V43I polymerases. P-values are based on Student’s t-test. The mean ± SD relative polymerase activities under different concentrations of ribavirin and gunaosine were determined from triplicate samples. The experiment was repeated twice independently. (d) Mutation frequencies of recombinant Wuhan95 or VN04 wild-type and PB1-V43I viruses in the HA gene were determined by clonal sequencing. Data was expressed as the percentage of clones with 0 or ≥1 mutations in the HA gene. One of the three independently repeated experiments is shown. Fisher’s exact test was performed to determine the P-values.

Mentions: As ribavirin is a purine analog, it is possible that the PB1-V43I mutant which exhibited altered selectivity to ribavirin would possess altered binding affinity for guanosine triphosphate (GTP) or adenosine triphosphate (ATP). We evaluated the effect of guanosine treatment on the polymerase activity of both wild-type PB1 and PB-V43I proteins in IAV mini-genome assay. The VN04 polymerase complex was used for the assay as the V43I mutation does not significantly decrease its activity. We observed that an increasing concentration of guanosine leads to reduced polymerase activity (as reflected by the expressed firefly luciferase activity) for both the wild-type as well as PB1-V43I polymerase complexes (Fig. 5a). The reduction of polymerase activity is likely through the biased intracellular nucleoside triphosphate (NTP) concentrations, as the addition of 25 to 500 μM guanosine to the Jurkat cells has been reported to increase intracellular GTP but deplete ATP concentrations 23. Interestingly, we observed that the PB1-V43I protein exhibited more significant reduction of polymerase activity under increasing concentration of guanosine (Fig. 5a), suggesting that the PB1-V43I showed increased selectivity for nucleosides over the wild-type PB1 protein under the biased intracellular NTP concentrations. The observed greater reduction of the polymerase activity (as reflected by expressed firefly luciferase activity) by the PB1-V43I protein under high guanosine concentrations (leading to biased intracellular NTP concentration) can be due to direct inhibition of RdRP as a result of increased nucleoside selectivity, or due to mutagenesis effect under the biased intracellular NTP concentration, leading to increased deleterious mutations in the luciferase mRNA and the synthesis of nonfunctional luciferase proteins. To clarify the potential mechanism, we quantified the firefly luciferase mRNA copy numbers from cells co-transfected with the PB1 plasmid carrying the V43I mutation under high guanosine concentrations. The reduced polymerase activity (as reflected by reduced firefly luciferase activity) was correlated with lower firefly luciferase mRNA levels from cells co-transfected with the PB1 plasmid carrying the V43I mutation (Fig. 5b). This observation further supports that the reduced polymerase activity is through decreased RNA synthesis. However, we cannot rule out the possibility that the reduced luciferase mRNA copy numbers can also be explained by reduced promoter binding, cap-binding, or cap-cleavage by the viral polymerase.


Generation and characterization of influenza A viruses with altered polymerase fidelity.

Cheung PP, Watson SJ, Choy KT, Fun Sia S, Wong DD, Poon LL, Kellam P, Guan Y, Malik Peiris JS, Yen HL - Nat Commun (2014)

Effects of PB1-V43I mutation on guanosine selectivity and mutational frequencies(a) Effect of increasing concentrations of guanosine on VN04 wild-type or PB1-V43I polymerase complex using mini-genome assay. P-values are based on Student’s t-test. The error bars denote standard deviation calculated for samples performed in triplicates. The experiment was repeated twice independently. (b) Firefly luciferase mRNA expression (copy numbers) under increasing concentration of guanosine. P-values are based on Student’s t-test. (c) Guanosine and ribavirin competition assay to determine the effect of guaosine in reversing the inhibitory effects of ribavirin on wild-type versus PB1-V43I polymerases. P-values are based on Student’s t-test. The mean ± SD relative polymerase activities under different concentrations of ribavirin and gunaosine were determined from triplicate samples. The experiment was repeated twice independently. (d) Mutation frequencies of recombinant Wuhan95 or VN04 wild-type and PB1-V43I viruses in the HA gene were determined by clonal sequencing. Data was expressed as the percentage of clones with 0 or ≥1 mutations in the HA gene. One of the three independently repeated experiments is shown. Fisher’s exact test was performed to determine the P-values.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4155405&req=5

Figure 5: Effects of PB1-V43I mutation on guanosine selectivity and mutational frequencies(a) Effect of increasing concentrations of guanosine on VN04 wild-type or PB1-V43I polymerase complex using mini-genome assay. P-values are based on Student’s t-test. The error bars denote standard deviation calculated for samples performed in triplicates. The experiment was repeated twice independently. (b) Firefly luciferase mRNA expression (copy numbers) under increasing concentration of guanosine. P-values are based on Student’s t-test. (c) Guanosine and ribavirin competition assay to determine the effect of guaosine in reversing the inhibitory effects of ribavirin on wild-type versus PB1-V43I polymerases. P-values are based on Student’s t-test. The mean ± SD relative polymerase activities under different concentrations of ribavirin and gunaosine were determined from triplicate samples. The experiment was repeated twice independently. (d) Mutation frequencies of recombinant Wuhan95 or VN04 wild-type and PB1-V43I viruses in the HA gene were determined by clonal sequencing. Data was expressed as the percentage of clones with 0 or ≥1 mutations in the HA gene. One of the three independently repeated experiments is shown. Fisher’s exact test was performed to determine the P-values.
Mentions: As ribavirin is a purine analog, it is possible that the PB1-V43I mutant which exhibited altered selectivity to ribavirin would possess altered binding affinity for guanosine triphosphate (GTP) or adenosine triphosphate (ATP). We evaluated the effect of guanosine treatment on the polymerase activity of both wild-type PB1 and PB-V43I proteins in IAV mini-genome assay. The VN04 polymerase complex was used for the assay as the V43I mutation does not significantly decrease its activity. We observed that an increasing concentration of guanosine leads to reduced polymerase activity (as reflected by the expressed firefly luciferase activity) for both the wild-type as well as PB1-V43I polymerase complexes (Fig. 5a). The reduction of polymerase activity is likely through the biased intracellular nucleoside triphosphate (NTP) concentrations, as the addition of 25 to 500 μM guanosine to the Jurkat cells has been reported to increase intracellular GTP but deplete ATP concentrations 23. Interestingly, we observed that the PB1-V43I protein exhibited more significant reduction of polymerase activity under increasing concentration of guanosine (Fig. 5a), suggesting that the PB1-V43I showed increased selectivity for nucleosides over the wild-type PB1 protein under the biased intracellular NTP concentrations. The observed greater reduction of the polymerase activity (as reflected by expressed firefly luciferase activity) by the PB1-V43I protein under high guanosine concentrations (leading to biased intracellular NTP concentration) can be due to direct inhibition of RdRP as a result of increased nucleoside selectivity, or due to mutagenesis effect under the biased intracellular NTP concentration, leading to increased deleterious mutations in the luciferase mRNA and the synthesis of nonfunctional luciferase proteins. To clarify the potential mechanism, we quantified the firefly luciferase mRNA copy numbers from cells co-transfected with the PB1 plasmid carrying the V43I mutation under high guanosine concentrations. The reduced polymerase activity (as reflected by reduced firefly luciferase activity) was correlated with lower firefly luciferase mRNA levels from cells co-transfected with the PB1 plasmid carrying the V43I mutation (Fig. 5b). This observation further supports that the reduced polymerase activity is through decreased RNA synthesis. However, we cannot rule out the possibility that the reduced luciferase mRNA copy numbers can also be explained by reduced promoter binding, cap-binding, or cap-cleavage by the viral polymerase.

Bottom Line: We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses.However, a decrease in viral population diversity at day 3 post inoculation is associated with a tenfold reduced lethality and neurotropism in mice.Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Li Ka Shing Faculty of Medicine, Centre of Influenza Research, School of Public Health, The University of Hong Kong, No. 21 Sassoon Road, Pokfulam, Hong Kong SAR, China.

ABSTRACT
Genetic diversity of influenza A viruses (IAV) acquired through the error-prone RNA-dependent RNA polymerase (RdRP) or through genetic reassortment enables perpetuation of IAV in humans through epidemics or pandemics. Here, to assess the biological significance of genetic diversity acquired through RdRP, we characterize an IAV fidelity variant derived from passaging a seasonal H3N2 virus in the presence of ribavirin, a purine analogue that increases guanosine-to-adenosine mutations. We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses. The H5N1 PB1-V43I-recombinant virus replicates to comparable titres as the wild-type virus in vitro or in the mouse lungs. However, a decrease in viral population diversity at day 3 post inoculation is associated with a tenfold reduced lethality and neurotropism in mice. Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis.

Show MeSH
Related in: MedlinePlus