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Replication and transcription activities of ribonucleoprotein complexes reconstituted from avian H5N1, H1N1pdm09 and H3N2 influenza A viruses.

Ngai KL, Chan MC, Chan PK - PLoS ONE (2013)

Bottom Line: By using a highly sensitive quantitative reverse transcription-polymerase chain reaction, an obvious enhancement in replication and transcription activities of ribonucleoproteins was observed by the introduction of lysine at residue 627 in the H5N1 PB2 subunit.Although less strongly in polymerase activity, E158G mutation appeared to alter the accumulation of H5N1 RNA levels in a temperature-dependent manner, suggesting a temperature-dependent mechanism in regulating transcription and replication exists.This information may help to predict the pandemic potential of newly emerged influenza strains, and provide a scientific basis for stepping up surveillance measures and vaccine production.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, New Territories, Hong Kong Special Administration Region, PR China.

ABSTRACT
Avian influenza viruses pose a serious pandemic threat to humans. Better knowledge on cross-species adaptation is important. This study examined the replication and transcription efficiency of ribonucleoprotein complexes reconstituted by plasmid co-transfection between H5N1, H1N1pdm09 and H3N2 influenza A viruses, and to identify mutations in the RNA polymerase subunit that affect human adaptation. Viral RNA polymerase subunits PB1, PB2, PA and NP derived from influenza viruses were co-expressed with pPolI-vNP-Luc in human cells, and with its function evaluated by luciferase reporter assay. A quantitative RT-PCR was used to measure vRNA, cRNA, and mRNA levels for assessing the replication and transcription efficiency. Mutations in polymerase subunit were created to identify signature of increased human adaptability. H5N1 ribonucleoprotein complexes incorporated with PB2 derived from H1N1pdm09 and H3N2 viruses increased the polymerase activity in human cells. Furthermore, single amino acid substitutions at PB2 of H5N1 could affect polymerase activity in a temperature-dependent manner. By using a highly sensitive quantitative reverse transcription-polymerase chain reaction, an obvious enhancement in replication and transcription activities of ribonucleoproteins was observed by the introduction of lysine at residue 627 in the H5N1 PB2 subunit. Although less strongly in polymerase activity, E158G mutation appeared to alter the accumulation of H5N1 RNA levels in a temperature-dependent manner, suggesting a temperature-dependent mechanism in regulating transcription and replication exists. H5N1 viruses can adapt to humans either by acquisition of PB2 from circulating human-adapted viruses through reassortment, or by mutations at critical sites in PB2. This information may help to predict the pandemic potential of newly emerged influenza strains, and provide a scientific basis for stepping up surveillance measures and vaccine production.

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Related in: MedlinePlus

Polymerase activity of H5N1 RNP complexes containing mutations E158G, T271A and E627K in PB2.293T cells were co-transfected with expression plasmids of NP, PA, PB1 and either wild type (WT) or PB2 mutants with the indicated amino acid substitution of E158G, T271A or E627K, together with pPolI-vNP-Luc and a reporter plasmid pGL4.73. Cells were incubated at (A) 33°C, (B) 37°C. Polymerase activity was normalized with the expression of a reporter plasmid. Relative polymerase activity (%) was expressed as relative activity to WT. Results shown are means with standard deviations from three independent assays. *indicates P<0.05 when compared to WT.
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pone-0065038-g003: Polymerase activity of H5N1 RNP complexes containing mutations E158G, T271A and E627K in PB2.293T cells were co-transfected with expression plasmids of NP, PA, PB1 and either wild type (WT) or PB2 mutants with the indicated amino acid substitution of E158G, T271A or E627K, together with pPolI-vNP-Luc and a reporter plasmid pGL4.73. Cells were incubated at (A) 33°C, (B) 37°C. Polymerase activity was normalized with the expression of a reporter plasmid. Relative polymerase activity (%) was expressed as relative activity to WT. Results shown are means with standard deviations from three independent assays. *indicates P<0.05 when compared to WT.

Mentions: The effects of H5N1 PB2 mutations on polymerase activity are shown in Figure 3. At 33°C, E627K, E158G and T271A resulted in an increase in activity by 27.5-fold, 16.5-fold and 3.9-fold (P<0.001), respectively (Figure 3A). These mutations also led to an increase in activity at 37°C, though to a lesser extent compared to those at 33°C (Figure 3B). Among these mutations, E627K exhibited the most pronounced effect.


Replication and transcription activities of ribonucleoprotein complexes reconstituted from avian H5N1, H1N1pdm09 and H3N2 influenza A viruses.

Ngai KL, Chan MC, Chan PK - PLoS ONE (2013)

Polymerase activity of H5N1 RNP complexes containing mutations E158G, T271A and E627K in PB2.293T cells were co-transfected with expression plasmids of NP, PA, PB1 and either wild type (WT) or PB2 mutants with the indicated amino acid substitution of E158G, T271A or E627K, together with pPolI-vNP-Luc and a reporter plasmid pGL4.73. Cells were incubated at (A) 33°C, (B) 37°C. Polymerase activity was normalized with the expression of a reporter plasmid. Relative polymerase activity (%) was expressed as relative activity to WT. Results shown are means with standard deviations from three independent assays. *indicates P<0.05 when compared to WT.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672204&req=5

pone-0065038-g003: Polymerase activity of H5N1 RNP complexes containing mutations E158G, T271A and E627K in PB2.293T cells were co-transfected with expression plasmids of NP, PA, PB1 and either wild type (WT) or PB2 mutants with the indicated amino acid substitution of E158G, T271A or E627K, together with pPolI-vNP-Luc and a reporter plasmid pGL4.73. Cells were incubated at (A) 33°C, (B) 37°C. Polymerase activity was normalized with the expression of a reporter plasmid. Relative polymerase activity (%) was expressed as relative activity to WT. Results shown are means with standard deviations from three independent assays. *indicates P<0.05 when compared to WT.
Mentions: The effects of H5N1 PB2 mutations on polymerase activity are shown in Figure 3. At 33°C, E627K, E158G and T271A resulted in an increase in activity by 27.5-fold, 16.5-fold and 3.9-fold (P<0.001), respectively (Figure 3A). These mutations also led to an increase in activity at 37°C, though to a lesser extent compared to those at 33°C (Figure 3B). Among these mutations, E627K exhibited the most pronounced effect.

Bottom Line: By using a highly sensitive quantitative reverse transcription-polymerase chain reaction, an obvious enhancement in replication and transcription activities of ribonucleoproteins was observed by the introduction of lysine at residue 627 in the H5N1 PB2 subunit.Although less strongly in polymerase activity, E158G mutation appeared to alter the accumulation of H5N1 RNA levels in a temperature-dependent manner, suggesting a temperature-dependent mechanism in regulating transcription and replication exists.This information may help to predict the pandemic potential of newly emerged influenza strains, and provide a scientific basis for stepping up surveillance measures and vaccine production.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, New Territories, Hong Kong Special Administration Region, PR China.

ABSTRACT
Avian influenza viruses pose a serious pandemic threat to humans. Better knowledge on cross-species adaptation is important. This study examined the replication and transcription efficiency of ribonucleoprotein complexes reconstituted by plasmid co-transfection between H5N1, H1N1pdm09 and H3N2 influenza A viruses, and to identify mutations in the RNA polymerase subunit that affect human adaptation. Viral RNA polymerase subunits PB1, PB2, PA and NP derived from influenza viruses were co-expressed with pPolI-vNP-Luc in human cells, and with its function evaluated by luciferase reporter assay. A quantitative RT-PCR was used to measure vRNA, cRNA, and mRNA levels for assessing the replication and transcription efficiency. Mutations in polymerase subunit were created to identify signature of increased human adaptability. H5N1 ribonucleoprotein complexes incorporated with PB2 derived from H1N1pdm09 and H3N2 viruses increased the polymerase activity in human cells. Furthermore, single amino acid substitutions at PB2 of H5N1 could affect polymerase activity in a temperature-dependent manner. By using a highly sensitive quantitative reverse transcription-polymerase chain reaction, an obvious enhancement in replication and transcription activities of ribonucleoproteins was observed by the introduction of lysine at residue 627 in the H5N1 PB2 subunit. Although less strongly in polymerase activity, E158G mutation appeared to alter the accumulation of H5N1 RNA levels in a temperature-dependent manner, suggesting a temperature-dependent mechanism in regulating transcription and replication exists. H5N1 viruses can adapt to humans either by acquisition of PB2 from circulating human-adapted viruses through reassortment, or by mutations at critical sites in PB2. This information may help to predict the pandemic potential of newly emerged influenza strains, and provide a scientific basis for stepping up surveillance measures and vaccine production.

Show MeSH
Related in: MedlinePlus