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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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Comparison of in vitro polymerase activity of reconstituted RNP complexes.Activities of polymerase complexes with a single gene replacement following expression in human 293T cells. (A) H5N1 polymerase complexes substituted with one H3N2 gene, (B) H3N2 polymerase complexes substituted with one H5N1 gene, (C) H5N1 polymerase complexes substituted with one H1N1pdm09 gene, and (D) H1N1pdm09 polymerase complexes substituted with one H5N1 gene, were analyzed in 293T cells transfected with the indicated plasmids of NP, PA, PB1 and PB2 together with pPolI-vNP-Luc and a reporter plasmid pGL4.73. Cells were incubated at 33°C and 37°C. Polymerase activity was normalized with the expression of a reporter plasmid. Relative polymerase activity (%) was expressed as relative activity to the corresponding parental vRNPs. Results shown are means with standard deviations from three independent assays.
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pone-0065038-g002: Comparison of in vitro polymerase activity of reconstituted RNP complexes.Activities of polymerase complexes with a single gene replacement following expression in human 293T cells. (A) H5N1 polymerase complexes substituted with one H3N2 gene, (B) H3N2 polymerase complexes substituted with one H5N1 gene, (C) H5N1 polymerase complexes substituted with one H1N1pdm09 gene, and (D) H1N1pdm09 polymerase complexes substituted with one H5N1 gene, were analyzed in 293T cells transfected with the indicated plasmids of NP, PA, PB1 and PB2 together with pPolI-vNP-Luc and a reporter plasmid pGL4.73. Cells were incubated at 33°C and 37°C. Polymerase activity was normalized with the expression of a reporter plasmid. Relative polymerase activity (%) was expressed as relative activity to the corresponding parental vRNPs. Results shown are means with standard deviations from three independent assays.

Mentions: The polymerase activities of hybrid RNP complexes are shown in Figure 2. When the H5N1 PB2 was replaced by an H3N2 PB2 or H1N1pdm09 PB2, a significant increase in polymerase activity was observed. The effect was stronger at 33°C than 37°C (H3N2 PB1∶18.9-fold increase in activity at 33°C, P<0.001, and 4-fold at 37°C, P<0.001; H1N1pdm09 PB1∶16.1-fold at 33°C, P<0.001, and 3.6-fold at 37°C, P<0.001) (Figures 2A and 2C). In line with this, when the H3N2 PB2 or the H1N1pdm09 PB2 was replaced by H5N1 PB2, a significant decrease in polymerase activity was observed (Figure 2B and 2D).


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)

Comparison of in vitro polymerase activity of reconstituted RNP complexes.Activities of polymerase complexes with a single gene replacement following expression in human 293T cells. (A) H5N1 polymerase complexes substituted with one H3N2 gene, (B) H3N2 polymerase complexes substituted with one H5N1 gene, (C) H5N1 polymerase complexes substituted with one H1N1pdm09 gene, and (D) H1N1pdm09 polymerase complexes substituted with one H5N1 gene, were analyzed in 293T cells transfected with the indicated plasmids of NP, PA, PB1 and PB2 together with pPolI-vNP-Luc and a reporter plasmid pGL4.73. Cells were incubated at 33°C and 37°C. Polymerase activity was normalized with the expression of a reporter plasmid. Relative polymerase activity (%) was expressed as relative activity to the corresponding parental vRNPs. Results shown are means with standard deviations from three independent assays.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672204&req=5

pone-0065038-g002: Comparison of in vitro polymerase activity of reconstituted RNP complexes.Activities of polymerase complexes with a single gene replacement following expression in human 293T cells. (A) H5N1 polymerase complexes substituted with one H3N2 gene, (B) H3N2 polymerase complexes substituted with one H5N1 gene, (C) H5N1 polymerase complexes substituted with one H1N1pdm09 gene, and (D) H1N1pdm09 polymerase complexes substituted with one H5N1 gene, were analyzed in 293T cells transfected with the indicated plasmids of NP, PA, PB1 and PB2 together with pPolI-vNP-Luc and a reporter plasmid pGL4.73. Cells were incubated at 33°C and 37°C. Polymerase activity was normalized with the expression of a reporter plasmid. Relative polymerase activity (%) was expressed as relative activity to the corresponding parental vRNPs. Results shown are means with standard deviations from three independent assays.
Mentions: The polymerase activities of hybrid RNP complexes are shown in Figure 2. When the H5N1 PB2 was replaced by an H3N2 PB2 or H1N1pdm09 PB2, a significant increase in polymerase activity was observed. The effect was stronger at 33°C than 37°C (H3N2 PB1∶18.9-fold increase in activity at 33°C, P<0.001, and 4-fold at 37°C, P<0.001; H1N1pdm09 PB1∶16.1-fold at 33°C, P<0.001, and 3.6-fold at 37°C, P<0.001) (Figures 2A and 2C). In line with this, when the H3N2 PB2 or the H1N1pdm09 PB2 was replaced by H5N1 PB2, a significant decrease in polymerase activity was observed (Figure 2B and 2D).

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