Naturally occurring PAE206K point mutation in 2009 H1N1 pandemic influenza viruses impairs viral replication at high temperatures

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  Fig. 1. Polymerase activities of influenza A/Beijing/2009 and A/Kurume/2009 viruses in vitro. A 293T cells were transfected with PB1, PB2, PA, and NP protein-expressing plasmids of A/Kurume/2009 or A/Beijing/2009, together with pPOLI-HA-RT (WSN virus). The transfected cells were incubated at 37 ​°C for 24 ​h and then lysed. The lysates were analysed by Western blotting. B 293T cells were transfected with PB1, PB2, PA, and NP protein-expressing plasmids of A/Kurume/2009 or A/Beijing/2009, together with pPOLI-NA-RT (WSN virus). Total RNAs were extracted and the levels of the three RNA species were evaluated by primer extension analysis. C Viral polymerase activity in 293T cells were compared among wild-type and variant viruses harbouring PAK206E, PB2R521K, or PB1R353K/I435V. D Viral polymerase activity in 293T cells was compared among wild-type and variant viruses harbouring PAK206E of A/Beijing/2009. E Statistical analysis of HA levels in (D). HA values were standardized to the actin levels and normalized to those of HA in cells transfected with PB1, PB2, PA, and NP protein-expressing plasmids of A/Kurume/2009. Data are shown as mean ​± ​standard error of the mean of three independent…

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  Fig. 2. PAE206K reduces polymerase activity of influenza A virus in vitro. A 293T cells were transfected with A/WSN/1933 virus RNP reconstitution system plasmids (pcDNA-PB1, pcDNA-PB2, pcDNA-NP, and pPOLI-HA-RT), along with pcDNA-PA or pcDNA-PAE206K. HA levels were analysed by Western blotting. B Statistical analysis of HA levels in A. HA values were standardized to the actin levels and normalized to those of HA in cells transfected with control vector. C 293T cells were transfected with A/WSN/1933 virus RNP reconstitution system plasmids (pcDNA-PB1, pcDNA-PB2, pcDNA-NP, and pPOLI-NA-RT), along with pcDNA-PA or pcDNA-PAE206K. Total RNAs were extracted and the levels of the three RNA species were analysed by primer extension analysis. D Statistical analysis of viral RNAs in (C). Values of the viral RNAs were standardized to the levels of 5S rRNA and normalized to those of viral RNAs in cells transfected with control vector. Data are represented as mean ​± ​standard error of the mean of three independent experiments.

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  Fig. 3. PAE206K impairs replication of influenza A virus in vitro. A A549 (left) or MDCK (right) cells were infected with wild-type or PAE206K WSN ( H1N1 PAE206K) viruses at an MOI of 1 (left) or 0.01 (right) and cultured at 37 °C. Viral titres were measured by plaque assays at the indicated time points. Error bars represent the standard deviations of the results of three independent experiments. B A549 cells were infected with wild-type or PAE206K WSN viruses (MOI=1). At the indicated time post-infection, total RNAs were extracted and the levels of the three RNA species were analysed by primer extension analysis. C Statistical analysis of viral RNAs in (B). (D) A549 cells were infected with wild-type or PAE206K WSN viruses (MOI=1). At the indicated time post-infection, cells were harvested and lysed for Western blotting analysis. E Values of HA were standardized to the actin levels and normalized to those of HA in cells infected with wild-type virus. Data are represented as mean ± standard error of the mean from three independent experiments.

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  Fig. 4. Pathogenicity of wild-type and PAE206K WSN in vivo. BALB/c mice were intranasally inoculated with 30 ​μL PBS (Mock) or 104 ​PFU (in 30 ​μL) of wild-type or PAE206K viruses (n ​= ​11 per group). Mouse body weights (A) were monitored daily for 15 days and the survival rates (B) were calculated. C Lung sections were stained with haematoxylin and eosin. Magnification: ​×100. D Viral titres from mice lung on days 2 and 4 post-infection. Data are shown as mean ​± ​standard error of the mean (n ​= ​3).

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  Fig. 5. Replication Kinetics of wild-type and PAE206K WSN viruses at different temperatures. A Viral polymerase activity was determined in a vRNP reconstitution system in 293T cells at different temperatures (33, 37, and 39.5 ​°C). B Statistical analysis of the HA levels in (A). Values of HA were standardized to the actin levels and normalized to those of HA in cells transfected with wild-type vector. C MDCK cells were infected with wild-type or PAE206K WSN at a multiplicity of infection of 0.001 ​at different temperatures (33, 37, and 39.5 ​°C) in A549 ​cells. Virus titres were determined via plaque assays at 24 h post-infection. Data represent the mean ​± ​standard error of the mean of three independent experiments.

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  Fig. 6. Subcellular localization of PB1 and PA subunits (wild-type and PAE206K) transiently expressed in 293T cells at 37 and 39.5 ​°C. A 293T cells were transfected with PB1-GFP and wild-type PA or PAE206K for 24 ​h. Cells were fixed and analysed by immunofluorescence. Scale bar = 10 μm. B 293T cells were transfected with PA or PAE206K with PB1. The transfected cells were incubated at 37 ​°C or 39.5 ​°C for 40h and then lysed with RIPA buffer. The lysates were analysed by co-immunoprecipitation with PA antibody.

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