Comparison of RNP activities under thermal stress.

<p>(A) Brief protocol and incubation periods are indicated. (B) Representative analyzed polyacrylamide gel (6%) is shown. * represents statistical significance at p<0.05 in a Student's t-test (n = 3). 293T cells expressing influenza RNP were incubated at 37°C for 24 hours as pre-incubation. Pre-incubated cells were additionally incubated at 34, 37 and 42°C for 9 hours, respectively. Then total RNAs were extracted and analyzed by primer extension assay. A/WSN/33, A/Hong Kong/156/97, A/NT/60/68, A/Vietnam/1194/2004 and pandemic H1N1 2009 virus are abbreviated as WSN, HK, NT, VN and SW, respectively. 5s ribosomal RNA (rRNA) is indicated as an internal control. mRNA, cRNA and vRNA are viral messenger RNA, complementary viral RNA and viral RNA, respectively.</p

Promoter binding activity and replication initiation activity of HK PB2 mutants.

<p>(<b>A</b>) Partially purified polymerases analyzed by silver-stained 7.5% SDS-PAGE. Hybrid WSN (W) polymerase replaced only PB2 subunit with HK PB2 wild type (H) (lanes 2), HK PB2 mutants (lanes 3–6) and VN PB2 (V) (lane 7) was transiently expressed in 293T cells and partially purified by using TAP-tagged PA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032634#s2" target="_blank">materials and methods</a>). The numbers in HK PB2 mutants indicate mutated positions in HK PB2. The highlighted numbers are important for the rescue of the RNP activity. (<b>B</b>) UV cross-linking of model vRNA and cRNA promoters to hybrid polymerases. Purified and quantified polymerases were incubated with ³²P-labelled 3′ strand of the vRNA promoter in the presence of the unlabelled 5′ strand of the vRNA promoter (upper panel), or ³²P -labelled 3′ strand of the cRNA promoter in the presence of the unlabelled 5′ strand of the cRNA promoter (lower panel). Lane numbers correspond to the numbers in (A). The positions of the cross-linked products are indicated on the right. (<b>C</b>) ApG synthesis with a model vRNA promoter (upper panel) or cRNA promoter (lower panel). Lane numbers correspond to the numbers in (A). The position of the specific ApG product is indicated on the right. (<b>D and E</b>) Quantification of results obtained in panels B and C, respectively, by phosphorimaging. Data are expressed as percentages relative to VN PB2 (lane 7) (mean ± standard deviation; n = 3). Black bars, model vRNA promoter; white bars, model cRNA promoter. * shows statistical significance at P<0.01 in a Student's t-test.</p

Time course of RNP activity on thermal stress at 42°C.

<p>(A) Brief protocol and incubation periods are indicated. (B) Quantitation of cRNA (closed circle) and vRNA (opened circle) of WSN at 42°C is shown. Data have been normalized for total RNA using the 5S rRNA signal. Data are expressed as a percentage of wild type activity (with standard deviation). Quantitation was based on at lease three independent sets of data. (C) Representative analyzed polyacrylamide gel (6%) is shown (n = 3). 293T cells expressing influenza RNP were incubated at 37°C for 24 hours as pre-incubation. Then pre-incubated cells were transferred to 42°C. Total RNAs were extracted and analyzed at each time point by primer extension assay. (D) RNA polymerase subunits (PA, PB1 and PB2) and NP - transiently expressed in human 293T cells, analyzed by western blotting using specific anti-bodies of each subunit by 8% SDS-PAGE. A/WSN/33, A/HongKong/156/97 and pandemic H1N1 2009 virus are abbreviated as WSN, HK and SW, respectively. 5s ribosomal RNA (rRNA) is indicated as an internal control. mRNA, cRNA and vRNA are viral messenger RNA, complementary viral RNA and viral RNA, respectively.</p

Comparison of RNP activities containing hybrid polymerases between H5N1 strains and human strains in vivo.

<p>Polymerase subunits of human strains of A/WSN/1933 (H1N1) (<b>W</b>), A/Kurume/K0910/2009 (H1N1) (S) and A/NT/60/1968 (H3N2) (N) were replaced with the corresponding subunits of A/HongKong/156/1997 (H5N1) (H) (left 3 panels) or A/Vietnam/1194/2004 (H5N1) (V) (right 3 panels). The RNP was reconstituted in 293T cells by transfection of plasmids expressing various combinations of polymerase subunits, and WSN derived NP and vNA. Total RNA was extracted 30 h posttransfection. Transcript products of viral mRNA, cRNA and vRNA were measured by primer extension and its positions are indicated on the right. The expected size of mRNA, cRNA, vRNA and 5S rRNA as an internal control are 169–177 nt, 160 nt, 129 nt and 100 nt, respectively.</p

Effect of a transient heat shock on RNP activity at 42°C.

a<p>The replication (cRNA and vRNA) and transcription (mRNA) activities are indicated as a relative activity (%) ± S.D. from start point (0 hours) (n = 3).</p

Effect of transient heat shock at 42°C on RNP activity.

<p>(A) Brief protocol and incubation periods are indicated. (B) Representative analyzed polyacrylamide gel (6%) is shown (n = 3). 293T cells expressing influenza RNP were incubated at 37°C for 24 hours as pre-incubation. Pre-incubated cells were stimulated by 42°C for 15 min, and then continued to incubate at 37°C up to 6 hours. Total RNAs were extracted and analyzed by primer extension assay. A/WSN/33 and A/HongKong/156/97 are abbreviated as WSN and HK, respectively. 5s ribosomal RNA (rRNA) is indicated as an internal control. mRNA, cRNA and vRNA are viral messenger RNA, complementary viral RNA and viral RNA, respectively.</p

Relative RNP activities of each strain at different temperatures.

a<p>The replication (cRNA and vRNA) and transcription (mRNA) activities are indicated as a relative activity (%) ± S.D. to each strain set at 100% at 37°C (n = 3).</p

Alignment of PB2 subunit.

<p>(<b>A</b>) Functional map of PB2 subunit. (B) Alignment of amino acid residues in PB2 which are important for the accumulation of RNP. * shows influenza strains used in this study. Gray shading indicates high evolutionary conservation between influenza A, B and C virus sequences.</p

Effect of PA subunit under various thermal stresses.

<p>(A) Representative analyzed polyacrylamide gel (6%) is shown (n = 3). WSN PA subunit was replaced with that of each strain. 293T cells expressing influenza RNP were incubated at 37°C for 24 hours as pre-incubation. Pre-incubated cells were additionally incubated at 34°C, 37°C or 42°C for 9 hours. Total RNAs were extracted and analyzed by primer extension assay. A/WSN/33, A/HongKong/156/97, A/NT/60/68, A/Vietnam/1194/2004 and pandemic H1N1 2009 virus are abbreviated as W, H, N, V and S respectively. 5s ribosomal RNA (rRNA) is indicated as an internal control. mRNA, cRNA and vRNA are viral messenger RNA, complementary viral RNA and viral RNA, respectively. (B) Quantitation of cRNA (closed bar) and vRNA (opened bar) and standard deviations of bands in panel A expressed as a percentage of WSN strain at 37°C. ** represents statistical significance at p<0.01 in a Student's t-test (n = 3). Each RNA polymerase subunit is indicated as PB1, PB2 or PA.</p

Effects of mutations in the HK PB2 subunit on the impaired RNP activity reconstituted from hybrid polymerase.

<p>(<b>A</b>) Alignment of amino acid residues of PB2 subunit differing between HK and VN of H5N1 strains. All positions of HK PB2, except for 4 residues (asterisk) that were similar basic amino acids, were sequentially substituted for VN PB2 sequences. (<b>B</b>) Steady state level of NA vRNA, mRNA, and cRNA in WSN (W) RNP reconstituted from hybrid polymerase by replacing only PB2 subunit with the HK PB2 wild type (H) (lanes 2 and 9), HK PB2 mutants (lanes 3–7, 10–16, 18–20, 22–26 and 28–30) and VN PB2 (V) (lanes 17, 21, 27 and 31). The numbers in HK PB2 mutants indicate mutated positions in HK PB2. Arrows indicate HK PB2 mutants which showed significant increase of RNP activity. The positions found to be important for the rescue of the RNP activity are highlighted.</p