Localization of the mutants on the PA 3-D structure.

<p>The structure of the PA C-terminal region (PDB ID: 2ZNL) is shown in magenta, with the bound PB1 peptide shown in blue. (<b>A</b>) Residues corresponding to J10 and other J10-like mutants (L3, L5, L8, L9, L10, and D478) are highlighted in yellow. K378 is not shown as it is localized to an unresolved region. The position of J10 site is indicated by arrow. (<b>B</b>) Residues corresponding to L1, L2, and L4 mutations, which affect PA nuclear localization, are highlighted in yellow. (<b>C</b>) Residues of L6 mutant, which is localized to the nucleus but defective in all viral RNA synthesis, are highlighted in yellow.</p

Characterization of additional single alanine substitution at charged or bulky residues in the C-terminal region.

<p>(<b>A</b>) Plaque formation on MDCK cells. Effects of PA mutations on the polymerase activity. (<b>B</b>) Growth curve analysis of recombinant viruses on MDCK cells at moi of 0.001. (<b>C</b>) The effects of PA mutants on viral RNA synthesis were analyzed in a 5-plasmid system. Fold induction of luciferase activity over the control was shown in log scale. Results shown are the average of at least 3 independent experiments with error bars representing standard deviation. The levels of all three viral RNA species were analyzed by primer extension assay.</p

Rescue of recombinant viruses with single alanine substitution in the C-terminal region of PA.

<p>Rescue of recombinant viruses with single alanine substitution in the C-terminal region of PA.</p

Rescue of recombinant viruses with mutations within residues 497–518 of PA.

<p>N/A, not applicable.</p

Mutational analysis of residues 497–518 of PA on viral RNA synthesis.

<p>(<b>A</b>) The effects of PA mutants on viral RNA synthesis were analyzed in a 5-plasmid system. Fold induction of luciferase activity over the control was shown in log scale. Results shown are the average of at least 3 independent experiments with error bars representing standard deviation. (<b>B</b>) The levels of all three viral RNA species were analyzed by primer extension assay.</p

PA J10 mutant does not affect the formation of VLPs but decreases viral RNA packaging efficiency.

<p>The 293T cells were transfected with the 17-plasmid system to reconstitute the replication-defective VLPs, in which a full-length vRNA construct was replaced with a corresponding vRNA segment that encodes GFP and is packaged efficiently (PA-GFP, PB1-GFP, and PB2-GFP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029485#pone.0029485-Liang1" target="_blank">[26]</a>, with either the WT or J10 mutant PA (for both the RNA and protein-encoding vectors). The cells were metabolically labeled with ³⁵S for 24 h. (<b>A</b>) Cell lysates were immunoprecipitated with anti-H1N1 antiserum and separated by SDS-PAGE. The most abundant viral proteins, M1 and NP, are highlighted by arrows. C, negative control of which the PA plasmids were omitted from the transfection. (<b>B</b>) Supernatants were VLP-enriched by either chicken erythrocytes or anti-H1N1 antiserum and separated by SDS-PAGE. Only the chicken erythrocytes enriched VLPs are showing here. The viral proteins are highlighted by arrows. C, negative control of which the PA plasmids were omitted from the transfection. (<b>C</b>) Comparison of WT and J10 PA proteins in reporter vRNA packaging. The supernatants containing the replication-defective VLPs, prepared in the absence of metabolic labeling, were collected at 48 h post-transfection and used to infect fresh MDCK cells, with the helper virus at moi of 0.1. The infected MDCK cells were analyzed by flow cytometry for GFP expression at 18 hpi. The GFP-transferring unit per ml is shown.</p

Mutational effects on viral infectivity and PA polymerase activity.

1<p>Infectivity of viable recombinant virus is defined by viral titers at 20, 28, and 40 hpi time points during viral growth kinetic analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029485#pone-0029485-g001" target="_blank">Fig. 1C</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029485#pone-0029485-g004" target="_blank">Fig. 4B</a>). ++++, WT and <0.5 log difference; +++, lower than WT by 0.5–1 log; ++, lower than WT by 1–2 log; +, lower than WT by >2 log; −, no virus. If no viable virus is rescued, infectivity is defined as −.</p>2<p>The level of RNA synthesis is determined by the 5-plasmid assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029485#pone-0029485-g002" target="_blank">Fig. 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029485#pone-0029485-g004" target="_blank">Fig. 4C</a>). ++++, >20% WT; +++, 2–20% WT; ++, 0.1–2% WT; +, <0.1% WT; −, no detectable RNA synthesis.</p>3<p>Subcellular localization of PA protein in the presence of PB1 and PB2 proteins (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029485#pone-0029485-g003" target="_blank">Fig. 3</a>). Nuc, nucleus; Cyt, cytoplasm; n/a, data not available.</p

Kissing dimers (KDs) of all six SL1 constructs were incubated at ambient temperature for 2 h (left panels) or 18 h (right panels) with NCp7 in the RNA strand-to-protein ratios of 2:0, 2:2, 2:4 and 2:8

<p><b>Copyright information:</b></p><p>Taken from "Nucleocapsid protein-mediated maturation of dimer initiation complex of full-length SL1 stemloop of HIV-1: sequence effects and mechanism of RNA refolding"</p><p></p><p>Nucleic Acids Research 2007;35(6):2026-2034.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1874624.</p><p>© 2007 The Author(s)</p> RNA was ethanol-precipitated after phenol–chloroform extraction, dialyzed in the 1× dimerization buffer and separated in native PAGE in the TBE buffer. ‘LD’ indicates the position of the dimer bands, which correspond to the linear dimers at these conditions. KDs dissociate to monomers during PAGE (shown by ‘M’). Panels (– show results for SL1-wt, SL1-yb, SL1-yf, SL1-yy, SL1-nb and SL1-es, respectively

Electrophoretic behavior of various SL1 constructs; ‘M’ and ‘D’ indicate the position of monomers and dimers, respectively

<p><b>Copyright information:</b></p><p>Taken from "Nucleocapsid protein-mediated maturation of dimer initiation complex of full-length SL1 stemloop of HIV-1: sequence effects and mechanism of RNA refolding"</p><p></p><p>Nucleic Acids Research 2007;35(6):2026-2034.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1874624.</p><p>© 2007 The Author(s)</p> SL1 RNA was run in native 10% polyacrylamide gels at 100 V in either TBE or TBM buffer. () Kissing dimers (KDs) of SL1 in TBE buffer. () KDs of SL1 in TBM buffer. () KDs of SL1 constructs were incubated for 1 h at either 25 or 55°C as indicated and separated in PAGE run in TBE buffer. Only linear dimers of SL1 migrate as dimers in TBE buffer

Native PAGE experiments on SL1-wt RNA kissing dimer (KD) in the presence of sub-stoichiometric amounts of NCp7, run in the TBE buffer

<p><b>Copyright information:</b></p><p>Taken from "Nucleocapsid protein-mediated maturation of dimer initiation complex of full-length SL1 stemloop of HIV-1: sequence effects and mechanism of RNA refolding"</p><p></p><p>Nucleic Acids Research 2007;35(6):2026-2034.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1874624.</p><p>© 2007 The Author(s)</p> KD of SL1-wt was incubated with NCp7 at ambient temperature for 18 h in the RNA strand-to-protein ratio of 2:0, 2:2, 2:1.6, 2:0.8, 2:0.4 and 2:0.2, as indicated. The upper band in each lane (‘LD’) corresponds to mature linear dimer, while the lower monomer band (‘M’) emanates from the residual KD that dissociates during PAGE in the TBE buffer. Increasing intensity of the monomer band and simultaneous decrease in the dimer band across the lanes correlates with the amount of NCp7 present in each complex