Pathogenicity of the 1918 pandemic influenza virus in non-human primates.

<p>Taubenberger's group isolated viral genome RNA fragments from formalin-fixed, paraffin-embedded autopsy tissue from victims of the 1918 pandemic and determined the coding sequences of all eight RNA segments of the 1918 pandemic virus. Based on these sequences, a virus bearing all eight RNA segments of the 1918 virus was generated by reverse genetics and its pathogenicity was determined in a non-human primate model. The 1918 virus caused a highly pathogenic respiratory infection in non-human primates that culminated in acute respiratory distress and a fatal outcome. The infected animals mounted an immune response, characterized by dysregulation of the antiviral response, indicating that atypical host innate immune responses may contribute to lethality. The infection of nonhuman primates with the 1918 pandemic virus caused severe damage in the lungs (a), whereas no appreciable lesions were observed in the lungs of animals infected with a contemporary human H1N1 virus (b).</p

The role of the viral RNA polymerase complex in the replication properties of the 1918 virus in ferrets.

<p>The 1918 wild-type virus and a virus possessing the 1918 viral RNA polymerase complex genes (i.e., PA, PB1, PB2, and NP) replicated efficiently in both the upper and lower respiratory tracts of ferrets, whereas the replication of the contemporary human H1N1 virus was restricted to the upper respiratory tract, suggesting a role for the viral RNA polymerase complex in the optimal growth of the 1918 virus in the lower respiratory tract of ferrets. +++, high replicative ability; ++, moderate replicative ability; −, no virus detected.</p

The role of the HA gene in the pathogenicity of the 1918 virus in mice.

<p>Mice infected with a contemporary human H1N1 influenza virus showed no symptoms. In contrast, a virus possessing the HA gene derived from the 1918 virus in the genetic background of the contemporary human H1N1 virus was lethal to mice.</p

Data_Sheet_1_Host Factor Nucleoporin 93 Is Involved in the Nuclear Export of Influenza Virus RNA.DOCX

<p>Influenza virus replication relies on the functions of host factors. In our previous study, we identified host factors involved in virus replication and began analyses of their roles in this process. In this study, we focused on Nucleoporin 93 (NUP93) and revealed its importance in influenza virus replication. NUP93 knockdown mediated by siRNAs reduced viral replication and decreased the efficiency of the early step of the viral life cycle. NUP93 did not appear to be important for virus binding, internalization, or the nuclear import of viral ribonucleoprotein (vRNP); however, in NUP93-depleted cells, viral RNA accumulated in the nucleus. These results suggest that NUP93 is involved in the nuclear export of viral RNA.</p

Viral particle production of GagPol constructs containing the p6 domain.

<p>(A) Schematic representation of the GagPol and GagPR constructs and their amino acid sequences of the p6* and p6 domains. The p6* domain was replaced by the p1+p6 domain (lacking the 12 C-terminal amino acids), and the resultant constructs were referred to as Gag(p6)Pol and Gag(p6)PR. The GagPol-HA and Gag(p6*)PR-HA constructs contain the authentic p6* domain (upper), and the Gag(p6)Pol and Gag(p6)PR constructs contain the p6 domain instead of the p6* (lower). All constructs contained inactive PR and were expressed in the context of pNL43. (B-E) HeLa cells were transfected with Gag(p6)Pol, Gag(p6)PR, GagPol-HA, and Gag(p6*)PR-HA constructs. (B) Membrane affinity of the Gag(p6)Pol and Gag(p6)PR proteins. Cells were subjected to membrane flotation centrifugation followed by Western blotting using anti-p24 antibody. (C) Intracellular localization of the Gag(p6)Pol and Gag(p6)PR proteins. Cells were immunostained with anti-p24 antibody (green), and nuclei were stained with TO-PRO-3 (blue). All micrographs are shown at the same magnification. In each sample, approximately 100 antigen-positive cells (from 3 independent experiments) were subjected to distribution pattern analysis. (D) Intracellular expression and viral particle production of the Gag(p6)Pol and Gag(p6)PR proteins. The Gag-FLAG/Pol-HA construct was used as a positive control. Cells and purified viral particles were subjected to Western blotting using anti-p24 antibody. Arrows indicate GagPol and GagPR. (E) Electron microscopy of cells transfected with Gag(p6*)PR-HA and Gag(p6)PR. The cells were stained with uranyl acetate and lead citrate. Arrowheads show pedestal-like structures. Bars, 500 nm.</p

Mutations in NA That Induced Low pH-Stability and Enhanced the Replication of Pandemic (H1N1) 2009 Influenza A Virus at an Early Stage of the Pandemic

<div><p>An influenza A virus that originated in pigs caused a pandemic in 2009. The sialidase activity of the neuraminidase (NA) of previous pandemic influenza A viruses are stable at low pH (≤5). Here, we identified the amino acids responsible for this property. We found differences in low-pH stability at pH 5.0 among pandemic (H1N1) 2009 viruses, which enhanced the replication of these viruses. Low-pH-stable NA enhancement of virus replication may have contributed to the rapid worldwide spread and adaptation to humans of pandemic (H1N1) 2009 viruses during the early stages of the 2009 pandemic.</p></div

Viral particle production of HIV-1 GagPol and its C-terminally truncated derivatives.

<p>(A) Schematic representation of the pNL43 derivatives tagged with the FLAG and HA sequences. The pNL43 derivative containing inactive PR was used as wild type (WT). The FLAG and HA sequences were inserted in-frame in the C-terminal p6 domain of Gag and the C-terminus of GagPol, respectively (referred to as Gag-FLAG/Pol-HA) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047828#pone.0047828-Haraguchi1" target="_blank">[44]</a>. For expression of Gag-FLAG alone (without GagPol), the FLAG sequence was inserted in the C-terminal p6 domain of Gag and termination codons (asterisk) were placed in-frame in the <i>pol</i> frame. For expression of GagPol-HA (without Gag), the HA sequence was added to the C-terminus of the GagPol protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047828#pone.0047828-Haraguchi1" target="_blank">[44]</a>. For the C-terminal truncations of GagPol, the HA sequence followed by a termination codon (asterisk) was inserted at the PR/RT or RT/IN junction of GagPol [referred to as Gag(p6*)p66-HA and Gag(p6*)PR-HA, respectively]. GagPol-HA and the derivatives contained the frameshift mutation, and all constructs contained inactive PR. LTR, long terminal repeat. (B) N-myristoylation and viral particle production of the truncated GagPol proteins. HeLa cells were singly transfected with the GagPol-HA and its C-terminally truncated constructs, or doubly transfected with a combination of the GagPol-HA and Gag-FLAG constructs at a Gag-to-GagPol DNA ratio of 1∶10. Total DNA amounts were normalized to 8 µg with pUC plasmid. Gag-FLAG/Pol-HA and myr(-)GagPol-HA containing the myristoylation (G2A) mutation were used as positive and negative controls, respectively. Cells were labeled with [³H]myristic acid for 3 hr and were subjected to SDS-PAGE followed by fluorography. Arrowheads indicate myristoylated GagPol-HA, Gag(p6*)p66-HA, and Gag(p6*)PR-HA, respectively. The cells and purified viral particles were subjected to Western blotting using anti-HIV-1 p24 antibody. The intensity of the band corresponding to each construct in fluorographed gels and Western blots was measured by ImageJ software. For each construct, the band intensity in fluorographed gels was divided by the band intensity in Western blots. The myristoylation ratio of Gag was set at 1.0, and the myristoylation ratios of the constructs relative to the ratio of Gag were calculated.</p

Membrane affinity and plasma membrane targeting of GagPol with C-terminal truncations.

<p>(A and B) HeLa cells were transfected with the Gag-FLAG, GagPol-HA, and C-terminally truncated constructs. Gag-FLAG was used as a positive control. (A) Intracellular localization of the truncated GagPol proteins. At 24 hr post-transfection, cells were immunostained with anti-FLAG (red) or anti-HA (green) antibodies and nuclei were stained with TO-PRO-3 (blue). Bottom panels show confocal images overlaid with differential interference contrast images. All micrographs are shown at the same magnification. In each sample, approximately 100 antigen-positive cells (from 3 or 4 independent experiments) were subjected to analysis of the antigen distribution pattern. (B) Membrane affinity of the truncated GagPol proteins. Cells were subjected to membrane flotation centrifugation followed by Western blotting using anti-p24 antibody. Representative blots in three independent experiments were shown.</p

Plasma membrane targeting and viral particle production of GagPol with the Fyn(10) N-terminal sequence.

<p>(A) Schematic representation of GagPol-HA and its derivatives containing the Fyn(10) N-terminal sequence and the p6 domain. The initiation codon of GagPol-HA was replaced by Fyn(10) [referred to as Fyn(10)GagPol-HA] and the p6* domain was further replaced by the p6 domain [referred to as Fyn(10)Gag(p6)Pol-HA]. All constructs contained inactive PR. The letter <b>m</b> indicates a myristoylation site, and <b>palm</b> indicates a palmitoylation site. (B and C) HeLa cells were transfected with GagPol-HA, Fyn(10)GagPol-HA, and Fyn(10)Gag(p6)Pol-HA. (B) Intracellular localization of GagPol-HA derivatives. Cells were immunostained with anti-HA antibody (green or red) and nuclei were stained with TO-PRO-3 (blue). All micrographs are shown at the same magnification. (C) Intracellular expression and viral particle production. The Gag-FLAG/Pol-HA construct was used as a positive control. Cells and purified viral particles were subjected to Western blotting using anti-HA antibody.</p

Membrane affinity and viral particle production of Gag with C-terminal extensions of noncognate proteins.

<p>(A) Schematic representation of the pNL43 derivatives in which the Pol region was replaced by noncognate proteins. The Pol region was replaced with ß-gal, GFP, and 4GFP [referred to as Gag(p6*)ß-gal, Gag(p6*)GFP, and Gag-4GFP, respectively]. The Gag(p6*)ß-gal and Gag(p6*)GFP contained the same frameshift mutation as described before. (B-D) HeLa cells were transfected with GagPol-HA, Gag(p6*)ß-gal, Gag(p6*)GFP, Gag-4GFP, and Gag-FLAG/Pol-HA. (B) Membrane affinity. At 24 hr post-transfection, cells were subjected to membrane flotation centrifugation followed by Western blotting using anti-p24 antibody. (C) Intracellular localization. The cells transfected with GagPol-HA or Gag(p6*)ß-gal were immunostained with anti-p24 antibody (green), and nuclei were stained with TO-PRO-3 (blue). All micrographs are shown at the same magnification. In each sample, approximately 100 antigen-positive cells (from 3 or 4 independent experiments) were subjected to analysis of their distribution patterns. (D) Intracellular expression and viral particle production. Cells and purified viral particles were subjected to Western blotting using anti-p24 antibody. Gag-FLAG/Pol-HA was used as a positive control.</p