Oxidative damage in telomeric DNA disrupts recognition by TRF1 and TRF2

The ends of linear chromosomes are capped by protein–DNA complexes termed telomeres. Telomere repeat binding factors 1 and 2 (TRF1 and TRF2) bind specifically to duplex telomeric DNA and are critical components of functional telomeres. Consequences of telomere dysfunction include genomic instability, cellular apoptosis or senescence and organismal aging. Mild oxidative stress induces increased erosion and loss of telomeric DNA in human fibroblasts. We performed binding assays to determine whether oxidative DNA damage in telomeric DNA alters the binding activity of TRF1 and TRF2 proteins. Here, we report that a single 8-oxo-guanine lesion in a defined telomeric substrate reduced the percentage of bound TRF1 and TRF2 proteins by at least 50%, compared with undamaged telomeric DNA. More dramatic effects on TRF1 and TRF2 binding were observed with multiple 8-oxo-guanine lesions in the tandem telomeric repeats. Binding was likewise disrupted when certain intermediates of base excision repair were present within the telomeric tract, namely abasic sites or single nucleotide gaps. These studies indicate that oxidative DNA damage may exert deleterious effects on telomeres by disrupting the association of telomere-maintenance proteins TRF1 and TRF2

Mutational Analyses of the Influenza A Virus Polymerase Subunit PA Reveal Distinct Functions Related and Unrelated to RNA Polymerase Activity

Influenza A viral polymerase is a heterotrimeric complex that consists of PA, PB1, and PB2 subunits. We previously reported that a di-codon substitution mutation (G507A-R508A), denoted J10, in the C-terminal half of PA had no apparent effect on viral RNA synthesis but prevented infectious virus production, indicating that PA may have a novel role independent of its polymerase activity. To further examine the roles of PA in the viral life cycle, we have now generated and characterized additional mutations in regions flanking the J10 site from residues 497 to 518. All tested di-codon mutations completely abolished or significantly reduced viral infectivity, but they did so through disparate mechanisms. Several showed effects resembling those of J10, in that the mutant polymerase supported normal levels of viral RNA synthesis but nonetheless failed to generate infectious viral particles. Others eliminated polymerase activity, in most cases by perturbing the normal nuclear localization of PA protein in cells. We also engineered single-codon mutations that were predicted to pack near the J10 site in the crystal structure of PA, and found that altering residues K378 or D478 each produced a J10-like phenotype. In further studies of J10 itself, we found that this mutation does not affect the formation and release of virion-like particles per se, but instead impairs the ability of those particles to incorporate each of the eight essential RNA segments (vRNAs) that make up the viral genome. Taken together, our analysis identifies mutations in the C-terminal region of PA that differentially affect at least three distinct activities: protein nuclear localization, viral RNA synthesis, and a trans-acting function that is required for efficient packaging of all eight vRNAs

Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned

Oxidative damage in telomeric DNA disrupts recognition by TRF1 and TRF2.

The ends of linear chromosomes are capped by protein-DNA complexes termed telomeres. Telomere repeat binding factors 1 and 2 (TRF1 and TRF2) bind specifically to duplex telomeric DNA and are critical components of functional telomeres. Consequences of telomere dysfunction include genomic instability, cellular apoptosis or senescence and organismal aging. Mild oxidative stress induces increased erosion and loss of telomeric DNA in human fibroblasts. We performed binding assays to determine whether oxidative DNA damage in telomeric DNA alters the binding activity of TRF1 and TRF2 proteins. Here, we report that a single 8-oxo-guanine lesion in a defined telomeric substrate reduced the percentage of bound TRF1 and TRF2 proteins by at least 50%, compared with undamaged telomeric DNA. More dramatic effects on TRF1 and TRF2 binding were observed with multiple 8-oxo-guanine lesions in the tandem telomeric repeats. Binding was likewise disrupted when certain intermediates of base excision repair were present within the telomeric tract, namely abasic sites or single nucleotide gaps. These studies indicate that oxidative DNA damage may exert deleterious effects on telomeres by disrupting the association of telomere-maintenance proteins TRF1 and TRF2

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

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

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

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