Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283-6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.This work was supported in part by grants GR09/0023 (A. N.), GR09/0039 (J. A. M.) and GR09/0040 (I. C.) from Instituto de Salud Carlos III under a special research programme on pandemic flu. Additionally, the Biología Viral Unit is supported currently by grant SAF2012-31217 from Plan Nacional I+D+i.S

Apoptosis, toll-like, RIG-I-like and NOD-like receptors are pathways jointly induced by diverse respiratory bacterial and viral pathogens

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here.The authors gratefully acknowledge financial support from the “CIBER de Enfermedades Respiratorias” (CIBERES), an initiative of the “Instituto de Salud Carlos III” (ISCIII), Spain. Research activities in the participating laboratories received further funding from the following sources: Centro Nacional de Microbiología, ISCIII, PI15CIII/00024 and MINECO (SAF2015- 67033-R); Centro Nacional de Biotecnología, MINECO (BFU2014-57797-R); Hospital Universitari Germans Trias I Pujol, Spanish Society of Pneumology and Thoracic Surgery (SEPAR 054/2011); Departamento de Bioquímica y Biología Molecular I, MINECO (SAF2015-65307-R); Centro de Investigaciones Biológicas, MINECO (SAF2012-39444-C01/02); Fundación de Investigación Sanitaria de las Islas Baleares, MINECO (SAF2012-39841); Instituto de Agrobiotecnología, MINECO (SAF2015-66520-R); Instituto de Química Física Rocasolano, MINECO (BFU2015-70052-R) and the Marie Curie Initial Training Network GLYCOPHARM (PITN-GA- 2012-317297). Subprograma Estatal de Formación (BES-2013- 065355)

Identification of Polo-like kinases as potential novel drug targets for influenza A virus

In recent years genome-wide RNAi screens have revealed hundreds of cellular factors required for influenza virus infections in human cells. The long-term goal is to establish some of them as drug targets for the development of the next generation of antivirals against influenza. We found that several members of the polo-like kinases (PLK), a family of serine/threonine kinases with well-known roles in cell cycle regulation, were identified as hits in four different RNAi screens and we therefore studied their potential as drug target for influenza. We show that knockdown of PLK1, PLK3, and PLK4, as well as inhibition of PLK kinase activity by four different compounds, leads to reduced influenza virus replication, and we map the requirement of PLK activity to early stages of the viral replication cycle. We also tested the impact of the PLK inhibitor BI2536 on influenza virus replication in a human lung tissue culture model and observed strong inhibition of virus replication with no measurable toxicity. This study establishes the PLKs as potential drug targets for influenza and contributes to a more detailed understanding of the intricate interactions between influenza viruses and their host cells

Nuclear and cytosolic hCLE-associated proteins.

<p>Table includes proteins where at least 2 non-redundant peptides were identified, with a FDR <1% at peptide level that are present exclusively in hCLE purified samples or have at least 3-fold enrichment of total number of confidently identified peptides in hCLE purified fractions compared to the control (ctr.) samples (marked with asterisk). Their molecular weight, the total number of confidently identified peptides and the number of non-redundant ones, as well as the protein Mascot scores, is shown.</p

hCLE/C14orf166 Associates with DDX1-HSPC117-FAM98B in a Novel Transcription-Dependent Shuttling RNA-Transporting Complex

<div><p>hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nuclear RNA metabolism and is present in cytoplasmic RNA granules involved in localized translation. Here we have studied whether hCLE shares common interactors in the nucleus and the cytosol, which could shed light on its participation in the sequential phases of RNA metabolism. Nuclear and cytoplasmic purified hCLE-associated factors were identified and proteins involved in mRNA metabolism, motor-related proteins, cytoskeletal and translation-related factors were found. Purified hCLE complexes also contain RNAs and as expected some hCLE-interacting proteins (DDX1, HSPC117, FAM98B) were found both in the nucleus and the cytoplasm. Moreover, endogenous hCLE fractionates in protein complexes together with DDX1, HSPC117 and FAM98B and silencing of hCLE down-regulates their nuclear and cytosolic accumulation levels. Using a photoactivatable hCLE-GFP protein, nuclear import and export of hCLE was observed indicating that hCLE is a shuttling protein. Interestingly, hCLE nuclear import required active transcription, as did the import of DDX1, HSPC117 and FAM98B proteins. The data indicate that hCLE probably as a complex with DDX1, HSPC117 and FAM98B shuttles between the nucleus and the cytoplasm transporting RNAs suggesting that this complex has a prominent role on nuclear and cytoplasmic RNA fate.</p></div

Transcription inhibition increases the cytosolic accumulation of hCLE, DDX1, HSPC117 and FAM98B.

<p>Cultured HEK293T cells were incubated in the absence or the presence of Actinomycin D (Act. D) during 1 h. Nuclear and cytoslic extracts were prepared and used for Western blot assays to detect the indicated proteins. (N); nuclear fraction, (C), cytosolic fraction. RNAP II and α-actin were used as markers for subcellular fractionation.</p

Transcription inhibition retains DDX1 and HSPC117 in the cytoplasm.

<p>Cultured HEK293T cells were incubated in the absence (Control) or the presence of Actynomicin D (Act. D) during 30 min, washed, fixed and processed for immunofluorescence using antibodies anti-hCLE, DDX1, HSPC117 and DAPI.</p

Nuclear and cytosolic hCLE-associated proteins.

<p>Table includes proteins where at least 2 non-redundant peptides were identified, with a FDR <1% at peptide level that are present exclusively in hCLE purified samples or have at least 3-fold enrichment of total number of confidently identified peptides in hCLE purified fractions compared to the control (ctr.) samples (marked with asterisk). Their molecular weight, the total number of confidently identified peptides and the number of non-redundant ones, as well as the protein Mascot scores, is shown.</p

hCLE forms dimers that are resistant to denaturing conditions.

<p>(A); Western blot against hCLE of total extracts of HEK293T cells using denaturing (left) or native conditions (right). (B); Diagram showing the scheme of the fusion protein hCLE-TAP with its tags for affinity purification. (C); left, HEK293T cells were transfected with the hCLE-TAP expressing plasmid and total cell extract was used for affinity purification, a sample of the purified protein was analyzed by silver staining in SDS-PAGE gels. Right; The hCLE-CBC purified protein was dialyzed and analyzed in non-denaturing gels followed by Western blot detection. (D). The hCLE-CBD purified protein was subjected to silver staining and bands corresponding to different sizes were excised and analyzed by MS-MS technique. The lines denote hCLE identification by MS-MS technique. (*), denotes hCLE monomer and (**) hCLE dimer.</p

Identification of hCLE as monomer and dimer in denaturing gel conditions.

<p><b><i>AC</i></b><b>:</b> Accession Number of the top protein from NCBInr protein Database (non-identical NCBI protein database).</p><p><b><i>MW (Mr):</i></b> Nominal molecular weight of each protein.</p><p><b><i>pI:</i></b> Calculated pI value.</p><p><b><i>Pept (MS[MSMS]):</i></b> number of matched peptides from the top scoring protein in peptide mass fingerprinting and number of MS/MS spectra that were matched to this protein.</p><p><b><i>Score:</i></b> Mascot protein score. This number reflects the combined scores of all observed mass spectra that can be matched to amino acid sequences within that protein. A higher score indicates a more confident match.</p><p><b><i>Seq Cov:</i></b> Percentage of the database protein sequence covered by matching peptides.</p