IFN-γ-Inducible Irga6 Mediates Host Resistance against Chlamydia trachomatis via Autophagy

Chlamydial infection of the host cell induces Gamma interferon (IFNγ), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNγ-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogen's elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNγ-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNγ, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg5−/− MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNγ-induced Atg5−/− cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6−/−) MEFs, in which chlamydial growth is enhanced, do not respond to IFNγ even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction

Molecular evolution of viral multifunctional proteins: the case of Potyvirus HC-Pro

[EN] Our knowledge on the mode of evolution of the multifunctional viral proteins remains incomplete. To tackle this problem, here, we have investigated the evolutionary dynamics of the potyvirus multifunctional protein HC-Pro, with particular focus on its functional domains. The protein was partitioned into the three previously described functional domains, and each domain was analyzed separately and assembled. We searched for signatures of adaptive evolution and evolutionary dependencies of amino acid sites within and between the three domains using the entire set of available potyvirus sequences in GenBank. Interestingly, we identified strongly significant patterns of co-occurrence of adaptive events along the phylogenetic tree in the three domains. These patterns suggest that Domain I, whose main function is to mediate aphid transmission, has likely been coevolving with the other two domains, which are involved in different functions but all requiring the capacity to bind RNA. By contrast, episodes of positive selection on Domains II and III did not correlate, reflecting a trade-off between their evolvability and their evolutionary dependency likely resulting from their functional overlap. Covariation analyses have identified several groups of amino acids with evidence of concerted variation within each domain, but interdomain significant covariations were only found for Domains II and III, further reflecting their functional overlappingThis work was supported by grants BFU2012-30805 (SFE) and BFU2012-36346 (MAF) from the Spanish Direccio´n General de Investigacio´n Cientı´fica y Te´cnica and by an EMBO Short-Term Fellowship and the Mentoring Program from the Foundation for Polish Science (BHJ).Hasiów-Jaroszewska, B.; Fares Riaño, MA.; Elena Fito, SF. (2014). Molecular evolution of viral multifunctional proteins: the case of Potyvirus HC-Pro. Journal of Molecular Evolution. 78(1):75-86. https://doi.org/10.1007/s00239-013-9601-0S7586781Adams MJ, Antoniw JF, Beaudoin F (2005) Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Mol Plant Pathol 6:471–487Atreya CD, Atryea P, Thornbury DW, Pirone TP (1992) Site-directed mutations in the potyvirus HC-Pro gene affect helper component activity, virus accumulation and symptoms expression in infected tobacco plants. Virology 191:106–111Blanc S, López-Moya JJ, Wang R, García-Lampasona S, Thornbury DW, Pirone TP (1997) A specific interaction between coat protein and helper component correlates with aphid transmission of a potyvirus. Virology 231:141–147Blanc S, Ammar ED, García-Lampasona S, Dolja VV, Llave C, Baker J, Pirone TP (1998) Mutations in the potyvirus helper component protein: effects on interactions with virions and aphid stylets. J Gen Virol 79:3119–3122Cantó T, López-Moya JJ, Serra-Yodi MT, Díaz-Ruiz JR, López-Abella D (1995) Different helper component mutations associated with lack of aphid transmissibility in two isolates of potato virus. Phytopathology 85:1519–1524Carrington JC, Freed DD, Sanders TC (1989) Autocatalytic processing of the potyvirus helper component proteinase in Escherichia coli and in vitro. J Virol 63:4459–4463Chung BY, Miller WA, Atkins JF, Firth AE (2008) An overlapping essential gene in the Potyviridae. Proc Natl Acad Sci USA 105:5897–5902Cronin S, Verchot J, Haldeman-Cahill R, Schaad MC, Carrington JC (1995) Long distance movement factor: a transport function of the potyvirus helper component-proteinase. Plant Cell 7:549–559Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl Acids Res 32:1792–1797Elena SF, Rodrigo G (2012) Towards and integrated molecular model of plant-virus interactions. Curr Opin Virol 2:713–718Fares MA (2004) SWAPSC: sliding-window analysis procedure to detect selective constraints. Bioinformatics 20:2867–2868Fares MA, McNally D (2006) CAPS: coevolution analysis using protein sequences. Bioinformatics 22:2821–2822Fares MA, Travers AA (2006) A novel method for detecting intramolecular coevolution: adding a further dimension to selective constrains analyses. Genetics 173:9–23Fares MA, Elena SF, Ortiz J, Moya A, Barrio E (2002) A sliding window-based method to detect selective constraints in protein-coding genes and its application to RNA viruses. J Mol Evol 55:509–521Gibbs A, Ohshima K (2010) Potyviruses and the digital revolution. Annu Rev Phytopathol 48:205–223Guo D, Mertis A, Saarma M (1999) Self-association and mapping of interaction domains of helper component of Potato virus A potyvirus. J Gen Virol 80:1127–1131Guo B, Lin J, Ye K (2011) Structure of the autocatalytic cysteine protease domain of potyvirus helper-component proteinase. J Biol Chem 286:21937–21943Haikonen T, Rajamäki ML, Tian YP, Valkonen JPT (2013) Mutation of a short variable region in HC-Pro protein of Potato virus A affects interactions with microtubule-associated protein and induces necrotic responses in tobacco. Mol Plant Microbe Interact 26:721–733Hall TA (1999) BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98Hughes AL (2009) Small effective population sizes and rare nonsynonymous variants in potyviruses. Virology 393:127–134Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195–202Kasschau KD, Carrington JC (1995) Requirement for HC-Pro processing during genome amplification of Tobacco etch potyvirus. Virology 209:268–273Kasschau KD, Carrington JC (2001) Long-distance movement and replication maintenance functions correlate with silencing suppression activity of potyviral HC-Pro. Virology 285:71–81Kasschau KD, Cronin S, Carrington JC (1997) Genome amplification and long-distance movement functions associated with the central domain of Tobacco etch potyvirus helper component-proteinase. Virology 228:251–262Kosakovsky Pond SL, Frost SDW (2005a) DATAMONKEY: rapid detection of selective pressure on individual sites of codon alignments. Bioinformatics 21:2531–2533Kosakovsky Pond SL, Frost SDW (2005b) Not so different after all: a comparison of methods for detecting amino acid sites under selection. Mol Biol Evol 22:1208–1222Kosakovsky Pond SL, Posada D, Gravenor MB, Woelk CH, Frost SDW (2006) Automated phylogenetic detection of recombination using a genetic algorithm. Mol Biol Evol 23:1891–1901Lakatos L, Csorba T, Pantaleo V, Chapman EJ, Carrington JC, Liu YP, Dojla VV, Calvino LF, López-Moya JJ, Burgyan J (2006) Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors. EMBO J 25:2768–2780Lalić J, Elena SF (2012) Magnitude and sign epistasis among deleterious mutations in a positive-sense plant RNA virus. Heredity 109:71–77Leigh JW, Susko E, Baumgartner M, Roger AJ (2008) Testing congruence in phylogenomic analysis. Syst Biol 57:104–115Li WH (1993) Unbiased estimation of the rates of synonymous and nonsynonymous substitution. J Mol Evol 36:96–99Llave C, Kasschau KD, Carrington JC (2000) Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. Proc Natl Acad Sci USA 97:13401–13406Maia S, Haenni AL, Bernardi F (1996) Potyviral HC-Pro: a multifunctional protein. J Gen Virol 77:1335–1341Martin DP, Lemey P, Lott M, Moulton V, Posada D, Lefeuvre P (2010) RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics 26:2462–2463Moroni E, Morra G, Colombo G (2012) Molecular dynamics simulations of Hsp90 with an eye to inhibitor design. Pharmaceuticals 5:944–962Peng YH, Kadoury D, Gaol-On A, Huet H, Wang Y, Raccah B (1998) Mutations in HC-Pro gene of Zucchini yellow mosaic potyvirus: effects on aphid transmission and binding to purified virions. J Gen Virol 79:897–904Plisson C, Drucker M, Blanc S, German-Retana S, Le Gall O, Thomas D, Bron P (2003) Structural characterization of HC-Pro a plant virus multifunctional protein. J Biol Chem 278:23753–23761Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817–818Revers F, Le Gall O, Candresse T, Maule J (1999) New advances in understanding the molecular biology of plant/potyvirus interaction. Mol Plant Microbe Interact 12:367–376Riechmann JL, Lain S, García JA (1992) Highlights and prospects of potyvirus molecular biology. J Gen Virol 73:1–16Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725–738Ruiz-Ferrer V, Boskovic J, Alfonso C, Rivas G, Llorca O, López-Abella D, López-Moya JJ (2005) Structural analysis of Tobacco etch potyvirus HC-pro oligomers involved in aphid transmission. J Virol 79:3758–3765Shiboleth YM, Haronsky E, Leibman D, Arazi T, Wassenegger M, Whitham SA, Gaba V, Gal-On A (2007) The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development. J Virol 81:13135–13148Smoot M, Ono K, Ruschelnski J, Wang PL, Ideker T (2011) CYTOSCAPE 2.8: new features for data integration and network visualization. Bioinformatics 27:431–432Syller J (2006) The roles and mechanisms of helper component proteins encoded by potyviruses and caulimoviruses. Physiol Mol Plant Pathol 67:119–130Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739Torres-Barceló C, Martín S, Daròs JA, Elena SF (2008) From hypo- to hypersuppression: effect of amino acid substitutions on the RNA-silencing suppressor activity of Tobacco etch potyvirus HC-Pro. Genetics 180:1039–1049Torres-Barceló C, Daròs JA, Elena SF (2010a) Compensatory molecular evolution of HC-Pro, an RNA-silencing suppressor from a plant RNA virus. Mol Biol Evol 27:543–551Torres-Barceló C, Daròs JA, Elena SF (2010b) HC-Pro hypo- and hypersuppressor mutants: differences in viral siRNA accumulation in vivo and siRNA binding activity in vitro. Arch Virol 155:251–254Urcuqui-Inchima S, Walter J, Drugeon G, German-Retans S, Haeni AL, Candresse T, Bernardi F, Le Gall O (1999) Potyvirus HC-Pro self-interaction in the yeast two hybrid system and delineation of the interaction domain involved. Virology 258:95–99Urcuqui-Inchima S, Maia IG, Arruda P, Haenni AL, Bernardi F (2000) Deletion mapping of the potyviral helper component-proteinase reveals two regions involved in RNA binding. Virology 268:104–111Urcuqui-Inchima S, Haenni AL, Bernardi F (2001) Potyvirus proteins: a wealth of functions. Virus Res 74:157–175Varrelmann M, Maiss E, Pilot R, Palkovics L (2007) Use of pentapeptide-insertion scanning mutagenesis for functional mapping of the Plum pox virus helper component proteinase suppressor of gene silencing. J Gen Virol 88:10051015Ward CW, Shukla DD (1991) Taxonomy of potyviruses: current problems and some solutions. Intervirology 32:269–296Wu S, Zhang Y (2007) LOMETS: a local meta-threading-server for protein structure prediction. Nucl Acids Res 35:3375–3382Yang Z, Bielawski JP (2000) Statistical methods for detecting molecular adaptation. Trends Ecol Evol 15:496–503Yap YK, Duangjit J, Panyim S (2009) N-terminal of Papaya ringspot virus type-W (PRSV-W) helper component proteinase (HC-Pro) is essential for PRSV systemic infection in zucchini. Virus Genes 38:461–467Zheng H, Yan F, Lu Y, Sun L, Lin L, Cai L, Hou M, Chen J (2010) Mapping the self-interaction domains of TuMV HC-pro and the subcellular localization of the protein. Virus Genes 42:110–11

Nuclear Translocation of β-Catenin during Mesenchymal Stem Cells Differentiation into Hepatocytes Is Associated with a Tumoral Phenotype

Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/β-catenin inactivation. Hepatocytes with nuclear translocation of β-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase β-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin β-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/β-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype

Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection

The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse.Deutscher Akademischer Austausch Dienst (DAAD); International Graduate School in Development Health and Disease (IGS-DHD); Deutsche For-schungsgemeinschaft (SFBs 635, 670, 680); Max-Planck-Gesellschaft (Max Planck Fellowship)

Platelet-Activating Factor Receptor Plays a Role in Lung Injury and Death Caused by Influenza A in Mice

Influenza A virus causes annual epidemics which affect millions of people worldwide. A recent Influenza pandemic brought new awareness over the health impact of the disease. It is thought that a severe inflammatory response against the virus contributes to disease severity and death. Therefore, modulating the effects of inflammatory mediators may represent a new therapy against Influenza infection. Platelet activating factor (PAF) receptor (PAFR) deficient mice were used to evaluate the role of the gene in a model of experimental infection with Influenza A/WSN/33 H1N1 or a reassortant Influenza A H3N1 subtype. The following parameters were evaluated: lethality, cell recruitment to the airways, lung pathology, viral titers and cytokine levels in lungs. The PAFR antagonist PCA4248 was also used after the onset of flu symptoms. Absence or antagonism of PAFR caused significant protection against flu-associated lethality and lung injury. Protection was correlated with decreased neutrophil recruitment, lung edema, vascular permeability and injury. There was no increase of viral load and greater recruitment of NK1.1+ cells. Antibody responses were similar in WT and PAFR-deficient mice and animals were protected from re-infection. Influenza infection induces the enzyme that synthesizes PAF, lyso-PAF acetyltransferase, an effect linked to activation of TLR7/8. Therefore, it is suggested that PAFR is a disease-associated gene and plays an important role in driving neutrophil influx and lung damage after infection of mice with two subtypes of Influenza A. Further studies should investigate whether targeting PAFR may be useful to reduce lung pathology associated with Influenza A virus infection in humans

Blood transcriptomics of drug-na\uefve sporadic Parkinson's disease patients

BACKGROUND: Parkinson's disease (PD) is a chronic progressive neurodegenerative disorder that is clinically defined in terms of motor symptoms. These are preceded by prodromal non-motor manifestations that prove the systemic nature of the disease. Identifying genes and pathways altered in living patients provide new information on the diagnosis and pathogenesis of sporadic PD. METHODS: Changes in gene expression in the blood of 40 sporadic PD patients and 20 healthy controls ("Discovery set") were analyzed by taking advantage of the Affymetrix platform. Patients were at the onset of motor symptoms and before initiating any pharmacological treatment. Data analysis was performed by applying Ranking-Principal Component Analysis, PUMA and Significance Analysis of Microarrays. Functional annotations were assigned using GO, DAVID, GSEA to unveil significant enriched biological processes in the differentially expressed genes. The expressions of selected genes were validated using RT-qPCR and samples from an independent cohort of 12 patients and controls ("Validation set"). RESULTS: Gene expression profiling of blood samples discriminates PD patients from healthy controls and identifies differentially expressed genes in blood. The majority of these are also present in dopaminergic neurons of the Substantia Nigra, the key site of neurodegeneration. Together with neuronal apoptosis, lymphocyte activation and mitochondrial dysfunction, already found in previous analysis of PD blood and post-mortem brains, we unveiled transcriptome changes enriched in biological terms related to epigenetic modifications including chromatin remodeling and methylation. Candidate transcripts as CBX5, TCF3, MAN1C1 and DOCK10 were validated by RT-qPCR. CONCLUSIONS: Our data support the use of blood transcriptomics to study neurodegenerative diseases. It identifies changes in crucial components of chromatin remodeling and methylation machineries as early events in sporadic PD suggesting epigenetics as target for therapeutic intervention