Modularity and evolutionary constraints in a baculovirus gene regulatory network

Abstract Background The structure of regulatory networks remains an open question in our understanding of complex biological systems. Interactions during complete viral life cycles present unique opportunities to understand how host-parasite network take shape and behave. The Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is a large double-stranded DNA virus, whose genome may encode for 152 open reading frames (ORFs). Here we present the analysis of the ordered cascade of the AgMNPV gene expression. Results We observed an earlier onset of the expression than previously reported for other baculoviruses, especially for genes involved in DNA replication. Most ORFs were expressed at higher levels in a more permissive host cell line. Genes with more than one copy in the genome had distinct expression profiles, which could indicate the acquisition of new functionalities. The transcription gene regulatory network (GRN) for 149 ORFs had a modular topology comprising five communities of highly interconnected nodes that separated key genes that are functionally related on different communities, possibly maximizing redundancy and GRN robustness by compartmentalization of important functions. Core conserved functions showed expression synchronicity, distinct GRN features and significantly less genetic diversity, consistent with evolutionary constraints imposed in key elements of biological systems. This reduced genetic diversity also had a positive correlation with the importance of the gene in our estimated GRN, supporting a relationship between phylogenetic data of baculovirus genes and network features inferred from expression data. We also observed that gene arrangement in overlapping transcripts was conserved among related baculoviruses, suggesting a principle of genome organization. Conclusions Albeit with a reduced number of nodes (149), the AgMNPV GRN had a topology and key characteristics similar to those observed in complex cellular organisms, which indicates that modularity may be a general feature of biological gene regulatory networks.

Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells

Characterization of Human Respiratory Syncytial Virus (HRSV) protein interactions with host cell components is crucial to devise antiviral strategies. Viral nucleoprotein, phosphoprotein and matrix protein genes were optimized for human codon usage and cloned into expression vectors. HEK-293T cells were transfected with these vectors, viral proteins were immunoprecipitated, and co-immunoprecipitated cellular proteins were identified through mass spectrometry. Cell proteins identified with higher confidence scores were probed in the immunoprecipitation using specific antibodies. The results indicate that nucleoprotein interacts with arginine methyl-transferase, methylosome protein and Hsp70. Phosphoprotein interacts with Hsp70 and tropomysin, and matrix with tropomysin and nucleophosmin. Additionally, we performed immunoprecipitation of these cellular proteins in cells infected with HRSV, followed by detection of co-immunoprecipitated viral proteins. The results indicate that these interactions also occur in the context of viral infection, and their potential contribution for a HRSV replication model is discussed.

Proline dehydrogenase regulates redox state and respiratory metabolism in Trypanosoma Cruzi

Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ(1)-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a Km of 16.58±1.69 µM and a Vmax of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle.

Meccanismi evolutivi dei parapoxvirus: caratterizzazione genomica di pseudocowpoxvirus e messa a punto di sistemi per lo studio delle ricombinazioni

The Poxviruses are a family of double stranded DNA (dsDNA) viruses that cause disease in many species, both vertebrate and invertebrate. Their genomes range in size from 135 to 365 kbp and show conservation in both organization and content. In particular, the central genomic regions of the chordopoxvirus subfamily (those capable of infecting vertebrates) contain 88 genes which are present in all the virus species characterised to date and which mostly occur in the same order and orientation. In contrast, however, the terminal regions of the genomes frequently contain genes that are species or genera-specific and that are not essential for the growth of the virus in vitro but instead often encode factors with important roles in vivo including modulation of the host immune response to infection and determination of the host range of the virus. The Parapoxviruses (PPV), of which Orf virus is the prototypic species, represent a genus within the chordopoxvirus subfamily of Poxviridae and are characterised by their ability to infect ruminants and humans. The genus currently contains four recognised species of virus, bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) both of which infect cattle, orf virus (OV) that infects sheep and goats, and parapoxvirus of red deer in New Zealand (PVNZ). The ORFV genome has been fully sequenced, as has that of BPSV, and is ~138 kb in length encoding ~132 genes. The vast majority of these genes allow the virus to replicate in the cytoplasm of the infected host cell and therefore encode proteins involved in replication, transcription and metabolism of nucleic acids. These genes are well conserved between all known genera of poxviruses. There is however another class of genes, located at either end of the linear dsDNA genome, that encode proteins which are non-essential for replication and generally dictate host range and virulence of the virus. The non-essential genes are often the most variable within and between species of virus and therefore are potentially useful for diagnostic purposes. Given their role in subverting the host-immune response to infection they are also targets for novel therapeutics. The function of only a relatively small number of these proteins has been elucidated and there are several genes whose function still remains obscure principally because there is little similarity between them and proteins of known function in current sequence databases. It is thought that by selectively removing some of the virulence genes, or at least neutralising the proteins in some way, current vaccines could be improved. The evolution of poxviruses has been proposed to be an adaptive process involving frequent events of gene gain and loss, such that the virus co-evolves with its specific host. Gene capture or horizontal gene transfer from the host to the virus is considered an important source of new viral genes including those likely to be involved in host range and those enabling the virus to interfere with the host immune response to infection. Given the low rate of nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in poxviruses is intimately linked to DNA replication with both viral and cellular proteins participate in this recombination-dependent replication. It has been shown, in other poxvirus genera, that recombination between isolates and perhaps even between species does occur, thereby providing another mechanism for the acquisition of new genes and for the rapid evolution of viruses. Such events may result in viruses that have a selective advantage over others, for example in re-infections (a characteristic of the PPV), or in viruses that are able to jump the species barrier and infect new hosts. Sequence data related to viral strains isolated from goats suggest that possible recombination events may have occurred between OV and PCPV (Ueda et al. 2003). The recombination events are frequent during poxvirus replication and comparative genomic analysis of several poxvirus species has revealed that recombinations occur frequently on the right terminal region. Intraspecific recombination can occur between strains of the same PPV species, but also interspecific recombination can happen depending on enough sequence similarity to enable recombination between distinct PPV species. The most important pre-requisite for a successful recombination is the coinfection of the individual host by different virus strains or species. Consequently, the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology. At present there are no available data on the replication dynamics of PPV in permissive and non permissive hosts and reguarding co-infetions there are no information on the interference mechanisms occurring during the simultaneous replication of viruses of different species. This work has been carried out to set up permissive substrates allowing the replication of different PPV species, in particular keratinocytes monolayers and organotypic skin cultures. Furthermore a method to isolate and expand ovine skin stem cells was has been set up to indeep further aspects of viral cellular tropism during natural infection. The study produced important data to elucidate the replication dynamics of OV and PCPV virus in vitro as well as the mechanisms of interference that can arise during co-infection with different viral species. Moreover, the analysis carried on the genomic right terminal region of PCPV 1303/05 contributed to a better knowledge of the viral genes involved in host interaction and pathogenesis as well as to locate recombination breakpoints and genetic homologies between PPV species. Taken together these data filled several crucial gaps for the study of interspecific recombinations of PPVs which are thought to be important for a better understanding of the viral evolution and to improve the biosafety of antiviral therapy and PPV-based vectors.

Untersuchungen über Wechselwirkungen zwischen Proteinen der Leber und dem großen Hüllprotein des Hepatitis-B-Virus

Zusammenfassung:Im Infektionszyklus des Hepatitis-B-Virus spielt das große L-Hüllprotein mit seiner einzigartigen PräS1-Domäne eine zentrale Rolle. Es vermittelt die Bindung und Aufnahme in die Leberzelle, die Verpackung der Nukleokapside in die Virushülle, die Regulation der cccDNA-Amplifikation und eine transkriptionelle Aktivierung in der Wirtszelle. Zur Erfüllung seiner vielfältigen Aufgaben benötigt das L-Protein Unterstützung durch Wirtzellfaktoren, von denen einige im Rahmen dieser Untersuchung durch Verwendung von PräS1-Konstrukten als Fängerproteine im Hefe-Zwei-Hybrid-System identifiziert wurden. Mehrere Klone, die im Hefe-Zwei-Hybrid-Test mit dem C-terminalen PräS1-Fängerprotein (Aminosäure 44-108) isoliert worden waren, enthielten Teile der cDNA von gamma2-Adaptin, einem mutmaßlichen Mitglied der Clathrin-Adaptor-Proteine. Diese sind für intrazelluläre Membrantransportprozesse mittels clathrinumhüllter Vesikel verantwortlich. Unter den interagierenden Klonen, die mit dem N-terminalen Konstrukt des L-Proteins (Aminosäure 1-70) isoliert worden waren, befand sich überproportional häufig eine cDNA, die der schweren Kette H4 der Inter-Alpha-Trypsin-Inhibitor-Familie homolog war. H4 besitzt vermutlich bei der 'Akute-Phase-Reaktion', die Entzündungen folgt, und bei der Stabilisierung der extrazellulären Matrix physiologische Bedeutung. Weitere Klone kodierten für die Serinprotease C1r. Diese ist Bestandteil des C1-Komplex, der ersten Komponente des klassischen Komplementsystems. Die Spezifität der Bindung zwischen den positiven Klonen und der PräS1-Domäne wurde in weiteren biochemischen Interaktionstests bestätigt, sodaß H4, C1r und gamma2-Adaptin als Wirtszellfaktoren in der Physiologie des Hepatitis-B-Virus wahrscheinlich eine Rolle spielen.Abstract:Little is known about host cell factors necessary for hepatitis B virus assembly and infectivity. Central to virogenesis is the large L envelope protein that mediates hepatocyte receptor binding, envelopment of viral capsids, regulation of supercoiled DNA amplification and transcriptional transactivation. To assess its multiple functions and host-protein assistance involved, we here initiated a yeast two-hybrid screen using the L-specific preS1 domain as bait to screen a human liver cDNA library for L-interacting proteins. One of the most prominent cDNAs interacting with aminoacid sequence 44-108 of L-protein encodes for gamma2-adaptin, a novel clathrin adaptor-related protein responsible for protein sorting and trafficking. Among the clones interacting with the N-terminal construct of L-protein (aminoacid sequence 1-70), a frequently isolated cDNA corresponds to the gene for inter-alpha-trypsin family heavy chain H4, likely to be involved in acute inflammatory phase response and stabilization of extracellular matrices. Some other interacting clones were found to carry the cDNA for the serine protease C1r, a subunit of the C1 complex which initiates the classical complement cascade. The specificity of the interaction between the positive clones and the preS1 domain was further confirmed in independent biochemical experiments. Taken together, the results suggest a role for H4, C1r and gamma2-adaptin as host-cell factors in L-mediated process of viral biogenesis and/or pathogenesis.

Funktionsanalyse von Gamma2-Adaptin: ein zellulärer Adaptor der Hepatitis-B-Virus-Morphogenese

Im Replikationszyklus umhüllter Viren entstehen neue Viruspartikel durch die Knospung an Membranen der Wirtszelle. An diesem Prozess sind verschiedene zelluläre Faktoren und Mechanismen beteiligt, speziell die ESCRT-Proteinkomplexe, welche die Vesikelbildung an den MVBs steuern. Auch bei HBV ist davon auszugehen, dass Komponenten der Wirtszelle an der Umhüllung und Freisetzung der Virionen beteiligt sind, allerdings sind diese noch weitgehend unbekannt. Ziel dieser Arbeit war es daher, die zellulären Faktoren genauer zu charakterisieren und ihre Funktion bei der Virusumhüllung aufzuklären. Den Ausgangspunkt für die hier durchgeführten Untersuchungen bildeten vorangegangene Arbeiten, in denen die spezifische Interaktion des L-Hüllproteins von HBV mit g2-Adaptin nachgewiesen werden konnte. Diese ist für die Morphogenese von HBV essentiell, allerdings ist die zelluläre ebenso wie die virusspezifische Funktion von g2-Adaptin bislang unbekannt. Im Rahmen dieser Arbeit sollte daher untersucht werden, wo und wie g2-Adaptin in der Zelle funktionell ist, um daraus Rückschlüsse auf die Vorgänge bei der Morphogenese von HBV ziehen zu können. Die Grundlage für die Charakterisierung von g2-Adaptin bildete seine Ähnlichkeit zu zellulären Clathrin-Adaptorproteinen. So konnte hier gezeigt werden, dass auch g2-Adaptin ein Clathrin-Bindungsmotiv besitzt, welches eine Interaktion mit Clathrin ermöglicht. Außerdem konnte ein Ubiquitin-Interaktions-Motiv (UIM) identifiziert werden, das die Bindung an ubiquitinierte Proteine vermittelt. Diese Beobachtung deutet darauf hin, dass g2-Adaptin zu einer Gruppe monomerer Adaptorproteine zählen könnte, welche als Ubiquitin-Rezeptoren in der Zelle funktionell sind. Die folgenden Analysen zeigten eine weitere Gemeinsamkeit, da auch g2-Adaptin selbst durch Ubiquitin modifiziert wird, wobei die Ubiquitinierung von einem intakten UIM abhängt. Dieser als Coupled Monoubiquitination bezeichnete Prozess wird hierbei durch die Ubiquitin-Ligase Nedd4 vermittelt, die direkt mit g2-Adaptin interagiert. Dabei konnte nachgewiesen werden, dass die C2-Domäne von Nedd4 ebenfalls mit Ubiquitin modifiziert ist, wodurch der Kontakt zum UIM von g2-Adaptin erfolgt. Die meisten der bislang bekannten Ubiquitin-bindenden Adaptorproteine, spielen bei der Vesikelentstehung an verschiedenen zellulären Membranen eine Rolle, wo sie an der Sortierung der vorwiegend ubiquitinierten Membranproteine beteiligt sind und zelluläre Komponenten rekrutieren, welche die Vesikelabschnürung vermitteln. Die Adaptorproteine sind dabei meist mit der jeweiligen Membran assoziiert, was auch für g2-Adaptin nachgewiesen werden konnte. Diese Membranbindung wird durch den N-terminalen Proteinbereich von g2-Adaptin vermittelt und erfolgt unabhängig von den Ubiquitin-bindenden Eigenschaften und von Nedd4. Allerdings scheint die Ubiquitin-Modifikation von g2-Adaptin ausschließlich in membrangebundener Form zu erfolgen. An welchen Membranen g2-Adaptin lokalisiert ist, wurde in Immunfluoreszenzstudien untersucht, wobei eine enge Assoziation von g2-Adaptin mit späten Endosomen bzw. MVBs zu beobachten war. Bei weiteren Analysen konnte auch ein funktioneller Einfluss auf die Vesikelentstehung an den MVBs nachgewiesen werden, da durch die Depletion von g2-Adaptin stark vergrößerte, defekte MVBs induziert wurden. Dies deutet darauf hin, dass g2-Adaptin als Ubiquitin-Rezeptor an diesen Prozessen beteiligt sein könnte. Ebenso wie andere Adaptorproteine könnte es hier an die Cargo-Proteine binden, diese durch den Kontakt zu Clathrin lokal konzentrieren und die Vesikelabschnürung durch die Rekrutierung der MVB-Maschinerie vermitteln. Möglicherweise stellt g2-Adaptin hierbei den bislang nicht identifizierten Adaptor dar, der die Verbindung zwischen Nedd4 und der MVB-Kaskade herstellt. Eine ähnliche Funktion für g2-Adaptin ist auch bei der Morphogenese von HBV denkbar. Aufgrund der durchgeführten Lokalisationsstudien ist anzunehmen, dass die Umhüllung der HBV-Partikel direkt an den MVBs erfolgt. Vermutlich bindet g2-Adaptin hier an das L-Hüllprotein, wobei es durch die Rekrutierung von Clathrin zu einer lokalen Anreicherung der Hüllproteine kommt. g2-Adaptin interagiert zudem in UIM-abhängiger Weise mit dem Nukleokapsid, wobei der Kontakt direkt erfolgen könnte oder durch die Ubiquitin-Ligase Nedd4 vermittelt wird, welche über eine Late-Domäne ebenfalls mit dem Nukleokapsid verbunden ist. Anscheinend gelangt das Nukleokapsid durch den Einfluss von g2-Adaptin und Nedd4 zum Ort der Virusmorphogenese, wo die eigentliche Umhüllung und die Abschnürung der Viruspartikel erfolgen. Vermutlich sind auch hier Komponenten der MVB-Maschinerie beteiligt, die womöglich durch g2-Adaptin rekrutiert werden.

Charakterisierung zellulärer Interaktionspartner des großen Hüllproteins des Hepatitis-B-Virus

Im Mittelpunkt dieser Arbeit stand das große L-Hüllprotein (L) des Hepatitis B - Virus. L bildet eine ungewöhnliche duale Topologie in der ER-Membran aus, welche auch im reifen Viruspartikel erhalten bleibt. In einem partiellen, posttranslationalen Reifungsprozess wird die sogenannte PräS-Region von der zytosolischen Seite der Membran aus in das ER-Lumen transloziert. Aufgrund seiner dualen Topologie und der damit verbundenen Multifunktionalität übernimmt L eine Schlüsselfunktion im viralen Lebenszyklus. Ein Schwerpunkt dieser Arbeit lag deshalb darin, neue zelluläre Interaktionspartner des L-Hüllproteins zu identifizieren. Ihre Analyse sollte helfen, das Zusammenspiel des Virus mit der Wirtszelle besser zu verstehen. Hierfür wurde das Split - Ubiquitin Hefe - Zwei - Hybrid System eingesetzt, das die Interaktionsanalyse von Membranproteinen und Membran-assoziierten Proteinen ermöglicht. Zwei der neu identifizierten Interaktionspartner, der v-SNARE Bet1 und Sec24A, die Cargo-bindende Untereinheit des CoPII-vermittelten vesikulären Transports, wurden weitergehend im humanen Zellkultursystem untersucht. Sowohl für Bet1 als auch für Sec24A konnte die Interaktion mit dem L-Hüllprotein bestätigt und der Bindungsbereich eingegrenzt werden. Die Depletion des endogenen Bet1 reduzierte die Freisetzung L-haltiger, nicht aber S-haltiger subviraler Partikel (SVP) deutlich. Im Gegensatz zu Bet1 interagierte Sec24A auch mit dem mittleren M- und kleinen S-Hüllprotein von HBV. Die Inhibition des CoPII-vermittelten vesikulären Transportweges durch kombinierte Depletion der vier Sec24 Isoformen blockierte die Freisetzung sowohl L- als auch S-haltiger SVP. Dies bedeutet, dass die HBV - Hüllproteine das ER CoPII-vermittelt verlassen, wobei sie aktiv Kontakt zur Cargo-bindenden Untereinheit Sec24A aufnehmen. Der effiziente Export der Hüllproteine aus dem ER ist für die Virusmorphogenese und somit für den HBV - Lebenszyklus essentiell. rnEin weiterer Schwerpunkt dieser Arbeit basierte auf der Interaktion des L-Hüllproteins mit dem ER-luminalen Chaperon BiP. In der vorliegenden Arbeit wurde überprüft, ob BiP, ähnlich wie das zytosolische Chaperon Hsc70, an der Ausbildung der dualen Topologie des L-Hüllproteins beteiligt ist. Hierfür wurde BiP durch die ektopische Expression seiner Ko-Chaperone BAP und ERdj4 in seiner Substrat-bindenen Kapazität manipuliert. ERdj4, ein Mitglied der Hsp40 - Proteinfamilie, stimuliert die ATPase-Aktivität von BiP, was die Substratbindung stabilisiert. Der Nukleotid - Austauschfaktor BAP hingegen vermittelt die Auflösung des BiP - Substrat - Komplexes. Die Auswirkung der veränderten in vivo-Aktivität von BiP auf die posttranslationale PräS-Translokation wurde mit Proteaseschutz - Versuchen untersucht. Die ektopische Expression des positiven als auch des negativen Regulators von BiP resultierte in einer drastischen Reduktion der posttranslationalen PräS-Translokation. Ein vergleichbarer Effekt wurde nach Manipulation des BiP ATPase - Zyklus durch Depletion der zellulären ATP - Konzentration beobachtet. Dies spricht dafür, dass das ER-luminale Chaperon BiP, zusammen mit Hsc70, eine zentrale Rolle in der Ausbildung der dualen Topologie des L-Hüllproteins spielt. rnZwei weitere Proteine, Sec62 und Sec63, die sich für die posttranslationale Translokation in der Hefe als essentiell erwiesen haben, wurden in die Analyse der dualen Topologie des L-Hüllproteins einbezogen. Interessanterweise konnte eine rein luminale Ausrichtung der PräS-Region nach kombinierter Depletion des endogenen Sec62 und Sec63 beobachtet werden. Dies deutet an, dass sowohl Sec62 als auch Sec63 an der Ausbildung der dualen Topologie des L-Hüllproteins beteiligt sind. In Analogie zur Posttranslokation der Hefe könnte Sec62 als Translokon-assoziierter Rezeptor für Substrate der Posttranslokation, und damit der PräS-Region, dienen. Sec63 könnte mit seiner J-Domäne BiP zum Translokon rekrutieren und daraufhin dessen Substrat-bindende Aktivität stimulieren. BiP würde dann, einer molekularen Ratsche gleich, die PräS-Region durch wiederholtes Binden und Freisetzen aktiv in das ER-Lumen hereinziehen, bis eine stabile duale Topologie des L-Hüllproteins ausgebildet ist. Die Bedeutung von Sec62 und Sec63 für den HBV - Lebenszyklus wird dadurch untermauert, dass sowohl die ektopische Expression als auch die Depletion des endogenen Sec63 die Freisetzung L-haltiger SVP deutlich reduziert. rn

Transcriptional responses of the Helicobacter pylori cag pathogenicity island

The severity of Helicobacter pylori infections largely depends on the genetic diversity of the infecting strain, and particularly on the presence of the cag pathogenicity island (cag-PAI). This virulence locus encodes a type-IV secretion system able to translocate in the host cell at least the cag-encoded toxin CagA and peptidoglycan fragments, that together are responsible for the pathogenic phenotype in the host. Little is known about the bacterial regulators that underlie the coordinated expression of cag gene products, needed to assemble a functional secretion system apparatus. To fill this gap, a comprehensive analysis of the transcriptional regulation of the cag-PAI operons was undertaken. To pursue this goal, a robust tool for the analysis of gene expression in H. pylori was first implemented. A bioluminescent reporter system based on the P. luminescens luxCDABE operon was constructed and validated by comparisons with transcriptional analyses, then it was systematically used for the comprehensive study and mapping of the cag promoters. The identification of bona fide cag promoters had permitted to pinpoint the set of cag transcriptional units of the PAI. The responses of these cag transcriptional units to metabolic stress signals were analyzed in detail, and integrated with transcription studies in deletion mutants of important H. pylori virulence regulators and protein-DNA interaction analyses to map the binding sites of the regulators. Finally, a small regulatory RNA cncR1 encoded by the cag-PAI was identified, and the 5’- and 3’-ends of the molecule were mapped by primer extension analyses, northern blot and studies with lux reporter constructs. To identify regulatory effects exerted by cncR1 on the H. pylori gene expression, the cncR1 knock out strain was derived and compared to the parental wild type strain by a macroarray approach. Results suggest a negative effect exerted by cncR1 on the regulome of the alternative sigma54 factor.

An interferon regulatory factor element controls the major immune modulator of murine cytomegalovirus

Immune modulation by herpesviruses, such as cytomegalovirus, is critical for the establishment of acute and persistent infection confronting a vigorous antiviral immune response of the host. Therefore, the action of immune-modulatory proteins has long been the subject of research, with the final goal to identify new strategies for antiviral therapy.rnIn the case of murine cytomegalovirus (mCMV), the viral m152 protein has been identified to play a major role in targeting components of both the innate and the adaptive immune system in terms of infected host-cell recognition in the effector phase of the antiviral immune response. On the one hand, it inhibits cell surface expression of RAE-1 and thereby prevents ligation of the activating natural killer (NK)-cell receptor NKG2D. On the other hand, it decreases cell surface expression of peptide-loaded MHC class I molecules thereby preventing antigen presentation to CD8 T cells. Ultimately, the outcome of CMV infection is determined by the interplay between viral and cellular factors.rnIn this context, the work presented here has revealed a novel and intriguing connection between viral m152 and cellular interferon (IFN), a key cytokine of the immune system: rnthe m152 promoter region contains an interferon regulatory factor element (IRFE) perfectly matching the consensus sequence of cellular IRFEs.rnThe biological relevance of this regulatory element was first suggested by sequence comparisons revealing its evolutionary conservation among various established laboratory strains of mCMV and more recent low-passage wild-derived virus isolates. Moreover, search of the mCMV genome revealed only three IRFE sites in the complete sequence. Importantly, the functionality of the IRFE in the m152 promoter was confirmed with the use of a mutant virus, representing a functional deletion of the IRFE, and its corresponding revertant virus. In particular, m152 gene expression was found to be inhibited in an IRFE-dependent manner in infected cells. Essentially, this inhibition proved to have a severe impact on the immune-modulatory function of m152, first demonstrated by a restored direct antigen presentation on infected cells for CD8 T-cell activation. Even more importantly, this effect of IRFE-mediated IFN signaling was validated in vivo by showing that the protective antiviral capacity of adoptively-transferred, antigen-specific CD8 T cells is also significantly restored by the IRFE-dependent inhibition of m152. Somewhat curious and surprising, the decrease in m152 protein simultaneously prevented an enhanced activation of NK cells in acute-infected mice, apparently independent of the RAE-1/NKG2D ligand/receptor interaction but rather due to reduced ‘missing-self’ recognition.rnTaken together, this work presents a so far unknown mechanism of IFN signaling to control mCMV immune modulation in acute infection.rnrn

Differential roles for endothelial ICAM-1, ICAM-2, and VCAM-1 in shear-resistant T cell arrest, polarization, and directed crawling on blood-brain barrier endothelium

Endothelial ICAM-1 and ICAM-2 were shown to be essential for T cell diapedesis across the blood-brain barrier (BBB) in vitro under static conditions. Crawling of T cells prior to diapedesis was only recently revealed to occur preferentially against the direction of blood flow on the endothelial surface of inflamed brain microvessels in vivo. Using live cell-imaging techniques, we prove that Th1 memory/effector T cells predominantly crawl against the direction of flow on the surface of BBB endothelium in vitro. Analysis of T cell interaction with wild-type, ICAM-1-deficient, ICAM-2-deficient, or ICAM-1 and ICAM-2 double-deficient primary mouse brain microvascular endothelial cells under physiological flow conditions allowed us to dissect the individual contributions of endothelial ICAM-1, ICAM-2, and VCAM-1 to shear-resistant T cell arrest, polarization, and crawling. Although T cell arrest was mediated by endothelial ICAM-1 and VCAM-1, T cell polarization and crawling were mediated by endothelial ICAM-1 and ICAM-2 but not by endothelial VCAM-1. Therefore, our data delineate a sequential involvement of endothelial ICAM-1 and VCAM-1 in mediating shear-resistant T cell arrest, followed by endothelial ICAM-1 and ICAM-2 in mediating T cell crawling to sites permissive for diapedesis across BBB endothelium.

Characterisation of gp34, a GPI-anchored protein expressed by schizonts of Theileria parva and T. annulata

Using bioinformatics tools, we searched the predicted Theileria annulata and T. parva proteomes for putative schizont surface proteins. This led to the identification of gp34, a GPI-anchored protein that is stage-specifically expressed by schizonts of both Theileria species and is downregulated upon induction of merogony. Transfection experiments in HeLa cells showed that the gp34 signal peptide and GPI anchor signal are also functional in higher eukaryotes. Epitope-tagged Tp-gp34, but not Ta-gp34, expressed in the cytosol of COS-7 cells was found to localise to the central spindle and midbody. Overexpression of Tp-gp34 and Ta-gp34 induced cytokinetic defects and resulted in accumulation of binucleated cells. These findings suggest that gp34 could contribute to important parasite-host interactions during host cell division.

Structural basis for the regulation of cysteine-protease activity by a new class of protease inhibitors in Plasmodium

Plasmodium cysteine proteases are essential for host-cell invasion and egress, hemoglobin degradation, and intracellular development of the parasite. The temporal, site-specific regulation of cysteine-protease activity is a prerequisite for survival and propagation of Plasmodium. Recently, a new family of inhibitors of cysteine proteases (ICPs) with homologs in at least eight Plasmodium species has been identified. Here, we report the 2.6 A X-ray crystal structure of the C-terminal, inhibitory domain of ICP from P. berghei (PbICP-C) in a 1:1 complex with falcipain-2, an important hemoglobinase of Plasmodium. The structure establishes Plasmodium ICP as a member of the I42 class of chagasin-like protease inhibitors but with large insertions and differences in the binding mode relative to other family members. Furthermore, the PbICP-C structure explains why host-cell cathepsin B-like proteases and, most likely, also the protease-like domain of Plasmodium SERA5 (serine-repeat antigen 5) are no targets for ICP.

Canine distemper virus infects canine keratinocytes and immune cells by using overlapping and distinct regions located on one side of the attachment protein

The morbilliviruses measles virus (MeV) and canine distemper virus (CDV) both rely on two surface glycoproteins, the attachment (H) and fusion proteins, to promote fusion activity for viral cell entry. Growing evidence suggests that morbilliviruses infect multiple cell types by binding to distinct host cell surface receptors. Currently, the only known in vivo receptor used by morbilliviruses is CD150/SLAM, a molecule expressed in certain immune cells. Here we investigated the usage of multiple receptors by the highly virulent and demyelinating CDV strain A75/17. We based our study on the assumption that CDV-H may interact with receptors similar to those for MeV, and we conducted systematic alanine-scanning mutagenesis on CDV-H throughout one side of the beta-propeller documented in MeV-H to contain multiple receptor-binding sites. Functional and biochemical assays performed with SLAM-expressing cells and primary canine epithelial keratinocytes identified 11 residues mutation of which selectively abrogated fusion in keratinocytes. Among these, four were identical to amino acids identified in MeV-H as residues contacting a putative receptor expressed in polarized epithelial cells. Strikingly, when mapped on a CDV-H structural model, all residues clustered in or around a recessed groove located on one side of CDV-H. In contrast, reported CDV-H mutants with SLAM-dependent fusion deficiencies were characterized by additional impairments to the promotion of fusion in keratinocytes. Furthermore, upon transfer of residues that selectively impaired fusion induction in keratinocytes into the CDV-H of the vaccine strain, fusion remained largely unaltered. Taken together, our results suggest that a restricted region on one side of CDV-H contains distinct and overlapping sites that control functional interaction with multiple receptors.

The distribution of E-cadherin expression in listeric rhombencephalitis of ruminants indicates its involvement in Listeria monocytogenes neuroinvasion

AIM: To investigate the expression of E-cadherin, a major host cell receptor for Listeria monocytogenes (LM) internalin A, in the ruminant nervous system and its putative role in brainstem invasion and intracerebral spread of LM in the natural disease. METHODS: Immunohistochemistry and double immunofluorescence was performed on brains, cranial nerves and ganglia of ruminants with and without natural LM rhombencephalitis using antibodies against E-cadherin, protein gene product 9.5, myelin-associated glycoprotein and LM. RESULTS: In the ruminant brain, E-cadherin is expressed in choroid plexus epithelium, meningothelium and restricted neuropil areas of the medulla, but not in the endothelium. In cranial nerves and ganglia, E-cadherin is expressed in satellite cells and myelinating Schwann cells. Expression does not differ between ruminants with or without listeriosis and does not overlap with the presence of microabscesses in the medulla. LM is observed in phagocytes, axons, Schwann cells, satellite cells and ganglionic neurones. CONCLUSION: Our results support the view that the specific ligand-receptor interaction between LM and host E-cadherin is involved in the neuropathogenesis of ruminant listeriosis. They suggest that oral epithelium and Schwann cells expressing E-cadherin provide a port of entry for free bacteria offering a site of primary intracellular replication, from where the bacterium may invade the axonal compartment by cell-to-cell spread. As E-cadherin expression in the ruminant central nervous system is weak, only very locally restricted and not related to the presence of microabscesses, it is likely that further intracerebral spread is independent of E-cadherin and relies primarily on axonal spread.

Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.