Charakterisierung des Core-Proteins von Pestiviren

Die subzelluläre Lokalisierung und die RNS bindende Aktivität des Core-Proteins von Pestiviren wurden untersucht und folgende Ergebnisse erzielt: 1.- Das Gen des Core-Proteins von KSPV und BVDV wurde in einen prokaryotischen Expressionsvektor kloniert, das Core-Protein in Bakterien (E. coli) exprimiert und durch „Immobilized Metal Affinity Chromatography“ (IMAC) in hohem Maß gereinigt. Dieses Protein fand Anwendung als Antigen zur Herstellung von mAks und stellte die Basis eines RNS-Bindungsassays dar. 2.- Ein System zum Screening von Hybridom Überständen wurde entwickelt, mit dem acht anti Core-Protein mAk produzierende Hybridome isoliert wurden. Die Anwendung der hergestellten mAks ermöglichte die Detektion des Core-Proteins von 13 Pestiviren verschiedener Spezies [BVDV-1 (7 Stämme); BVDV-2 (2 Stämme); BDV (2 Stämme); KSPV; Giraffe] in infizierten Zellen und aufkonzentrierten Virionen. 3.- Ein Disulfid verbrücktes Core-Protein Homodimer wurde in Virionen von BVDV-2 Stämmen identifiziert und mittels reverser Genetik charakterisiert. Wie bei den Flaviviren kommt das Core-Protein in Virionen von Pestiviren sehr wahrscheinlich als Dimer vor. 4.- In infizierten Zellen kolokalisierte das Core-Protein mit dem endoplasmatischen Retikulum und mit den Mitochondrien. Auch wurde dieses Protein in Assoziation mit den „RNA-processing bodies“ („P-Bodies“) nachgewiesen, welche eine wichtige Rolle bei der Regulation der Translation spielen. Eine Kolokalisierung des Core-Proteins mit Komponenten der pestiviralen Replikase, nämlich NS3 und NS5B, wurde festgestellt. Das NS5B (virale RNS-abhängige RNS-Polymerase) wurde, wie die drei Strukturproteine Erns, E1 und E2, überwiegend ER assoziiert nachgewiesen. Aufgrund seiner Verteilung könnte das Core-Protein in der infizierten Zelle Prozesse wie z.B. Translation beeinflussen. Die Lokalisierung der Nichtstruktur- bzw. Strukturproteine deutet darauf hin, dass „assembly“ und Replikation bei Pestiviren in demselben Kompartiment stattfinden. 5.- Ein Agarose RNS-Bindungsassay wurde zur Untersuchung der RNS-Bindungsaktivität des Core-Proteins entwickelt. Das Core-Protein band RNS sequenzunabhängig und somit kann die Inkorporation genomischer RNS in die pestiviralen Virionen aufgrund einer spezifischen RNS bindenden Aktivität des Core-Proteins ausgeschlossen werden. Sehr wahrscheinlich wird es durch das zeitlich koordinierte Erfolgen der Replikation des viralen Genoms und des Virus „assembly“ ermöglicht.The subcellular localization and RNA binding activity of the core-protein from pestiviruses were studied and the following results were obtained: 1.- The gene of the core-protein from CSFV and BVDV was cloned in a prokaryotic expression vector. After expression in bacteria (E. coli) the protein was purified by „Immobilized Methal Affinity Chromatography“ (IMAC), and used as antigen to produce mAbs and also in a RNA-binding assay. 2.- A system for the screening from hybridoma supernatants was developed which allowed the isolation of eight hybridomas that produce an core-protein mAbs. The generated mAbs proved to be useful to detect the core-protein of different Pestivirus species [BVDV-1 (7 strains); BVDV-2 (2 strains); BDV (2 strains); CSFV; giraffe] in infected cells and concentrated virions. 3.- A core-protein disulfide bond-linked homodimer was identified in virions from BVDV-2 strains and was characterized by reverse genetics. It is very probable that, like the core-protein from flaviviruses, the pestiviral core-protein is found as a dimer in virions. 4.- In infected cells the core-protein colocalized with the endoplasmic reticulum und mitochondria. This protein was also found in association with „RNA-processing bodies“ („P-Bodies“), which play an important role in the regulation of translation. The core-protein also colocalized with components of the pestiviral replicase, namely NS3 and NS5B. The NS5B (viral RNA-dependent RNA polymerase), and the three structural proteins Erns, E1 und E2 associated mainly with the endoplasmic reticulum. Due to its distribution in the infected cell the core-protein could influence processes like i.e. translation. The localization patterns of the nonstructural and structural proteins indicate that in the case of pestiviruses assembly and replication could take place in the same compartment. 5.- An agarose RNA-binding assay was developed to study the RNA-binding activity of the core-protein. The core-protein bound RNA in a sequence independent manner, which excludes the possibility that the incorporation of viral genomes in the virions is due to a sequence specific interaction of the core-protein with the pestiviral RNA. Instead the specific incorporation of the viral genome very likely occurs due to the compartimentalized and time coordinated replication of the viral genome and the virus assembly

Isolation and characterization of new Puumala orthohantavirus strains from Germany

Orthohantaviruses are re-emerging rodent-borne pathogens distributed all over the world. Here, we report the isolation of a Puumala orthohantavirus (PUUV) strain from bank voles caught in a highly endemic region around the city Osnabrück, north-west Germany. Coding and non-coding sequences of all three segments (S, M, and L) were determined from original lung tissue, after isolation and after additional passaging in VeroE6 cells and a bank vole-derived kidney cell line. Different single amino acid substitutions were observed in the RNA-dependent RNA polymerase (RdRP) of the two stable PUUV isolates. The PUUV strain from VeroE6 cells showed a lower titer when propagated on bank vole cells compared to VeroE6 cells. Additionally, glycoprotein precursor (GPC)-derived virus-like particles of a German PUUV sequence allowed the generation of monoclonal antibodies that allowed the reliable detection of the isolated PUUV strain in the immunofluorescence assay. In conclusion, this is the first isolation of a PUUV strain from Central Europe and the generation of glycoprotein-specific monoclonal antibodies for this PUUV isolate. The obtained virus isolate and GPC-specific antibodies are instrumental tools for future reservoir host studies

Immunization with GP1 but Not Core-like Particles Displaying Isolated Receptor-Binding Epitopes Elicits Virus-Neutralizing Antibodies against Junín Virus

New World arenaviruses are rodent-transmitted viruses and include a number of pathogens that are responsible for causing severe human disease. This includes Junín virus (JUNV), which is the causative agent of Argentine hemorrhagic fever. The wild nature and mobility of the rodent reservoir host makes it difficult to control the disease, and currently passive immunization with high-titer neutralizing antibody-containing plasma from convalescent patients is the only specific therapy. However, dwindling supplies of naturally available convalescent plasma, and challenges in developing similar resources for other closely related viruses, have made the development of alternative antibody-based therapeutic approaches of critical importance. In this study, we sought to induce a neutralizing antibody response in rabbits against the receptor-binding subunit of the viral glycoprotein, GP1, and the specific peptide sequences in GP1 involved in cellular receptor contacts. While these specific receptor-interacting peptides did not efficiently induce the production of neutralizing antibodies when delivered as a particulate antigen (as part of hepatitis B virus core-like particles), we showed that recombinant JUNV GP1 purified from transfected mammalian cells induced virus-neutralizing antibodies at high titers in rabbits. Further, neutralization was observed across a range of unrelated JUNV strains, a feature that is critical for effectiveness in the field. These results underscore the potential of GP1 alone to induce a potent neutralizing antibody response and highlight the importance of epitope presentation. In addition, effective virus neutralization by rabbit antibodies supports the potential applicability of this species for the future development of immunotherapeutics (e.g., based on humanized monoclonal antibodies). Such information can be applied in the design of vaccines and immunogens for both prevention and specific therapies against this and likely also other closely related pathogenic New World arenaviruses.Fil: Roman Sosa, Gleyder. Ulm University Hospital; AlemaniaFil: Leske, Anne. Friedrich-Loeffler-Institut; AlemaniaFil: Ficht, Xenia. Ulm University Hospital; AlemaniaFil: Dau, Tung Huy. Friedrich-Loeffler-Institut; AlemaniaFil: Holzerland, Julia. Friedrich-Loeffler-Institut; AlemaniaFil: Hoenen, Thomas. Friedrich-Loeffler-Institut; AlemaniaFil: Beer, Martin. Friedrich-Loeffler-Institut; AlemaniaFil: Kammerer, Robert. Friedrich-Loeffler-Institut; AlemaniaFil: Schirmbeck, Reinhold. Friedrich-Loeffler-Institut; AlemaniaFil: Rey, Felix A.. Friedrich-Loeffler-Institut; AlemaniaFil: Cordo, Sandra Myriam. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Groseth, Allison. Friedrich-Loeffler-Institut; Alemani

Immunization with GP1 but Not Core-like Particles Displaying Isolated Receptor-Binding Epitopes Elicits Virus-Neutralizing Antibodies against Junín Virus

New World arenaviruses are rodent-transmitted viruses and include a number of pathogens that are responsible for causing severe human disease. This includes Junín virus (JUNV), which is the causative agent of Argentine hemorrhagic fever. The wild nature and mobility of the rodent reservoir host makes it difficult to control the disease, and currently passive immunization with high-titer neutralizing antibody-containing plasma from convalescent patients is the only specific therapy. However, dwindling supplies of naturally available convalescent plasma, and challenges in developing similar resources for other closely related viruses, have made the development of alternative antibody-based therapeutic approaches of critical importance. In this study, we sought to induce a neutralizing antibody response in rabbits against the receptor-binding subunit of the viral glycoprotein, GP1, and the specific peptide sequences in GP1 involved in cellular receptor contacts. While these specific receptor-interacting peptides did not efficiently induce the production of neutralizing antibodies when delivered as a particulate antigen (as part of hepatitis B virus core-like particles), we showed that recombinant JUNV GP1 purified from transfected mammalian cells induced virus-neutralizing antibodies at high titers in rabbits. Further, neutralization was observed across a range of unrelated JUNV strains, a feature that is critical for effectiveness in the field. These results underscore the potential of GP1 alone to induce a potent neutralizing antibody response and highlight the importance of epitope presentation. In addition, effective virus neutralization by rabbit antibodies supports the potential applicability of this species for the future development of immunotherapeutics (e.g., based on humanized monoclonal antibodies). Such information can be applied in the design of vaccines and immunogens for both prevention and specific therapies against this and likely also other closely related pathogenic New World arenaviruses

Core Protein of Pestiviruses Is Processed at the C Terminus by Signal Peptide Peptidase

The core protein of pestiviruses is released from the polyprotein by viral and cellular proteinases. Here we report on an additional intramembrane proteolytic step that generates the C terminus of the core protein. C-terminal processing of the core protein of classical swine fever virus (CSFV) was blocked by the inhibitor (Z-LL)(2)-ketone, which is specific for signal peptide peptidase (SPP). The same effect was obtained by overexpression of the dominant-negative SPP D(265)A mutant. The presence of (Z-LL)(2)-ketone reduced the viability of CSFV almost 100-fold in a concentration-dependent manner. Reduction of virus viability was also observed in infection experiments using a cell line that inducibly expressed SPP D(265)A. The position of SPP cleavage was determined by C-terminal sequencing of core protein purified from virions. The C terminus of CSFV core protein is alanine(255) and is located in the hydrophobic center of the signal peptide. The intramembrane generation of the C terminus of the CSFV core protein is almost identical to the processing scheme of the core protein of hepatitis C viruses

Classical Swine Fever Virus Glycoprotein E(rns) Is an Endoribonuclease with an Unusual Base Specificity

The glycoprotein E(rns) of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for E(rns). Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5′ of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that E(rns) is a highly active endoribonuclease (k(cat)/K(m) = 2 × 10(6) to 10 × 10(6) M(−1) s(−1)) with a strong affinity to NpU containing single-stranded RNA substrates (K(m) = 85 to 260 nM)

A Kinetic Study of CD83 Reveals an Upregulation and Higher Production of sCD83 in Lymphocytes from Pregnant Mice

For the normal development of pregnancy, a balance between immune tolerance and defense is crucial. However, the mechanisms mediating such a balance are not fully understood. CD83 is a transmembrane protein whose expression has been linked to anti-inflammatory functions of T and B cells. The soluble form of CD83, released by cleavage of the membrane-bound protein, has strong anti-inflammatory properties and was successfully tested in different mouse models. It is assumed that this molecule contributes to the establishment of immune tolerance. Therefore, we postulated that the expression of CD83 is crucial for immune tolerance during pregnancy in mice. Here, we demonstrated that the membrane-bound form of CD83 was upregulated in T and B cells during allogeneic murine pregnancies. An upregulation was also evident in the main splenic B cell subtypes: marginal zone, follicular zone, and transitional B cells. We also showed that there was an augmentation in the number of CD83+ cells toward the end of pregnancy within splenic B and CD4+ T cells, while CD83+ dendritic cells were reduced in spleen and inguinal lymph nodes of pregnant mice. Additionally, B lymphocytes in late-pregnancy presented a markedly higher sensitivity to LPS in terms of CD83 expression and sCD83 release. Progesterone induced a dosis-dependent upregulation of CD83 on T cells. Our data suggest that the regulation of CD83 expression represents a novel pathway of fetal tolerance and protection against inflammatory threats during pregnancy

Biosynthesis of Classical Swine Fever Virus Nonstructural Proteins ▿

Proteolytic processing of polyproteins is considered a crucial step in the life cycle of most positive-strand RNA viruses. An enhancement of NS2-3 processing has been described as a major difference between the noncytopathogenic (non-CP) and the cytopathogenic (CP) biotypes of pestiviruses. The effects of accelerated versus delayed NS2-3 processing on the maturation of the other nonstructural proteins (NSP) have never been compared. In this study, we analyzed the proteolytic processing of NSP in Classical swine fever virus (CSFV). Key to the investigation was a panel of newly developed monoclonal antibodies (MAbs) that facilitated monitoring of all nonstructural proteins involved in virus replication (NS2, NS3, NS4A, NS5A, and NS5B). Applying these MAbs in Western blotting and radioimmunoprecipitation allowed an unambiguous identification of the mature proteins and precursors in non-CP CSFV-infected cells. Furthermore, the kinetics of processing were determined by pulse-chase analyses for non-CP CSFV, CP CSFV, and a CP CSFV replicon. A slow but constant processing of NS4A/B-5A/B occurred in non-CP CSFV-infected cells, leading to balanced low-level concentrations of mature NSP. In contrast, the turnover of the polyprotein precursors was three times faster in CP CSFV-infected cells and in cells transfected with a CP CSFV replicon, causing a substantial increase of mature NSP concentrations. We conclude that a delayed processing not only of NS3 but further of all NSP represents a hallmark of regulation in non-CP pestiviruses