Membrantopologie und Prozessierung der Glykoproteine Gp2/3/4 des Equinen Arteritis Virus

Equine arteritis virus (EAV) is an enveloped RNA virus from the family Arteriviridae, which causes equine viral arteritis, mainly associated with abortions in mares. The trimer composed of the minor envelope glycoproteins Gp2, Gp3 and Gp4 is required for cell entry. These glycoproteins are predicted to be type I membrane protein with cleavable signal peptide (SP). To determine membrane topology in cells, the localisation of YFP fused C-terminally to the glycoproteins was analysed. In contrast to Gp2b and Gp4, the fluorescent tag of Gp3 localised in the lumen of the ER suggesting that the signal peptide (SP) functions as an uncleaved signal anchor (type II topology). The analysis of the glycosylation of Gp3 revealed that an overlapping sequon NNTT, adjacent to the SP cleavage site, was modified at both asparagines, although not in every Gp3 molecule. The presence of at least one glycosylation site in this region prevented SP cleavage in Gp3. Deletion of the overlapping sequon and blocking the glycosylation allowed SP cleavage, indicating that carbohydrate attachment inhibits processing of a potentially cleavable SP. The same phenomenon was observed in recombinant viruses, but surprisingly the infectivity of the EAV lacking the SP was not impaired. Membrane fractionation of transfected cells revealed that the uncleaved SP of the Gp3 was not responsible for membrane attachment of the protein. The membrane anchoring was achieved by the hydrophobic C-terminus, as its deletion allowed secretion of the protein to the supernatant. As a result of my study I propose a new topology model of Gp3, where the uncleaved SP is completely translocated into the ER lumen. The protein is anchored in the membrane via its hydrophobic C-terminus, which however does not span the membrane completely. Additionally, in order to express the Gp2/4 heterodimer on the plasma membrane for functional studies on this complex, the transmembrane regions and cytoplasmic tails were exchanged with corresponding regions of haemagglutinin of Influenza A. The proteins were able to form disulphide-linked Gp2/4 heterodimers, but were not targeted to the cell surface. In addition, single cysteines in the ectodomain of Gp4 were mutated, to determine the responsible one for the covalent linkage to Gp2. These mutations in transiently expressed Gp2/4, did not abolish dimer formation, but led to non-infectious virions in the viral context. The presented data about the minor glycoproteins of the EAV may facilitate studies on the Arterivirus entry. The SP retention mechanism observed in Gp3 could also apply for the other mammalian proteins. Future studies are needed to reveal the molecular basis of the unusual translocation and double sequon glycosylation of the Gp3.Das Equine Arteritisvirus (EAV) ist ein umhülltes RNA Virus aus der Familie der Arteriviridae, welches die equine virale Arteritis hervorruft, die mit Fehlgeburten in Stuten einhergeht. Der trimere Komplex, gebildet aus den Glykoproteinen Gp2, Gp3 und Gp4 ist für den Eintritt des Virus in die Zelle verantwortlich. Von diesen Glykoproteinen nimmt man an, dass sie Typ I-Membranproteine sind, die über ein spaltbares Signalpeptid (SP) verfügen. Um die Orientierung der Proteine in der Membran zu bestimmen, wurde die Lokalisierung eines YFPs, welches C-terminal mit den Glykoproteine fusioniert wurde, untersucht. Im Gegensatz zu Gp2 und Gp4, befindet sich der Fluorophor von Gp3 im Lumen des ERs, was darauf schließen lässt, dass das Signalpeptid als nicht gespaltener Signalanker fungiert (Typ II-Topologie). Die Untersuchung des Glykosylierungsstatus von Gp3 ergab, dass die überlappende Glykosylierungssequenz NNTT, welche sich neben der Spaltstelle des SPs befindet, an beiden Asparaginen modifiziert wird; jedoch nicht in jedem Gp3 Molekül. Das Vorkommen von mindestens einer Glykosylierung in dieser Region ist in der Lage die Signalpeptidspaltung zu verhindern. Die Deletion sowie das Blockieren der Glykosylierung ermöglichen jedoch die Signalpeptidspaltung, was darauf schließen lässt, dass das Anhängen der Zuckerketten die Prozessierung des potentiell spaltbaren SPs verhindert. Dieses Phänomen konnte auch in rekombinanten Viren gezeigt werden, jedoch hatte die Abspaltung des Signalpeptides überraschenderweise keinen Einfluss auf die Infektivität des Virus. Die Untersuchung der Membranbindung in transfizierten Zellen ergab, dass das ungespaltene SP nicht für die Membranbindung verantwortlich ist, sondern der hydrophobe C-Terminus, da seine Deletion die Sekretion des Proteins in den Überstand ermöglichte. Als Ergebnis meiner Untersuchungen stelle ich ein neues Modell für die Topologie von Gp3 auf, in dem das ungeschnittene Signalpeptid vollständig in das Lumen des ERs transloziert wird. Die Membranverankerung findet dabei durch die hydrophobe C-terminale Domäne statt, welche jedoch die Membran nicht komplett durchspannt. Zusätzlich wurden Konstrukte erstellt, in denen die Transmembrandomänen sowie der zytoplasmatische Teil von Gp2 und Gp4 durch die entsprechende Region vom Hämagglutinin der Influenzaviren ersetzt wurde. Diese waren in der Lage Gp2/4 Heterodimere zu bilden, wurde jedoch nicht an die Plasmamembran transportiert. Um das für die kovalente Bindung von Gp4 zu Gp2 verantwortliche Cystein zu identifizieren, wurden einzelne Cysteine in der Ektodomäne des Gp4s mutiert und in Zellen exprimiert. Keine der Mutationen was in der Lage die Dimerisierung zu verhindern, allerdings führten die Mutationen im viralen Kontext zu nicht infektiösen Viruspartikeln. Die präsentierten Daten über EAV könnten die Erforschung des Eintritts der Arteriviren erleichtern. Zudem könnte die Beibehaltung des Signalpeptids, wie es für Gp3 beobachtet wurde, auch für andere Säugerproteine zutreffen. Jedoch sind weitere Studien nötig, um den ungewöhnlichen Mechanismus der Translokation und der Nutzung der überlappenden Glykosylierungssequenz von Gp3 aufzuklären

Expression of the Heterotrimeric GP2/GP3/GP4 Spike of an Arterivirus in Mammalian Cells

Equine arteritis virus (EAV), an enveloped positive-strand RNA virus, is an important pathogen of horses and the prototype member of the Arteiviridae family. Unlike many other enveloped viruses, which possess homotrimeric spikes, the spike responsible for cellular tropism in Arteriviruses is a heterotrimer composed of 3 glycoproteins: GP2, GP3, and GP4. Together with the hydrophobic protein E they are the minor components of virus particles. We describe the expression of all 3 minor glycoproteins, each equipped with a different tag, from a multi-cassette system in mammalian BHK-21 cells. Coprecipitation studies suggest that a rather small faction of GP2, GP3, and GP4 form dimeric or trimeric complexes. GP2, GP3, and GP4 co-localize with each other and also, albeit weaker, with the E-protein. The co-localization of GP3-HA and GP2-myc was tested with markers for ER, ERGIC, and cis-Golgi. The co-localization of GP3-HA was the same regardless of whether it was expressed alone or as a complex, whereas the transport of GP2-myc to cis-Golgi was higher when this protein was expressed as a complex. The glycosylation pattern was also independent of whether the proteins were expressed alone or together. The recombinant spike might be a tool for basic research but might also be used as a subunit vaccine for horses

Avian hepatitis E virus is widespread among chickens in Polandand belongs to genotype 2

AbstractBig liver and spleen disease, caused by avian hepatitis E virus, has been reported in Poland, but the prevalence of the virushas not yet been investigated. In this study, 1034 serum samples from 57 breeder broiler and laying hen flocks were screenedfor the presence of anti-aHEV antibodies. In a random serology study, 56.1% of flocks were positive. Seroprevalence washigher in laying hen flocks than in broiler breeder flocks. Phylogenetic analysis of partial ORF1 and ORF2 sequences revealedthat all Polish isolates belonged to genotype 2. This is the first time this genotype has been detected in Central Europe

Production of Recombinant EAV with Tagged Structural Protein Gp3 to Study Artervirus Minor Protein Localization in Infected Cells

Equine arteritis virus (EAV) is a prototype member of the Arterivirus family, comprising important pathogens of domestic animals. Minor glycoproteins of Arteriviruses are responsible for virus entry and cellular tropism. The experimental methods for studying minor Arterivirus proteins are limited because of the lack of antibodies and nested open reading frames (ORFs). In this study, we generated recombinant EAV with separated ORFs 3 and 4, and Gp3 carrying HA-tag (Gp3-HA). The recombinant viruses were stable on passaging and replicated in titers similar to the wild-type EAV. Gp3-HA was incorporated into the virion particles as monomers and as a Gp2/Gp3-HA/Gp4 trimer. Gp3-HA localized in ER and, to a lesser extent, in the Golgi, it also co-localized with the E protein but not with the N protein. The co-localization of Gp3-HA and the E protein with ERGIC was reduced. Moreover, EAV with Gp3-HA could become a valuable research tool for identifying host cell factors during infection and the role of Gp3 in virus attachment and entry

Molecular characterisation of equid alphaherpesvirus 1 strains isolated from aborted fetuses in Poland

Abstract Background Equid alphaherpesvirus 1 (EHV-1) is one of the main infectious causative agents of abortion in mares and can also be associated with stillbirth, neonatal foal death, rhinopneumonitis in young horses and a neurological disorder called equine herpesvirus myeloencephalopathy (EHM). The neuropathogenicity of the virus was shown to be significantly higher in EHV-1 strains that carry a single nucleotide point (SNP) mutation in the ORF30, which encodes a catalytic subunit of viral DNA polymerase (ORF30 D752). Another gene, ORF68 is frequently used for phylogenetic analysis of EHV-1. Methods 27 EHV-1 strains isolated from aborted equine fetuses in Poland, collected between 1993 and 2017, were subjected to PCR targeting the open reading frames (ORFs) 30 and 68 of the EHV-1 genome. PCR products obtained were sequenced and SNPs were analyzed and compared to sequences available in GenBank. Results None of the analyzed sequences belonged to the ORF30 D752neuropathogenic genotype: all EHV-1 belonged to the non-neuropathogenic variant N752. On the basis of ORF68 sequences, the majority of EHV-1 sequences (76.9%) cannot be assigned to any of the known groups; only six sequences (23.1%) clustered within groups II and IV. Conclusions EHV-1 strains obtained from abortion cases belong to the non-neuropathogenic genotype. Many EHV-1 ORF68 sequences have similar SNPs to those already described in Poland, but a clear geographical distribution was not observed. A single particular ORF68 sequence type was observed in strains isolated from 2001 onwards

Expression of the Heterotrimeric GP2/GP3/GP4 Spike of an Arterivirus in Mammalian Cells

Equine arteritis virus (EAV), an enveloped positive-strand RNA virus, is an important pathogen of horses and the prototype member of the Arteiviridae family. Unlike many other enveloped viruses, which possess homotrimeric spikes, the spike responsible for cellular tropism in Arteriviruses is a heterotrimer composed of 3 glycoproteins: GP2, GP3, and GP4. Together with the hydrophobic protein E they are the minor components of virus particles. We describe the expression of all 3 minor glycoproteins, each equipped with a different tag, from a multi-cassette system in mammalian BHK-21 cells. Coprecipitation studies suggest that a rather small faction of GP2, GP3, and GP4 form dimeric or trimeric complexes. GP2, GP3, and GP4 co-localize with each other and also, albeit weaker, with the E-protein. The co-localization of GP3-HA and GP2-myc was tested with markers for ER, ERGIC, and cis-Golgi. The co-localization of GP3-HA was the same regardless of whether it was expressed alone or as a complex, whereas the transport of GP2-myc to cis-Golgi was higher when this protein was expressed as a complex. The glycosylation pattern was also independent of whether the proteins were expressed alone or together. The recombinant spike might be a tool for basic research but might also be used as a subunit vaccine for horses

Short Beak and Dwarfism Syndrome in Ducks in Poland Caused by Novel Goose Parvovirus

Short beak and dwarfism syndrome (SBDS), which was previously identified only in mule ducks, is now an emerging disease of Pekin ducks in China and Egypt. The disease is caused by the infection of ducks with a genetic variant of goose parvovirus—novel goose parvovirus (nGPV). In 2019, SBDS was observed for the first time in Poland in eight farms of Pekin ducks. Birds in the affected flock were found to show growth retardation and beak atrophy with tongue protrusions. Morbidity ranged between 15% and 40% (in one flock), while the mortality rate was 4–6%. Co-infection with duck circovirus, a known immunosuppressive agent, was observed in 85.7% of ducks. The complete coding regions of four isolates were sequenced and submitted to GenBank. The phylogenetic analysis revealed a close relationship of Polish viral sequences with the Chinese nGPV. Genomic sequence alignments showed 98.57–99.28% identity with the nGPV sequences obtained in China, and 96.42% identity with the classical GPV (cGPV; Derzsy’s disease). The rate of amino acid mutations in comparison to cGPV and Chinese nGPV was higher in the Rep protein than in the Vp1 protein. To our knowledge, this is the first report of nGPV infection in Pekin ducks in Poland and Europe. It should be emphasized that monitoring and sequencing of waterfowl parvoviruses is important for tracking the viral genetic changes that enable adaptation to new species of waterbirds

Iceland in the eyes of young geologists - report from the scientific trip of SKNG UWr. (2018)

The geological field trip of the Student Science Association of Geologists at the University of Wroclaw took place in September of 2018. The main goal of a 17-day and a 2850 km long trip around Iceland was to study the field geology and to gain the knowledge on active extensional tectonics, volcanism and magmatic processes in a complex geotectonic setting of the Atlatnic Mid-ocean Ridge combined with the mantle plume, as exemplified by Iceland. The group visited several dozen spectacular geosites in the active volcanic systems of Askja, Hekla, Krafla, Laki, Torfajökull, few geothermal fields across the country along with many sites of general geological and touristic interest, like waterfalls, fiords, glacial lagoons and others. This fascinating field study tour greatly deepened the geological experience of participants, highlighting the diversity and complexity of natural tectonic and volcanic phenomena

Quantifying the Dynamics of Bacterial Biofilm Formation on the Surface of Soft Contact Lens Materials Using Digital Holographic Tomography to Advance Biofilm Research

The increase in bacterial resistance to antibiotics in recent years demands innovative strategies for the detection and combating of biofilms, which are notoriously resilient. Biofilms, particularly those on contact lenses, can lead to biofilm-related infections (e.g., conjunctivitis and keratitis), posing a significant risk to patients. Non-destructive and non-contact sensing techniques are essential in addressing this threat. Digital holographic tomography emerges as a promising solution. This allows for the 3D reconstruction of the refractive index distribution in biological samples, enabling label-free visualization and the quantitative analysis of biofilms. This tool provides insight into the dynamics of biofilm formation and maturation on the surface of transparent materials. Applying digital holographic tomography for biofilm examination has the potential to advance our ability to combat the antibiotic bacterial resistance crisis. A recent study focused on characterizing biofilm formation and maturation on six soft contact lens materials (three silicone hydrogels, three hydrogels), with a particular emphasis on Staphylococcus epidermis and Pseudomonas aeruginosa, both common culprits in ocular infections. The results revealed species- and time-dependent variations in the refractive indexes and volumes of biofilms, shedding light on cell dynamics, cell death, and contact lens material-related factors. The use of digital holographic tomography enables the quantitative analysis of biofilm dynamics, providing us with a better understanding and characterization of bacterial biofilms