Engineering and characterizing porcine reproductive and respiratory syndrome virus with separated and tagged genes encoding the minor glycoproteins

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen affecting pigs and belongs to the enveloped plus-stranded RNA virus family Arteriviridae. A unique feature of Arteriviruses is that the genes encoding the structural proteins overlap at their 3` and 5` ends. This impedes mutagenesis opportunities and precludes the binding of short peptides for antibody detection, as this would alter the amino acids encoded by the overlapping gene. In this study, we aimed to generate infectious PRRSV variants with separated genes encoding the minor glycoproteins Gp2, Gp3, and Gp4, accompanied by appended tags for detection. All recombinant genomes facilitate the release of infectious virus particles into the supernatant of transfected 293 T cells, as evidenced by immunofluorescence of infected MARC-145 cells using anti-nucleocapsid antibodies. Furthermore, expression of Gp2-Myc and Gp3-HA was confirmed through immunofluorescence and western blot analysis with tag-specific antibodies. However, after two passages of Gp2-Myc and Gp3-HA viruses, the appended tags were completely removed as indicated by sequencing the viral genome. Recombinant viruses with separated Gp2 and Gp3 genes remained stable for at least nine passages, while those with Gp3 and Gp4 genes separated reverted to wild type after only four passages. Notably, this virus exhibited significantly reduced titers in growth assays. Furthermore, we introduced a tag to the C-terminus of Gp4. The Gp4-HA virus was consistently stable for at least 10 passages, and the HA-tag was detectable by western blotting and immunofluorescence

Using Alphafold2 to Predict the Structure of the Gp5/M Dimer of Porcine Respiratory and Reproductive Syndrome Virus

Porcine reproductive and respiratory syndrome virus is a positive-stranded RNA virus of the family Arteriviridae. The Gp5/M dimer, the major component of the viral envelope, is required for virus budding and is an antibody target. We used alphafold2, an artificial-intelligence-based system, to predict a credible structure of Gp5/M. The short disulfide-linked ectodomains lie flat on the membrane, with the exception of the erected N-terminal helix of Gp5, which contains the antibody epitopes and a hypervariable region with a changing number of carbohydrates. The core of the dimer consists of six curved and tilted transmembrane helices, and three are from each protein. The third transmembrane regions extend into the cytoplasm as amphiphilic helices containing the acylation sites. The endodomains of Gp5 and M are composed of seven β-strands from each protein, which interact via β-strand seven. The area under the membrane forms an open cavity with a positive surface charge. The M and Orf3a proteins of coronaviruses have a similar structure, suggesting that all four proteins are derived from the same ancestral gene. Orf3a, like Gp5/M, is acylated at membrane-proximal cysteines. The role of Gp5/M during virus replication is discussed, in particular the mechanisms of virus budding and models of antibody-dependent virus neutralization

Towards Fair Multiparty Computation in Scriptless Distributed Ledger Systems

Fairness is one of the fundamental properties for multiparty computation (MPC) protocols. Although fair MPC protocols for general functions is shown to be impossible with a dishonest majority, a variant of fairness called ``fairness with penalty\u27\u27 has been explored recently. A MPC protocol provides fairness with penalty if either all participants can get the output, or the dishonest parties who break the protocol after getting the output will be financially penalized. Fairness with penalty is enabled by previous works leveraging the emerging distributed ledger systems (DLS), e.g. Bitcoin and Ethereum. They utilize the scripting functionality provided by the DLSs to make automatic penalty practical without relying on any trusted third party. However, there is also a significant number of DLSs that do not provide the scripting functionality. In this paper, we propose the ROSE protocol which enables fairness with penalty while only requiring the underlying DLS can verify and broadcast digital signatures on transactions. This requirement can be fulfilled by almost all DLSs, including the scriptless DLSs. To the best of our knowledge, it is still unknown how to realize fairness with penalty on scriptless DLSs before our work. We also provide a implementation of ROSE. The experimental results show that applying ROSE only brings little computation and communication overhead

Microbiota in monocultured Litopenaeus vannamei vs. polyculture with Trachinotus ovatus

The structures of the microbial community in the intestine, aquaculture water, and sediment of Litopenaeus vannamei, both in monoculture and mixed culture with Trachinotus ovatus, were analyzed by sequencing 16S rRNA amplicons. 1,120,500 valid reads were obtained from 21 samples, and 3,767 operational taxonomic units (OTUs) were classified. In the two culture modes, the abundance and diversity of bacterial in the sediment were significantly higher than in the L. vannamei intestine under the monoculture mode, in the water and intestines of L. vannamei and T. ovatus under the mix-culture mode (P 0.05). The dominant phyla in the sediment under two culture modes were Proteobacteria, Bacteroidetes, and Chloroflexi. The microbial community structure in the water and L. vannamei intestine were similar in both culture modes. The dominant phyla included Cyanobacteria, Proteobacteria, and Actinobacteria, with their abundances ranging from 80.88% to 97.10%. Proteobacteria was the dominant phylum in each group of samples, and the dominant genus in both culture modes was GpIIa. There was little difference in microbial community structures under the two culture modes; while the culture mode did not affect the core phyla/genera, there were differences in relative abundance. The experimental results provide a reference for the exploration of efficient and specific probiotic screening and microbial formulation techniques

FUS-NLS/Transportin 1 complex structure provides insights into the nuclear targeting mechanism of FUS and the implications in ALS

The C-terminal nuclear localization sequence of FUsed in Sarcoma (FUS-NLS) is critical for its nuclear import mediated by transportin (Trn1). Familial amyotrophic lateral sclerosis (ALS) related mutations are clustered in FUS-NLS. We report here the structural, biochemical and cell biological characterization of the FUS-NLS and its clinical implications. The crystal structure of the FUS-NLS/Trn1 complex shows extensive contacts between the two proteins and a unique α-helical structure in the FUS-NLS. The binding affinity between Trn1 and FUS-NLS (wide-type and 12 ALS-associated mutants) was determined. As compared to the wide-type FUS-NLS (K(D) = 1.7 nM), each ALS-associated mutation caused a decreased affinity and the range of this reduction varied widely from 1.4-fold over 700-fold. The affinity of the mutants correlated with the extent of impaired nuclear localization, and more importantly, with the duration of disease progression in ALS patients. This study provides a comprehensive understanding of the nuclear targeting mechanism of FUS and illustrates the significance of FUS-NLS in ALS

Membrantopology und Prozessierung des Glykoproteins 3 des Porzinen Reproduktiven und Respiratorischem Syndrom-Virus

The porcine reproductive and respiratory syndrome virus (PRRSV) causes one of the most important infectious disease of pigs. PRRSV infects pigs of all ages, where it causes reproductive failure in sows and respiratory problems in piglets. Usually, symptoms are mild, but lead to reduced weight gain, which causes huge financial losses in the pork industry worldwide. In China even highly pathogenic strains emerged that kill 90% of infected pigs. So far, vaccines failed to eliminate the virus, which is due to the large variation between strains and their ability to escape the immunity of the host. The glycoprotein GP3 consists of an N-terminal signal peptide, a 180 amino acids long and highly glycosylated domain, a hydrophobic conserved region (20 aa) and a variable unglycosylated C-terminal domain (50-60 aa). GP3 is supposed to form a complex with two other glycoproteins (GP2 and GP4) in virus particles, but secretion of the protein from infected cells has also been reported. Here I analyzed the membrane topology of GP3 from type-1 and -2 PRRSV strains. First, Ifound that the N-terminal signal peptide of GP3 (and also from lactate dehydrogenase-elevating virus) is cleaved despite the presence of a carbohydrate in its vicinity. This is in contrast to GP3 of equine arteritis virus where a carbohydrate attached at a similar position prevents processing. Second, I confirmed that a fraction of wild-type GP3 is secreted from transfected cells; GP3 from PRRSV-1 strains (Lelystad, Lena) to a greater extent than GP3 from PRRSV-2 strains (VR-2332, IAF-Klop, XH-GD). This secretion behavior is reversed after exchange of the variable C-terminal domain. In contrast to intracellular GP3, secreted GP3 contains complex-type carbohydrates, indicating that it passed through the secretory pathway. Since intracellular and secreted GP3 have identical SDS-PAGE mobility after deglycosylation, the secreted form is not derived from proteolytic cleavage. Next I used a fluorescence protease protection assay to show that the C terminus of GP3, fused to GFP, is resistant against proteolytic digestion in permeabilized cells. Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. Both experiments indicate that the C-terminal part of GP3 is translocated into the lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic region, but not of the variable C-terminus greatly enhances secretion of GP3. In addition, fusion of the hydrophobic region of GP3 to GFP promotes complete membrane anchorage of this (otherwise soluble) protein. Bioinformatics suggests that the hydrophobic region might form an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents and in its hydrophobic face enhances secretion of GP3. Exchanging the latter amino acids in the context of the viral genome did not affect release of virions, but released particles were not infectious. This is consistent with the proposed role of GP3 in virus entry. In sum, GP3 exhibits a very unusual hairpin-like membrane topology. The signal peptide is cleaved and the C-terminus is exposed to the lumen of the ER. Membrane attachment is caused by a short hydrophobic region, which might form an amphiphilic helix. This rather weak membrane anchoring might explain why a fraction of the protein is secreted. We speculate that secreted GP3 might function as a “decoy”, which distracts antibodies away from virus particles.Das Porzine Reproduktive und Respiratorische Syndrom-Virus (PRRSV) ist einer der bedeutensten Infektionskrankheiten bei Schweinen. PRRSV infiziert Schweine jeden Alters, verursacht Fehl- und Totgeburten bei Säuen und Atemwegserkrankungen bei Ferkeln. In den meisten Fällen sind die Symptome mild, allerdings bei deutlich reduzierter Gewichtszunahme. Dies führt weltweit zu erheblichen finanziellen Einbußen in der Schweinemast. In China sind sogar hoch-pathogene Stämme aufgetaucht, die über 90% der infizierten Schweine töteten. Bisher sind Impfstoffe daran gescheitert, das Virus zu eliminieren. Eine Ursache hierfür sind die starken Variationen zwischen den Stämmen und die Fähigkeit dem Immunsystem des Wirtes zu entkommen. Das Glykoprotein GP3 besteht aus einem N-terminalen Signalpeptid, einer 180 aminosäuren-langen stark glykosylierten Domäne, einem konservierten hydrophoben Bereich (20 Aminosäuren) und einer variablen nicht-glykosylierten C-terminalen Domäne (50-60 Aminosäuren). GP3 formt vermutlich im Viruspartikel einen Komplex mit zwei weiteren Glykoproteinen (GP2 und GP4); es wurde jedoch auch berichtet, dass infizierte Zellen GP3 sekretieren. In dieser Arbeit analysierte ich die Membrantopologie von GP3 aus PRRSV Typ-1 und Typ-2 Stämmen. Als Erstes habe ich entdeckt, dass das N-terminale Signalpeptid von PRRSV-GP3 (ebenso für GP3 vom laktatdehydrogenase-erhöhenden Virus) abgeschnitten wird, unabhängig davon, ob in unmittelbarer Nähe eine Kohlenhydratkette an das Protein gehängt wurde. Dieses Ergebnis unterscheidet sich vom GP3 des Equinen Arteritis-Virus, bei dem eine Kohlenhydratkette an ähnlicher Position die Prozessierung des Signalpeptids verhindert. Zum Zweiten, habe ich bestätigt, dass ein Anteil des GP3 (Wildtyp) von transfizierten Zellen sekretiert wird; GP3 von PRRSV-1 Stämmen (Lelystad, Lena) wird hierbei deutlich stärker sekretiert als GP3 von PRRSV-2 Stämmen (VR2332, IAF-Klop, XHGD). Der Effekt dreht sich um, wenn die variable C-terminalen Domäne entsprechend ausgetauscht wird. Im Unterschied zu intrazellulärem GP3 weist sekretiertes komplexprozessierte Kohlenhydratketten auf. Dies spricht dafür, dass diese Moleküle den sekretorischen Pfad in der Zelle komplett durchlaufen haben. Da intrazelluläres und sekretiertes GP3 nach der de-Glykosylierung die gleiche Wanderungsgeschwindigkeit in denaturierenden Proteingelen zeigten, wurde die sekretierte Form nicht proteolytisch gespalten. Als Nächstes verwendete ich einen über Fluoreszenz nachgewiesenen Protease-Zugangs-Versuch, um zu zeigen, dass der C-Terminus von GP3 verknüpft mit GFP in permeabilisierten Zellen gegen proteolytischen Verdau geschützt ist. Weiterhin habe ich nachgewiesen, dass in den C-Terminus eingefügte Glykosylierungsstellen genutzt werden. Beide Experimente deuten darauf hin, dass der C-terminale Teil des GP3's ins Lumen des Endoplasmatischen Retikulums transloziert wird. Die Deletion der hydrophoben Region verstärkte die Sekretion von GP3; die Deletion des variablen C-Terminus zeigte diesen Effekt nicht. Weiterhin führte die Verknüpfung der hydrophoben Region von GP3 mit GFP zur kompletten Membranbindung dieses üblicherweise löslichen Proteins. Bioinformatische Analysen sagen voraus, dass die hydrophobe Region eine amphipatische Helix bilden könnte. Dem entsprechend verhinderte der Austausch von einigen Aminosäuren im hydrophilen Bereich die Sekretion von GP3. Der Austausch dieser Aminosäuren im viralen Genom beeinflusste nicht die Freisetzung von Viruspartikeln, allerdings waren diese nicht infektiös. Dieser Befund stimmt mit der GP3 zugewiesenen Rolle beim Virus-Eintritt in die Zelle überein. Zusammengefasst: Gp3 weist eine ungewöhnliche Haarnadel-artige Membrantopology auf. Das Signalpeptid wird abgespalten und der C-Terminus befindet sich im Lumen des Endoplasmatischen Retikulums. Die Membranbindung wird durch eine kurze hydrophobe Region gewährleistet, die wahrscheinlich eine amphipatische Helix bildet. Diese eher schwache Membranverankerung könnte erklären, warum ein Teil dieses Proteins sekretiert wird. Wir spekulieren, dass sekretiertes GP3 als "Köder" benutzt wird, der Antikörper von den Viruspartikeln ablenken könnte

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

Palmitoylation of the envelope membrane proteins GP5 and M of porcine reproductive and respiratory syndrome virus is essential for virus growth

Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped positive-strand RNA virus in the Arteiviridae family, is a major pathogen affecting pigs worldwide. The membrane (glyco)proteins GP5 and M form a disulfide-linked dimer, which is a major component of virions. GP5/M are required for virus budding, which occurs at membranes of the exocytic pathway. Both GP5 and M feature a short ectodomain, three transmembrane regions, and a long cytoplasmic tail, which contains three and two conserved cysteines, respectively, in close proximity to the transmembrane span. We report here that GP5 and M of PRRSV-1 and -2 strains are palmitoylated at the cysteines, regardless of whether the proteins are expressed individually or in PRRSV-infected cells. To completely prevent S-acylation, all cysteines in GP5 and M have to be exchanged. If individual cysteines in GP5 or M were substituted, palmitoylation was reduced, and some cysteines proved more important for efficient palmitoylation than others. Neither infectious virus nor genome-containing particles could be rescued if all three cysteines present in GP5 or both present in M were replaced in a PRRSV-2 strain, indicating that acylation is essential for virus growth. Viruses lacking one or two acylation sites in M or GP5 could be rescued but grew to significantly lower titers. GP5 and M lacking acylation sites form dimers and GP5 acquires Endo-H resistant carbohydrates in the Golgi apparatus suggesting that trafficking of the membrane proteins to budding sites is not disturbed. Likewise, GP5 lacking two acylation sites is efficiently incorporated into virus particles and these viruses exhibit no reduction in cell entry. We speculate that multiple fatty acids attached to GP5 and M in the endoplasmic reticulum are required for clustering of GP5/M dimers at Golgi membranes and constitute an essential prerequisite for virus assembly