Intracellular Transport and Processing of the Marburg Virus Surface Protein in Vertebrate and Insect Cells

AbstractThe surface protein (GP) of Marburg virus (MBG) is synthesized as a 90-kDa precursor protein which is cotranslationally modified by the addition of high-mannose sugars (140 kDa). This step is followed by the conversion of the N-linked sugars to endoglycosidase H (endo H)-resistant species and the addition of O-linked oligosaccharides leading to a mature protein of 170–200 kDa approximately 30 min after pulse labeling. The mature form of GP is efficiently transported to the plasma membrane. GP synthesized using the T7 polymerase-driven vaccinia virus expression system was transported with essentially the same kinetics as the authentic GP. However, the protein that is shown to appear 30 min after pulse labeling at the plasma membrane was slightly smaller (160 kDa) than GP incorporated into the virions (170 kDa). Using a recombinant baculovirus, GP was expressed at high levels in insect cells. Three different species could be identified: a 90-kDa unglycosylated GP localized in the cytoplasm and two 140-kDa glycosylated proteins. Characterization of the glycosylated GPs revealed that processing of the oligosaccharides of GP was less efficient in insect cells than in mammalian cells. The majority of GP remained endo H sensitive containing high-mannose type N-linked glycans, whereas only a small fraction became endo H resistant carrying processed N-glycans and O-glycans. Tunicamycin treatment of the GP-expressing cells demonstrated that N-glycosylation is essential for the transport of the MBG surface protein

Charakterisierung von Oligomerisierungsdomänen des Marburg-Virus Nukleoprotein und deren funktionelle Bedeutung

Das phosphorylierte Nukleokapsidprotein NP ist Hauptbestandteil des Marburg-Virus (MARV)-Nukleokapsidkomplexes (NC). Neben NP sind drei weitere virale Strukturproteine, VP35, VP30, L, und die genomische RNA in den NC eingebunden. NP, VP35 und L können die virale Transkription und Replikation in einem in vitro Transkriptions/ Replikationssystem vermitteln. NP interagiert mit sich selbst, mit VP35 und VP30. Die Interaktion des NP mit sich selbst (Homooligomerisierung) führt zur Ausbildung von helikalen Strukturen, die das Grundgerüst des NC bilden. In MARV-infizierten Zellen sowie bei rekombinanter Expression des NP lagern sich die NC zu charakteristischen Einschlusskörpern (EK) zusammen. In der vorliegenden Arbeit wurden Homo- und Heterooligomerisierungsdomänen auf dem NP charakterisiert und strukturelle sowie funktionelle Merkmale untersucht. Durch Deletionsmutanten des NP wurden drei wichtige Aminosäurebereiche bestimmt, die die Homooligomerisierung beeinflussen: AS 118-234 (N), AS 320-400 (CC) und AS 522-695 (C). Für CC konnte direkt gezeigt werden, dass dieser Bereich notwendig und ausreichend zur Vermittlung der Homooligomerisierung ist. CC zeigt alle Merkmale zweier Coiled-Coil-Motive (C1: AS 320-50, C2: AS 371-400; CC). Die durch CC-vermittelte Interaktion konnte durch den monoklonalen a-NP Antikörper 2B10 gehemmt werden. Das von diesem Antikörper erkannte Epitop wurde auf die AS 391-410 eingegrenzt. Deletionen von CC zeigten, dass ein intaktes Doppel-Coiled-Coil-Motiv für die NP-abhängige virale Transkription unerlässlich ist. Die Bereiche N und C können miteinander interagieren. Wahrscheinlich findet der Kontakt von N und C intramolekular statt und reguliert die Aktivität von CC. Die kürzeste NP-Mutante, die zur Bildung von Einschlusskörpern fähig ist, umfasst die ersten 270 AS. Wahrscheinlich sind diese AS ausreichend, helikale Nukleokapsidähnliche Strukturen auszubilden, die sich in Einschlusskörpern aneinanderlagern. VP35 bindet in den ersten 390 AS des NP. Die Bindung zwischen beiden Proteinen ist offenbar abhängig von der Kooperation verschiedener NP-Bereiche. Erforderlich, aber nicht ausreichend für die Interaktion zwischen VP35 und NP ist CC. Sowohl die Bindung des NP an sich selbst als auch die Bindung an VP35 sind von der Phosphorylierung eines charakteristischen Serinclusters (AS 450-455) abhängig. Die VP35-Interaktion nimmt mit zunehmender Phosphorylierung des Serinclusters ab. Die Homooligomerisierung ist bei dephosphoryliertem oder komplett phosphorylierten Serincluster gering. Sind einige Serinreste nicht phosphoryliert ist die Homooligomerisierung wildtypisch. Die Phosphorylierung des Serinclusters reguliert auch die Funktion des NP während der viralen Transkription. NP mit dephosphoryliertem oder komplett phosphoryliertem Serincluster vermittelt keine Transkription mehr. Für die Funktion des NP ist unerlässlich, dass einige Serinreste des Serinclusters nicht phosphoryliert sind

New low-viscosity overlay medium for viral plaque assays

BACKGROUND: Plaque assays in cell culture monolayers under solid or semisolid overlay media are commonly used for quantification of viruses and antiviral substances. To overcome the pitfalls of known overlays, we tested suspensions of microcrystalline cellulose Avicel RC/CL™ as overlay media in the plaque and plaque-inhibition assay of influenza viruses. RESULTS: Significantly larger plaques were formed under Avicel-containing media, as compared to agar and methylcellulose (MC) overlay media. The plaque size increased with decreasing Avicel concentration, but even very diluted Avicel overlays (0.3%) ensured formation of localized plaques. Due to their low viscosity, Avicel overlays were easier to use than methylcellulose overlays, especially in the 96-well culture plates. Furthermore, Avicel overlay could be applied without prior removal of the virus inoculum thus facilitating the assay and reducing chances of cross-contamination. Using neuraminidase inhibitor oseltamivir carboxylate, we demonstrated applicability of the Avicel-based plaque reduction assay for testing of antiviral substances. CONCLUSION: Plaque assay under Avicel-containing overlay media is easier, faster and more sensitive than assays under agar- and methylcellulose overlays. The assay can be readily performed in a 96-well plate format and seems particularly suitable for high-throughput virus titrations, serological studies and experiments on viral drug sensitivity. It may also facilitate work with highly pathogenic agents performed under hampered conditions of bio-safety labs

Interaction of Polymerase Subunit PB2 and NP with Importin α1 Is a Determinant of Host Range of Influenza A Virus

We have previously reported that mutations in the polymerase proteins PB1, PB2, PA, and the nucleocapsid protein NP resulting in enhanced transcription and replication activities in mammalian cells are responsible for the conversion of the avian influenza virus SC35 (H7N7) into the mouse-adapted variant SC35M. We show now that adaptive mutations D701N in PB2 and N319K in NP enhance binding of these proteins to importin α1 in mammalian cells. Enhanced binding was paralleled by transient nuclear accumulation and cytoplasmic depletion of importin α1 as well as increased transport of PB2 and NP into the nucleus of mammalian cells. In avian cells, enhancement of importin α1 binding and increased nuclear transport were not observed. These findings demonstrate that adaptation of the viral polymerase to the nuclear import machinery plays an important role in interspecies transmission of influenza virus

Transfer of an Esterase-Resistant Receptor Analog to the Surface of Influenza C Virions Results in Reduced Infectivity Due to Aggregate Formation

AbstractA synthetic sialic acid,N-acetyl-9-thioacetamidoneuraminic acid (9-ThioAcNeu5Ac), is recognized by influenza C virus as a receptor determinant but—in contrast to the natural receptor determinant,N-acetyl-9-O-acetylneuraminic acid—is resistant to inactivation by the viral acetylesterase. This sialic acid analog was used to analyze the importance of the receptor-destroying enzyme of influenza C virus in keeping the viral surface free of receptor determinants. Enzymatic transfer of 9-ThioAcNeu5Ac to the surface of influenza C virions resulted in the loss of the hemagglutinating activity. The ability to agglutinate erythrocytes was restored when the synthetic sialic acid was released from the viral surface by neuraminidase treatment. Infectivity of influenza C virus containing surface-bound 9-ThioAcNeu5Ac was reduced about 20-fold. Sedimentation analysis as well as electron microscopy indicated that virions resialylated with the esterase-resistant sialic acid analog formed virus aggregates. These results indicate that the receptor-destroying enzyme of influenza C virus is required to avoid the presence of receptor determinants on the virion surface and thus to prevent aggregate formation and a reduction of the infectious titer

Neuraminidase Is Essential for Fowl Plague Virus Hemagglutinin to Show Hemagglutinating Activity

AbstractWhen hemagglutinin (HA) of fowl plague virus (FPV) was expressed in CV-1 cells by a simian virus 40 vector, hemadsorption was barely detectable, although HA was exposed at the cell surface. However, treatment of HA-expressing cells with Vibrio cholerae neuraminidase (VCNA) resulted in extensive hemadsorption. VCNA treatment enhanced the electrophoretic mobility of the HA1 subunit of HA, indicating the removal of sialic acid. When two oligosaccharides in the vicinity of the receptor binding site of FPV HA were deleted by site-specific mutagenesis, VCNA treatment was not required for hemadsorption. Mutants which retained one of these oligosaccharides and mutants in which oligosaccharides not adjacent to the receptor binding site were deleted needed VCNA treatment to show hemadsorption. VCNA treatment also enhanced hemadsorption of vector-expressed HA of the WSN strain, which had a complex-type oligosaccharide in the vicinity of the receptor binding site, but had no effect on hemadsorption of Hong Kong type HA, which has a high-mannose type oligosaccharide adjacent to the receptor binding site. These results indicate that sialic acid on oligosaccharides near the receptor binding site interferes with hemadsorption. Thus, the neuraminidase is essential for FPV HA to show hemagglutinating activity

The Hantaan Virus Glycoprotein Precursor Is Cleaved at the Conserved Pentapeptide WAASA

AbstractThe medium segment of the tripartite negative-stranded RNA genome of hantaviruses encodes for the predicted glycoprotein precursor GPC. We have demonstrated here the expression of the glycoprotein precursor of Hantaan virus following transfection of mammalian cells. The cleavage of the precursor into the glycoproteins G1 and G2 followed the rules for signal peptides and seemed to occur directly at the pentapeptide motif “WAASA.” Our data indicate that the signal peptidase complex is responsible for the proteolytic processing of the precursor GPC of Hantaan virus. The comparison of this region of the glycoprotein precursor, including the absolutely conserved WAASA motif, suggests a similar cleavage event for all hantavirus glycoproteins

Carbohydrate masking of an antigenic epitope of influenza virus haemagglutinin independent of oligosaccharide size

Comparison of the haemagglutinins (HA) of the pathogenic avian influenza viruses A/FPV/Dutch/27 (H7N7) and A/FPV/Rostock/34 (H7N1) revealed 94.7% nucleotide and 93.8% amino acid sequence homologies. Six of the seven N-glycosidic oligosaccharides of the Rostock HA are at the same positions as the six carbohydrates of the Dutch strain. The additional oligosaccharide side chain of the Rostock strain, which is of the complex type, is attached to asparagine149 in antigenic epitope B. The accessibility of this antigenic epitope has been analysed by using rabbit antisera raised against synthetic peptides comprising amino acids 143-162. The carbohydrates of the HA of the Rostock strain have been modified (i) to truncated cores by expression in insect cells using a baculovirus vector, (ii) to oligomannosidic side chains by growth in the presence of the trimming inhibitor methyldeoxynojirimycin and (iii) to a single N-acetylglucosamine residue by removal of the oligomannosidic sugar with endo-β-N-acetylglucosaminidase H. Neither the authentic nor the modified oligosaccharides allowed antibody binding, as indicated by enzyme-linked immunosorbent assay (ELISA) and Western blot analyses. Reactivity was observed, however, after complete removal of the carbohydrate from HA of the Rostock strain by digestion with peptide-N-glycosidase F. HA of the Dutch strain was reactive without prior peptide-N-glycosidase F treatment. These results demonstrate that a single N-acetylglucosamine at asparagine149 is sufficient to prevent recognition of the peptide epitop