Characterization of Langat virus antigenic determinants defined by monoclonal antibodies to E, NS1 and preM and identification of a protective, non-neutralizing preM-specific monoclonal antibody

The prM protein of Japanese encephalitis virus (JEV) contains a single potential N-linked glycosylation site, N 15 -X 16 -T 17 , which is highly conserved among JEV strains and closely related flaviviruses. To investigate the role of this site in JEV replication and pathogenesis, we manipulated the RNA genome by using infectious JEV cDNA to generate three prM mutants (N15A, T17A, and N15A/T17A) with alanine substiting for N 15 and/or T 17 and one mutant with silent point mutations introduced into the nucleotide sequences corresponding to all three residues in the glycosylation site. An analysis of these mutants in the presence or absence of endoglycosidases confirmed the addition of oligosaccharides to this potential glycosylation site. The loss of prM N glycosylation, without significantly altering the intracellular levels of viral RNA and proteins, led to an Ϸ20-fold reduction in the production of extracellular virions, which had protein compositions and infectivities nearly identical to those of wild-type virions; this reduction occurred at the stage of virus release, rather than assembly. This release defect was correlated with small-plaque morphology and an N-glycosylation-dependent delay in viral growth. A more conservative mutation, N15Q, had the same effect as N15A. One of the four prM mutants, N15A/T17A, showed an additional defect in virus growth in mosquito C6/36 cells but not human neuroblastoma SH-SY5Y or hamster BHK-21 cells. This cell type dependence was attributed to abnormal N-glycosylationindependent biogenesis of prM. In mice, the elimination of prM N glycosylation resulted in a drastic decrease in virulence after peripheral inoculation. Overall, our findings indicate that this highly conserved N-glycosylation motif in prM is crucial for multiple stages of JEV biology: prM biogenesis, virus release, and pathogenesis.Japanese encephalitis virus (JEV) is a member of the genus Flavivirus, which consists of Ϸ80 enveloped RNA viruses in the family Flaviviridae (5,22,36). The flaviviruses include many other clinically important human pathogens, such as dengue virus (DENV), yellow fever virus, West Nile virus (WNV), St. Louis encephalitis virus, Murray Valley encephalitis virus, and tick-borne encephalitis virus (TBEV). JEV is transmitted in an enzoonotic cycle between mosquito vectors and vertebrate hosts, with pigs and ardeid birds as the primary viremia-amplifying hosts and reservoirs, respectively, and with humans as incidental hosts (4). JEV is the most important cause of epidemic encephalitis in many Asian countries, leading to permanent neuropsychiatric sequelae and even death in children and young adults (13,60,64,66). Over the past two decades, JEV has spread throughout the Indonesian archipelago (9, 75) to the Australian territories (41,42,65), attracting increasing attention in the arena of international public health.JEV contains a single-stranded, positive-sense RNA genome of Ϸ11,000 nucleotides. The RNA genome has a cap structure at the 5Ј end and lacks a poly(A) tail at the 3Ј end (37)...

Section: Discussionmentioning

confidence: 99%

A Single N-Linked Glycosylation Site in the Japanese Encephalitis Virus prM Protein Is Critical for Cell Type-Specific prM Protein Biogenesis, Virus Particle Release, and Pathogenicity in Mice

Kim

Yun

Song

et al. 2008

“…To visualize intracellular expression of protein E or NS1, cells were fixed with acetone-methanol (1:1) and dried. After rehydration with phosphate-buffered saline, the monolayers were treated with a polyclonal rabbit anti-protein E serum or a monoclonal antibody directed against NS1 (17). Antibody-antigen reactions were visualized with fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin (Jackson Immune Research Laboratory) or anti-mouse immunoglobulin (Sigma) antibody.…”

Section: Methodsmentioning

confidence: 99%

Incorporation of Tick-Borne Encephalitis Virus Replicons into Virus-Like Particles by a Packaging Cell Line

Gehrke

Ecker

Aberle

et al. 2003

100

RNA replicons derived from flavivirus genomes show considerable potential as gene transfer and immunization vectors. A convenient and efficient encapsidation system is an important prerequisite for the practical application of such vectors. In this work, tick-borne encephalitis (TBE) virus replicons and an appropriate packaging cell line were constructed and characterized. A stable CHO cell line constitutively expressing the two surface proteins prM/M and E (named CHO-ME cells) was generated and shown to efficiently export mature recombinant subviral particles (RSPs). When replicon Nd⌬ME lacking the prM/M and E genes was introduced into CHO-ME cells, virus-like particles (VLPs) capable of initiating a single round of infection were released, yielding titers of up to 5 ؋ 10 7 /ml in the supernatant of these cells. Another replicon (Nd⌬CME) lacking the region encoding most of the capsid protein C in addition to proteins prM/M and E was not packaged by CHO-ME cells. As observed with other flavivirus replicons, both TBE virus replicons appeared to exert no cytopathic effect on their host cells. Sedimentation analysis revealed that the Nd⌬ME-containing VLPs were physically distinct from RSPs and similar to infectious virions. VLPs could be repeatedly passaged in CHO-ME cells but maintained the property of being able to initiate only a single round of infection in other cells during these passages. CHO-ME cells can thus be used both as a source for mature TBE virus RSPs and as a safe and convenient replicon packaging cell line, providing the TBE virus surface proteins prM/M and E in trans.Subgenomic replicons of positive-stranded RNA viruses contain all of the genetic elements needed to amplify themselves in susceptible host cells but lack some or all of the genes coding for structural proteins. Consequently, these RNAs are replicated in cells but are not packaged into viral particles. Replicons have proven to be valuable tools for studying replication independently of virion assembly and maturation (4,20,30). Moreover, they have great potential as molecular tools for gene expression and as vectors for therapeutic and prophylactic purposes (15,19,40). The delivery of replicon RNAs to host cells can be achieved in three different ways: (i) transfection with in vitro-transcribed replicon RNA, (ii) transfection with plasmid DNA encoding replicon sequences under the control of a cellular RNA polymerase II promoter, and (iii) infection with virus-like particles (VLPs) generated by encapsidation of replicon RNA with viral structural proteins provided in trans. VLPs are capable of initiating a single round of infection, providing an efficient and easy way to deliver the replicon vector into specific host cells, and are therefore particularly useful tools in gene therapy or for immunization purposes (19).Flaviviruses, members of the genus Flavivirus (family Flaviviridae), are positive-stranded RNA viruses and include important human pathogens such as yellow fever virus, the four serotypes of dengue virus, Japanese encephaliti...

“…The hemagglutination (HA) activity of RSPs was measured at pH 6.4 according to a method described previously by Clarke and Casals using goose erythrocytes (6). The maturation state (presence of prM) of RSPs was analyzed by Western blotting using prM-specific polyclonal sera (8,9) and by ELISA using prM-specific monoclonal antibody (MAb) 8H1 (14). The structural integrity and proper folding of E were confirmed by epitope mapping with a panel of E-protein-specific MAbs (8,9).…”

Section: Mutagenesismentioning

confidence: 99%

Mutational Analysis of the Zippering Reaction during Flavivirus Membrane Fusion

Pangerl

Heinz

Stiasny

2011

The current model of flavivirus membrane fusion is based on atomic structures of truncated forms of the viral fusion protein E in its dimeric prefusion and trimeric postfusion conformations. These structures lack the two transmembrane domains (TMDs) of E as well as the so-called stem, believed to be involved in an intra-and intermolecular zippering reaction within the E trimer during the fusion process. In order to gain experimental evidence for the functional role of the stem in flavivirus membrane fusion, we performed a mutagenesis study with recombinant subviral particles (RSPs) of tick-borne encephalitis virus, which have fusion properties similar to those of whole infectious virions and are an established model for viral fusion. Mutations were introduced into the stem as well as that part of E predicted to interact with the stem during zippering, and the effect of these mutations was analyzed with respect to fusion peptide interactions with target cells, E protein trimerization, trimer stability, and membrane fusion in an in vitro liposome fusion assay. Our data provide evidence for a molecular interaction between a conserved phenylalanine at the N-terminal end of the stem and a pocket in domain II of E, which appears to be essential for the positioning of the stem in an orientation that allows zippering and the formation of a structure in which the TMDs can interact as required for efficient fusion.Flaviviruses form a genus within the family Flaviviridae and include important human pathogens such as the dengue viruses, Japanese encephalitis virus, West Nile virus, yellow fever virus, and tick-borne encephalitis virus (TBEV) (41). These small enveloped viruses enter cells by receptor-mediated endocytosis and fuse their membrane with that of the endosome. Fusion is triggered by the acidic pH of this compartment and is mediated by the viral envelope protein E, a class II viral fusion protein (37). During virus assembly, heterodimers of E and the precursor membrane protein prM together with the nucleocapsid bud into the lumen of the endoplasmic reticulum (ER), thus leading to the formation of nonfusogenic immature particles (10,24). In the course of their exocytosis, the prM protein is processed by the host protease furin in the transGolgi network, and only the membrane-anchored part of M remains associated with secreted mature virions (42, 43). The maturation process results in the formation of smooth-surfaced particles with a herringbone-like arrangement of metastable E homodimers on the virus surface (18, 28).X-ray crystallography of carboxy-terminally truncated soluble E (sE) proteins revealed an organization into three structurally distinct domains (domain I [DI], DII, and DIII) linked by short flexible regions (Fig. 1A) (15,25,27,30,31,46). DII contains the highly conserved fusion peptide (FP) loop at its tip. In the full-length protein, DIII is followed by the ϳ50-amino-acid-long "stem" that connects the ectodomain to the membrane anchor and consists of two amphipathic alpha helices (H1 and H2) flanking...