Immune response to synthetic peptides of dengue prM protein

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: Discussionsupporting

confidence: 68%

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

“…The majority of flavivirus-neutralizing antibodies recognize the structural E protein, although some also bind to the prM/M pro-tein (16,21,58,73). Serotype-specific epitopes elicit antibodies with the strongest neutralizing activities (62,63), and protection in animals by antibodies correlates with neutralizing activity in vitro (6,20,25,43,53,63).…”

mentioning

confidence: 99%

Type- and Subcomplex-Specific Neutralizing Antibodies against Domain III of Dengue Virus Type 2 Envelope Protein Recognize Adjacent Epitopes

Sukupolvi-Petty

Austin

Purtha

et al. 2007

231

331

Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals.Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.Dengue fever (DF), the most prevalent arthropod-borne viral illness in humans, is caused by dengue virus (DENV). The four serotypes of DENV are transmitted to humans primarily by the mosquitoes Aedes aegypti and Aedes albopictus. DENV is a member of the Flaviviridae family and is related to the viruses that cause yellow fever and the Japanese, St. Louis, and West Nile encephalitides (8). Infection by DENV causes a spectrum of clinical disease, ranging from an acute, debilitating, selflimited febrile illness (DF) to a life-threatening hemorrhagic and capillary leak syndrome (dengue hemorrhagic fever/dengue shock syndrome). At present, no approved antiviral treatment or vaccine is available, and therapy is supportive in nature. DENV causes an estimated 25 to 100 million cases of DF and 250,000 cases of dengue hemorrhagic fever per year worldwide, with 2.5 billion people at risk for infection (27,48).DENV is an enveloped virus with a single-stranded, positivesense RNA genome (11). The 10.7-kilobase genome is translated as a single polyprotein, which is then cleaved into three structural proteins (C, prM/M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by virus-and host-encoded proteases. The 500-Å DENV mature virion has a well-organized outer protein she...

“…In the case of dengue virus, some anti-prM monoclonal antibodies cause a low level of neutralization in vitro, but this activity may be due to their cross-reactivities with E (20,40). On the other hand, it is well established that dengue virus prM and M actively induce a protective immune response, and passively administered anti-prM antibodies protect mice against a lethal challenge (9,20,40,80). The protective effect of a nonneutralizing anti-prM monoclonal antibody was also observed for a Langat virus challenge (39).…”

mentioning

confidence: 99%

Alterations of pr-M Cleavage and Virus Export in pr-M Junction Chimeric Dengue Viruses

Keelapang

Sriburi

Supasa

et al. 2004

114

During the export of flavivirus particles through the secretory pathway, a viral envelope glycoprotein, prM, is cleaved by the proprotein convertase furin; this cleavage is required for the subsequent rearrangement of receptor-binding E glycoprotein and for virus infectivity. Similar to many furin substrates, prM in vector-borne flaviviruses contains basic residues at positions P1, P2, and P4 proximal to the cleavage site; in addition, a number of charged residues are found at position P3 and between positions P5 and P13 that are conserved for each flavivirus antigenic complex. The influence of additional charged residues on pr-M cleavage and virus replication was investigated by replacing the 13-amino-acid, cleavage-proximal region of a dengue virus (strain 16681) with those of tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV) and by comparing the resultant chimeric viruses generated from RNA-transfected mosquito cells. Among the three chimeric viruses, cleavage of prM was enhanced to a larger extent in JEVpr/16681 than in YFVpr/16681 but was slightly reduced in TBEVpr/16681. Unexpectedly, JEVpr/16681 exhibited decreased focus size, reduced peak titer, and depressed replication in C6/36, PS, and Vero cell lines. The reduction of JEVpr/16681 multiplication correlated with delayed export of infectious virions out of infected cells but not with changes in specific infectivity. Binding of JEVpr/16681 to immobilized heparin and the heparin-inhibitable infection of cells were not altered. Thus, diverse pr-M junction-proximal sequences of flaviviruses differentially influence pr-M cleavage when tested in a dengue virus prM background. More importantly, greatly enhanced prM cleavability adversely affects dengue virus export while exerting a minimal effect on infectivity. Because extensive changes of charged residues at the pr-M junction, as in JEVpr/16681, were not observed among a large number of dengue virus isolates, these results provide a possible mechanism by which the sequence conservation of the pr-M junction of dengue virus is maintained in nature.The genus Flavivirus within the family Flaviviridae comprises about 73 enveloped RNA viruses that are transmitted by either mosquitoes or ticks or without a known vector (11). For these viruses, a single-stranded RNA genome of about 11 kb encodes a polyprotein, which is cleaved by cellular and viral enzymes into three structural proteins (C, prM/M, and E) and seven nonstructural proteins (54). Virions consist of two envelope proteins, E and prM/M, and an internal C protein, which binds genomic RNA. Differences in antigenicities of E allow the subdivision of flaviviruses into eight antigenic complexes and a number of unclassified viruses, which include the prototype yellow fever virus (YFV) (12). More recent assignments based on nucleotide sequence variations of the nonstructural gene NS5 generally agree with antigenic classifications (49).The assembly of flaviviruses in the endoplasmic reticulum is followed by modif...