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

Abstract

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)...

Sequence signatures in envelope protein may determine whether flaviviruses produce hemorrhagic or encephalitic syndromes

“…Though missing the Asn-67 glycosylation site, more virulent strains of WNV are glycosylated at the Asn-153 equivalent site while the envelope proteins of less virulent strains are not [35,36]. Recent studies on Japanese encephalitis virus have shown that a single glycosylation site in the prM protein is critical for viral biogenesis and pathogenicity in mice [37]. Whether the missing glycosylation at position 67 in other flaviviruses is similarly compensated needs further investigation.…”

N-glycosylation of the premembrane protein of Japanese encephalitis virus is critical for folding of the envelope protein and assembly of virus-like particles

“…Most previous studies have indicated important roles of Nglycosylations sites in E protein, but few have focused on prM N-glycosylation. Kim et al (2008) generated a recombinant JEV with N-glycosylation mutant in prM by reverse genetics and they found that JEV secretion was reduced at the stage of virus release, rather than assembly. However, the glycan in prM protein was located in the precursor (pr) section, which was cleaved in the trans-Golgi network during assembly.…”

“…N-glycosylation of E protein is known to be important in viral propagation, infectivity, secretion, and neuroinvasion (Beasley et al, 2005;Bryant et al, 2007;Lee et al, 2010;Shirato et al, 2004;Vorndam et al, 1993). However, little attention had been paid to the roles of the N-glycosylation in prM protein until Kim et al (2008) reported that glycosyla-tion of JEV prM protein was associated with viral release and pathogenesis. But how the N-glycan in prM protein affects the viral release and pathogenesis is still unclear.…”

Genetic Evolution of Japanese Encephalitis Virus

“…Apparently, the E protein is not the only site of the viral genome related to phenotype determination of the JEV. A single N-linked glycosylation site in the prM protein of the JEV was documented to be critical for the pathogenicity of the virus in mice (Kim et al, 2008). Factors affecting the genetic diversity of the JEV may be complex and remain to be worked out.…”