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A molecularly cloned, live-attenuated japanese encephalitis vaccine SA14-14-2 virus: a conserved single amino acid in the ij Hairpin of the Viral E glycoprotein determines neurovirulence in mice.

Yun SI, Song BH, Kim JK, Yun GN, Lee EY, Li L, Kuhn RJ, Rossmann MG, Morrey JD, Lee YM - PLoS Pathog. (2014)

Bottom Line: Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain.In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication.Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.

ABSTRACT
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, <1.5 PFU) from SA14-14-2MCV (LD50, >1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.

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E-244: A key neurovirulence factor located in the ij hairpin of the viral E glycoprotein.(A) RNA infectivity and replicability. BHK-21 cells were mock-transfected or transfected with RNAs transcribed from the parent or one of the 14 E-244 mutant cDNAs. RNA infectivity (in PFU/µg) at 4 dpt was estimated by infectious center assay, coupled with staining of cell monolayers using an α-JEV antiserum, and virus yield (in PFU/ml) at 22 hpt was determined by plaque titration on BHK-21 cells. (B) Viral growth. BHK-21 cells were infected at an MOI of 0.1 with the parent or one of the 14 E-244 mutant viruses. At the indicated time points, culture supernatants were used for virus titration on BHK-21 cells. (C) Neurovirulence. Groups of 3-week-old ICR mice (n = 10 per group) were infected IC with serial 10-fold dilutions of each virus stock, and the LD50 values were determined. (D) Homology model. The predicted model of the E ectodomain of JEV SA14-14-2 was built based on the crystal structure of the E ectodomain of WNV NY99 [79], and the model was then fitted into the cryo-EM structure of WNV NY99 [18]. Illustrated is an icosahedral asymmetric unit of the three E monomers on the viral membrane. Highlighted in the inset is the critical residue Glu at E-244 in the ij hairpin adjacent to the fusion loop of the viral E DII.
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ppat-1004290-g005: E-244: A key neurovirulence factor located in the ij hairpin of the viral E glycoprotein.(A) RNA infectivity and replicability. BHK-21 cells were mock-transfected or transfected with RNAs transcribed from the parent or one of the 14 E-244 mutant cDNAs. RNA infectivity (in PFU/µg) at 4 dpt was estimated by infectious center assay, coupled with staining of cell monolayers using an α-JEV antiserum, and virus yield (in PFU/ml) at 22 hpt was determined by plaque titration on BHK-21 cells. (B) Viral growth. BHK-21 cells were infected at an MOI of 0.1 with the parent or one of the 14 E-244 mutant viruses. At the indicated time points, culture supernatants were used for virus titration on BHK-21 cells. (C) Neurovirulence. Groups of 3-week-old ICR mice (n = 10 per group) were infected IC with serial 10-fold dilutions of each virus stock, and the LD50 values were determined. (D) Homology model. The predicted model of the E ectodomain of JEV SA14-14-2 was built based on the crystal structure of the E ectodomain of WNV NY99 [79], and the model was then fitted into the cryo-EM structure of WNV NY99 [18]. Illustrated is an icosahedral asymmetric unit of the three E monomers on the viral membrane. Highlighted in the inset is the critical residue Glu at E-244 in the ij hairpin adjacent to the fusion loop of the viral E DII.

Mentions: To probe the functional importance of the amino acid side chain at position E-244 for the viral replication and neurovirulence of SA14-14-2MCV, we performed site-directed mutagenesis, replacing G244 with 14 other amino acids of six different classes: (1) aliphatic A, V, and L; (2) hydroxyl S and T; (3) cyclic P; (4) aromatic F and W; (5) basic R and K; and (6) acidic and their amides D, E, N, and Q. We first tested the viability of synthetic RNAs transcribed in vitro from the corresponding mutant cDNAs by measuring their infectivity after transfection of BHK-21 cells. In all cases, the mutant RNA was as viable as the parent RNA, with a specific infectivity of 6.5–8.2×105 PFU/µg (Fig. 5A, RNA infectivity). However, three mutants (G244K, G244F, and G244W) were noticeably different from the parent and the other 11 mutants, as demonstrated by a ∼10-fold decrease in the yield of progeny virions released into culture medium during the first 22 hpt (Fig. 5A, virus yield) and a ∼2-2.5-fold reduction in the size of foci/plaques produced at 96 hpt (Fig. 5A, foci/plaques), although no significant difference was observed in the level of viral proteins (i.e., C, prM, E, and NS1) accumulated in RNA-transfected cells at 18 hpt (Fig. S5). As compared to G244K, the mutant G244R exhibited a barely marginal decrease in focus/plaque size and no detectable change in virus production (Fig. 5A). Overall, these findings were more evident when all mutant viruses were evaluated in multistep growth assays over the course of 96 h after infection at an MOI of 0.1, assessing their ability to grow and establish a productive infection (Fig. 5B). Our findings indicate that in BHK-21 cells, the amino acid side chain at position E-244 has no effect on the viability of the mutant RNAs, although it has a negative impact on the production and spread of infectious virions in the case of the three mutants G244K, G244F, and G244W.


A molecularly cloned, live-attenuated japanese encephalitis vaccine SA14-14-2 virus: a conserved single amino acid in the ij Hairpin of the Viral E glycoprotein determines neurovirulence in mice.

Yun SI, Song BH, Kim JK, Yun GN, Lee EY, Li L, Kuhn RJ, Rossmann MG, Morrey JD, Lee YM - PLoS Pathog. (2014)

E-244: A key neurovirulence factor located in the ij hairpin of the viral E glycoprotein.(A) RNA infectivity and replicability. BHK-21 cells were mock-transfected or transfected with RNAs transcribed from the parent or one of the 14 E-244 mutant cDNAs. RNA infectivity (in PFU/µg) at 4 dpt was estimated by infectious center assay, coupled with staining of cell monolayers using an α-JEV antiserum, and virus yield (in PFU/ml) at 22 hpt was determined by plaque titration on BHK-21 cells. (B) Viral growth. BHK-21 cells were infected at an MOI of 0.1 with the parent or one of the 14 E-244 mutant viruses. At the indicated time points, culture supernatants were used for virus titration on BHK-21 cells. (C) Neurovirulence. Groups of 3-week-old ICR mice (n = 10 per group) were infected IC with serial 10-fold dilutions of each virus stock, and the LD50 values were determined. (D) Homology model. The predicted model of the E ectodomain of JEV SA14-14-2 was built based on the crystal structure of the E ectodomain of WNV NY99 [79], and the model was then fitted into the cryo-EM structure of WNV NY99 [18]. Illustrated is an icosahedral asymmetric unit of the three E monomers on the viral membrane. Highlighted in the inset is the critical residue Glu at E-244 in the ij hairpin adjacent to the fusion loop of the viral E DII.
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Related In: Results  -  Collection

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Show All Figures
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ppat-1004290-g005: E-244: A key neurovirulence factor located in the ij hairpin of the viral E glycoprotein.(A) RNA infectivity and replicability. BHK-21 cells were mock-transfected or transfected with RNAs transcribed from the parent or one of the 14 E-244 mutant cDNAs. RNA infectivity (in PFU/µg) at 4 dpt was estimated by infectious center assay, coupled with staining of cell monolayers using an α-JEV antiserum, and virus yield (in PFU/ml) at 22 hpt was determined by plaque titration on BHK-21 cells. (B) Viral growth. BHK-21 cells were infected at an MOI of 0.1 with the parent or one of the 14 E-244 mutant viruses. At the indicated time points, culture supernatants were used for virus titration on BHK-21 cells. (C) Neurovirulence. Groups of 3-week-old ICR mice (n = 10 per group) were infected IC with serial 10-fold dilutions of each virus stock, and the LD50 values were determined. (D) Homology model. The predicted model of the E ectodomain of JEV SA14-14-2 was built based on the crystal structure of the E ectodomain of WNV NY99 [79], and the model was then fitted into the cryo-EM structure of WNV NY99 [18]. Illustrated is an icosahedral asymmetric unit of the three E monomers on the viral membrane. Highlighted in the inset is the critical residue Glu at E-244 in the ij hairpin adjacent to the fusion loop of the viral E DII.
Mentions: To probe the functional importance of the amino acid side chain at position E-244 for the viral replication and neurovirulence of SA14-14-2MCV, we performed site-directed mutagenesis, replacing G244 with 14 other amino acids of six different classes: (1) aliphatic A, V, and L; (2) hydroxyl S and T; (3) cyclic P; (4) aromatic F and W; (5) basic R and K; and (6) acidic and their amides D, E, N, and Q. We first tested the viability of synthetic RNAs transcribed in vitro from the corresponding mutant cDNAs by measuring their infectivity after transfection of BHK-21 cells. In all cases, the mutant RNA was as viable as the parent RNA, with a specific infectivity of 6.5–8.2×105 PFU/µg (Fig. 5A, RNA infectivity). However, three mutants (G244K, G244F, and G244W) were noticeably different from the parent and the other 11 mutants, as demonstrated by a ∼10-fold decrease in the yield of progeny virions released into culture medium during the first 22 hpt (Fig. 5A, virus yield) and a ∼2-2.5-fold reduction in the size of foci/plaques produced at 96 hpt (Fig. 5A, foci/plaques), although no significant difference was observed in the level of viral proteins (i.e., C, prM, E, and NS1) accumulated in RNA-transfected cells at 18 hpt (Fig. S5). As compared to G244K, the mutant G244R exhibited a barely marginal decrease in focus/plaque size and no detectable change in virus production (Fig. 5A). Overall, these findings were more evident when all mutant viruses were evaluated in multistep growth assays over the course of 96 h after infection at an MOI of 0.1, assessing their ability to grow and establish a productive infection (Fig. 5B). Our findings indicate that in BHK-21 cells, the amino acid side chain at position E-244 has no effect on the viability of the mutant RNAs, although it has a negative impact on the production and spread of infectious virions in the case of the three mutants G244K, G244F, and G244W.

Bottom Line: Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain.In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication.Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.

ABSTRACT
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, <1.5 PFU) from SA14-14-2MCV (LD50, >1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.

Show MeSH
Related in: MedlinePlus