Journal List > J Bacteriol Virol > v.36(2) > 1033835

Yun, Choi, Song, Kim, Kang, and Lee: Evidence for the Presence of a cis-acting Element in the Coding Region of the Japanese Encephalitis Virus Capsid Protein

Abstract

Japanese encephalitis virus (JEV) is a member of mosquito-borne flaviviruses. To investigate whether there is a cis-acting genetic element in the coding region of the JEV C protein, which is required for viral replication, we generated four mutants by introducing a various size of deletions in each structural protein-coding region, designated as pJEV/Rep/ΔCC/LUC, pJEV/Rep/ΔC/LUC, pJEV/Rep/ΔprM/LUC, and pJEV/Rep/ΔE/LUC, of these, all replicons except for pJEV/Rep/ΔCC/LUC were competent in replication. Since pJEV/Rep/ΔCC/LUC is the same as pJEV/Rep/ΔC/LUC except for an additional 5′ deletion (nt 132∼201) in the coding region of the C protein, this region appeared to be essential for RNA replication. This is consistent with the proposed cyclization sequence motif in the 5′ region of the C gene, which has been recently shown to be required for replication in other mosquito-borne flaviviruses such as DV, YFV, KUN, and WNV. Thus, our results suggest that a cis-acting genetic element in the coding region of the JEV C protein may play an important role in RNA replication. This study will facilitate the current understanding of JEV RNA replication.

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Figure 1.
Construction of JEV viral replicons. The structures of the JEV viral replicons are shown. Solid boxes (▪) indicate in-frame deletions that had been introduced into the genome of the infectious pJEV/FL/LUC construct. Four constructs, namely, pJEV/Rep/ΔCC/LUC, pJEV/Rep/ΔC/LUC, pJEV/Rep/ΔprM/LUC, and pJEV/Rep/ΔE/LUC, contain a single in-frame deletion in each structural gene of JEV. pJEV/Rep/ΔCC/LUC has a deletion that extends to the proposed cyclization sequence motif in the 5′ region of the C gene, unlike pJEV/Rep/ΔC/LUC. EMCV IRES, Internal ribosome entry site element of encephalomyocarditis virus; LUC, luciferase gene.
jbv-36-125f1.tif
Figure 2.
Generation of the synthetic RNA transcripts in vitro transcribed from pJEV/Rep/ΔCC/LUC, pJEV/Rep/ΔC/LUC, pJEV/Rep/ΔprM/LUC, and pJEV/Rep/ΔE/LUC cDNA templates. Each cDNA as indicated was linearized by XbaI digestion. Linearized cDNA templates were used for in vitro transcription reaction using the SP6 RNA polymerase. 1∼1.5 μl of the reaction mixtures was analyzed by 0.5% agarose gel electrophoresis. cDNA templates and RNA transcripts as indicated were visualized by ethidium bromide staining.
jbv-36-125f2.tif
Figure 3.
Expression of LUC. Naive BHK-21 cells (8×106) were transfected with 2 μg of the parent or JEV viral replicon RNAs that had been transcribed from each cDNA template and then seeded on 6-well plates at a density of 4×105 cells per well. At the indicated time points, the cell lysates were subjected to luciferase assays. The experiments were performed in triplicate and the mean values are shown. Solid circle, pJEV/Rep/ΔCC/LUC; open circle, pJEV/Rep/ΔC/LUC; open triangle, pJEV/Rep/ΔprM/LUC; open square, pJEV/Rep/ΔE/LUC. A dotted line indicates the level of background luminescence of naïve cells. hpt, hours post-transfection.
jbv-36-125f3.tif
Figure 4.
Expression of JEV NS1 protein. The transfected cells (4×105) as indicated were lysed with 1X sample loading buffer 48 hr post-transfection and the protein extracts were resolved on 10% SDS-polyacrylamide gels. The proteins were transferred onto the nitrocellulose membrane and immunoblotted with a JEV hyperimmune antiserum.
jbv-36-125f4.tif
Table 1.
Oligonucleotides used in this study
Oligonucleotide Sequence Polarity
DelF 5′-GATTTAATTAACCTGCAGGGGGCTGTT sense
C1R 5′-GATCTCGAGCCGGTTTTTACCGGGCCC antisense
C2F 5′-GATCTCGAGAAAAGAGGAGGAAATGAA sense
DelR 5′-TGTGGCCAAGAAGGCCAAAGCAATTGA antisense
C3R 5′-GATCTCGAGCATTACTACCCTCTTCAC antisense
prM1R 5′-GATCTCGAGGTCCGTGTTGTTAATGGT antisense
prM2F 5′-GATCTCGAGGATTCAACGAAAGCCACA sense
E1R 5′-GATCTCGAGGGCTCCACTGGCTCCTTC antisense
E2F 5′-GATCTCGAGACAACTTTGAAGGGAGCT sense
Table 2.
플라비바이러스의 cyclization sequence
Virus 5′CS 3′CS
DEN-1 UCAAUAUGCUG CAGCAUAUUGA
DEN-2 UCAAUAUGCUG CAGCAUAUUGA
DEN-3 UCAAUAUGCUG CAGCAUAUUGA
DEN-4 UCAAUAUGCUG CAGCAUAUUGA
JEV UCAAUAU–GUG CAC-AUAUUGA
WNV GUCAAUAUGCUA AGCAUAUUGAC
MVE GUCAAUAUGCUA AGCAUAUUGAC
YFV CCCUGGGCGUCAAUAUGGU ACCAUAUUGACGCCAGGG
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