J Bacteriol Virol.  2006 Jun;36(2):125-132. 10.4167/jbv.2006.36.2.125.

Evidence for the Presence of a cis-acting Element in the Coding Region of the Japanese Encephalitis Virus Capsid Protein

• Yun SI
• Choi YJ
• Song BH
• Kim JM
• Kang SY
• Lee YM

• KMID: 2055019
• DOI: http://doi.org/10.4167/jbv.2006.36.2.125

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/deltaCC/LUC, pJEV/Rep/deltaC/LUC, pJEV/Rep/deltaprM/LUC, and pJEV/Rep/deltaE/LUC, of these, all replicons except for pJEV/Rep/deltaCC/LUC were competent in replication. Since pJEV/Rep/deltaCC/LUC is the same as pJEV/Rep/ deltaC/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.

Keyword

Japanese encephalitis virus; cis-acting element; Capsid protein; Viral replication

MeSH Terms

Asian Continental Ancestry Group*
Capsid Proteins
Clinical Coding*
Cyclization
Encephalitis Virus, Japanese*
Encephalitis, Japanese*
Flavivirus
Humans
Replicon
RNA
Capsid Proteins
RNA

Figure

• 

  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.

• 

  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.

• 

  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.

• 

  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.

Reference

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