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Journal of Virology, August 2009, p. 7909-7930, Vol. 83, No. 16
0022-538X/09/$08.00+0     doi:10.1128/JVI.02541-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

3' cis-Acting Elements That Contribute to the Competence and Efficiency of Japanese Encephalitis Virus Genome Replication: Functional Importance of Sequence Duplications, Deletions, and Substitutions{triangledown}

Sang-Im Yun,{dagger} Yu-Jeong Choi,{dagger} Byung-Hak Song, and Young-Min Lee*

Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, South Korea

Received 10 December 2008/ Accepted 26 May 2009

The positive-strand RNA genome of Japanese encephalitis virus (JEV) terminates in a highly conserved 3'-noncoding region (3'NCR) of six domains (V, X, I, II-1, II-2, and III in the 5'-to-3' direction). By manipulating the JEV genomic RNA, we have identified important roles for RNA elements present within the 574-nucleotide 3'NCR in viral replication. The two 3'-proximal domains (II-2 and III) were sufficient for RNA replication and virus production, whereas the remaining four (V, X, I, and II-1) were dispensable for RNA replication competence but required for maximal replication efficiency. Surprisingly, a lethal mutant lacking all of the 3'NCR except domain III regained viability through pseudoreversion by duplicating an 83-nucleotide sequence from the 3'-terminal region of the viral open reading frame. Also, two viable mutants displayed severe genetic instability; these two mutants rapidly developed 12 point mutations in domain II-2 in the mutant lacking domains V, X, I, and II-1 and showed the duplication of seven upstream sequences of various sizes at the junction between domains II-1 and II-2 in the mutant lacking domains V, X, and I. In all cases, the introduction of these spontaneous mutations led to an increase in RNA production that paralleled the level of protein accumulation and virus yield. Interestingly, the mutant lacking domains V, X, I, and II-1 was able to replicate in hamster BHK-21 and human neuroblastoma SH-SY5Y cells but not in mosquito C6/36 cells, indicating a cell type-specific restriction of its viral replication. Thus, our findings provide the basis for a detailed map of the 3' cis-acting elements in JEV genomic RNA, which play an essential role in viral replication. They also provide experimental evidence for the function of 3' direct repeat sequences and suggest possible mechanisms for the emergence of these sequences in the 3'NCR of JEV and perhaps in other flaviviruses.

* Corresponding author. Mailing address: Department of Microbiology, College of Medicine & Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju-Si, South Korea. Phone: 82-43-261-2863. Fax: 82-43-272-1603. E-mail: ymlee{at}chungbuk.ac.kr

{triangledown} Published ahead of print on 3 June 2009.

{dagger} These authors contributed equally to this work.

Journal of Virology, August 2009, p. 7909-7930, Vol. 83, No. 16
0022-538X/09/$08.00+0     doi:10.1128/JVI.02541-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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