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Accumulation of a 3'-terminal genome fragment in Japanese encephalitis virus-infected mammalian and mosquito cells - PubMed

Accumulation of a 3'-terminal genome fragment in Japanese encephalitis virus-infected mammalian and mosquito cells

Kuo-Chih Lin et al. J Virol. 2004 May.

Abstract

Japanese encephalitis virus (JEV) contains a single positive-strand RNA genome nearly 11 kb in length and is not formally thought to generate subgenomic RNA molecules during replication. Here, we report the abundant accumulation of a 3'-terminal 521- to 523-nucleotide (nt) genome fragment, representing a major portion of the 585-nt 3' untranslated region, in both mammalian (BHK-21) and mosquito (C6/36) cells infected with any of nine strains of JEV. In BHK-21 cells, the viral genome was detected as early as 24 h postinfection, the small RNA was detected as early as 28 h postinfection, and the small RNA was 0.25 to 1.5 times as abundant as the genome on a molar basis between 28 and 48 h postinfection. In C6/36 cells, the genome and small RNA were present 5 days postinfection and the small RNA was 1.25 to 5.14 times as abundant as the genome. The 3'-terminal 523-nt small RNA contains a 5'-proximal stable hairpin (nt 6 to 56) that may play a role in its formation and the conserved flavivirus 3'-cyclization motif (nt 413 to 420) and the 3'-terminal long stable hairpin structure (nt 440 to 523) that have postulated roles in genome replication. Abundant accumulation of the small RNA during viral replication in both mammalian and mosquito cells suggests that it may play a biological role, perhaps as a regulator of RNA synthesis.

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Figures

FIG. 1.
FIG. 1.

Detection of small RNA in JEV-infected mammalian cells. Cytoplasmic RNA was extracted from uninfected BHK-21 cells and from cells infected with JEV RP-9 at the indicated times postinfection, and Northern analysis was done with a probe detecting nt 10950 to 10976 in the 3′ UTR. M., DIG-labeled RNA molecular size marker with the number of bases indicated on the left; Uninf., uninfected cells. The positions of the viral genome and the small RNA are indicated on the right. The molar ratios of small RNA to genome RNA are indicated at the bottom.

FIG. 2.
FIG. 2.

Primer extension analysis of the small RNA. The cytoplasmic RNAs shown in Fig. 1 were used as templates for the extension of the 32P-labeled 3JEV10543(−) probe. The DNA used for preparing the sequencing ladder was pGEM-T vector containing a 1.7-kb 3′-proximal cDNA insert from the JEV genome. The sizes of the extended products are indicated as bases.

FIG. 3.
FIG. 3.

Detection of the small RNA in both mammalian (BHK-21) and mosquito (C6/36) cells infected with different isolates of JEV. Cytoplasmic RNA was extracted from JEV-infected BHK-21 cells 48 h postinfection (lanes 2 to 8) and from JEV-infected C6/36 cells 5 days postinfection (lanes 10 to 16). Lanes 1 and 9 contain cytoplasmic RNA extracted from uninfected (Uninf.) cells. Northern analysis was done with a probe detecting nt 10950 to 10976 in the 3′ UTR.

FIG. 4.
FIG. 4.

mfold-predicted 5′-terminal hairpin on the small RNA. The stable bulged 5′-terminal hairpin (nt 6 to 56 of the small RNA, corresponding to nt 10459 to 10509 in the JEV RP9 genome), as it is predicted by the m-fold algorithm of Zuker, is depicted.

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