Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp.
Here, we report for the first time the successful use of cycloheximide (CHX) as an inhibitor to block de novo viral protein synthesis during WSSV (white spot syndrome virus) infection. Sixty candidate IE (immediate-early) genes were identified using a global analysis microarray technique. RT-PCR showed that the genes corresponding to ORF126, ORF242 and ORF418 in the Taiwan isolate were consistently CHX-insensitive, and these genes were designated ie1, ie2 and ie3, respectively. The sequences for these IE genes also appear in the two other WSSV isolates that have been sequenced. Three corresponding ORFs were identified in the China WSSV isolate, but only an ORF corresponding to ie1 was predicted in the Thailand isolate. In a promoter activity assay in Sf9 insect cells using EGFP (enhanced green fluorescence protein) as a reporter, ie1 showed very strong promoter activity, producing higher EGFP signals than the insect Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) ie2 promoter.
Liu
Wang-Jing
WJ
Institute of Zoology, National Taiwan University, Taipei 106, Taiwan, ROC.
Chang
Yun-Shiang
YS
Wang
Chung-Hsiung
CH
Kou
Guang-Hsiung
GH
Lo
Chu-Fang
CF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology*; D020869:Gene Expression Profiling; D015967:Gene Expression Regulation, Viral; D017781:Genes, Immediate-Early; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D033561:Penaeidae / Q000187:drug effects / Q000821:virology*; D011401:Promoter Regions, Genetic; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012758:Shellfish / Q000821:virology; D018411:Spodoptera; D046848:White spot syndrome virus 1 / Q000187:drug effects / Q000378:metabolism*
D017874:Immediate-Early Proteins; D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide