Internal initiation of mRNA translation in insect cell mediated by an internal ribosome entry site (IRES) from shrimp white spot syndrome virus (WSSV).
Internal initiation of mRNA translation can be mediated by internal ribosome entry site (IRES) elements which are located mainly in RNA viruses as well as certain mammalian and insect mRNA molecules. Thus far, only one DNA virus has been discovered to contain IRES element. In this investigation, an IRES element from white spot syndrome virus (WSSV), a DNA virus of marine shrimp, was demonstrated to direct the efficient translation of dicistronic mRNA in Trichoplusia ni insect cells. The IRES was inserted between glutathione S-transferase (GST) and green fluorescent protein (GFP) genes to construct a dicistronic cassette (GST-IRES-GFP). After transfection of this dicistronic cassette in insect cell, the Northern blot indicated that only one transcript corresponding to the mRNA of GST-IRES-GFP could be detected. However, the GST and GFP genes were simultaneously translated as revealed by Western blot and fluorescent microscopy, respectively. Based on sequence orientation and deletion analyses, the IRES element was 180 nucleotides (nt) in length and orientation-dependent. By comparison with that of cap-dependent initiation, the translation efficiency mediated by IRES was 98.77%. This finding promises that the WSSV IRES could be very useful to co-express two or more proteins due to its shorter length and higher translation efficiency.
Han
Fang
F
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration Xiamen 361005, PR China.
Zhang
Xiaobo
X
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D033202:Anostraca / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D018387:Codon, Initiator / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics; D007915:Lepidoptera / Q000235:genetics*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D046848:White spot syndrome virus 1 / Q000235:genetics*
D020121:5' Untranslated Regions; D018387:Codon, Initiator; D004251:DNA Transposable Elements