Internal initiation of mRNA translation can be mediated by internal ribosome entry site (IRES) elements which Lire located inainly 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 investiagation, 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. (c) 2006 Elsevier Inc. All rights reserved.