In order to evaluate the contribution of the remaining component of the eIF4F complex, the RNA helicase eIF4A, two different experimental in vitro approaches were used. Firstly, the effect on DHV-1 translation initiation of two different dominant negative mutants of eIF4A was evaluated. The dominant negative mutant PRRVAA contains a mutation in the conserved Ia region (PTRELA to PRRVAA) and is inactive in the ATP hydrolysis and the RNA unwinding activities. The dominant negative mutant R362Q contains a mutation in the conserved arginine in position 362 (R362Q) of the C-terminal region HRIGRXXR, and exhibits drastically reduced RNA binding and RNA helicase activity. The EMCV IRES, a class 2 IRES dependent on eIF4A activity, was used as negative control. Whereas the cap-dependent CAT expression and the EMCV-directed translation were efficiently inhibited, DHV-1 the IRES-directed translation was insensitive to eIF4A dominant negative mutant.
To further analyze the eIF4A requirement and confirm these results, in vitro experiments were carried out using increasing amounts of a small molecule inhibitor of eIF4A. The hippuristanol is a sterol isolated from the coral Isis hippuris and identified via a high throughput screening for general translation inhibitors. It has been shown to block the eIF4A-dependent translation by inhibiting its RNA binding, ATPase, and helicase activities by interaction with the C-terminal domain of the eIF4A. The in vitro translation efficiency of the different dicistronic RNAs was evaluated in the RRL in the presence and in the absence of hippuristanol. In this experiment, PTV IRES, a class IV IRES which direct translation initiation independently of eIF4A, was used as positive control. Cap-dependent translation was efficiently inhibited by the eIF4A inhibitor, while PTV and DHV-1 IRES-directed translation were unaffected in these conditions. These results indicate that, DHV-1 5′-UTR, as well as the class IV HCV-like IRESes, do not require functional eIF4A to initiate translation.
Discussion
Picornavirus mRNAs are translated by an internal initiation mechanism, in which the ribosome enters directly at an internal site within the mRNA rather than scanning from the physical 5′ end. So far, internal ribosome entry sites (IRESs) have been identified for all the picornaviruses as well as an increasing number of cellular mRNAs. The IRES elements in picornaviruses have been located within the 5′ non-coding region, where the sequence and the RNA secondary structure are well conserved among viral serotypes in each genera. Based on sequence alignments, the genome of DHV-1 showed a typical picornavirus genetic organization and a putative class IV IRES element within DHV-1 5′-UTR. Therefore, DHV-1 should use similar picornavirus-like strategies for the initiation of translation. In this paper, we demonstrate, for the first time, that the DHV-1 5′-UTR has typical IRES activity, and that it can drive the translation of a downstream reporter gene both in vitro and in vivo. According to the prediction of the IRES structure, the DHV-1 IRES belongs to the type IV HCV-like IRESes, but it shares some common features with the IRES of viruses included in different groups.