According to their secondary structure, picornavirus IRES elements can be divided into four groups that display distinct biological properties. The first group (class I) includes the IRES elements from entero- and rhinoviruses (e.g. poliovirus, PV), while the second includes cardio- and aphthoviruses IRES elements (e.g. encephalomyocarditis virus, EMCV). The IRES element from hepatitis A virus (HAV) represents the third type of IRES [21,13], while the fourth group of picornavirus IRES elements has been recently described and includes porcine teschovirus-1 (PTV-1) Talfan strain, simian virus 2, porcine enterovirus-8 (PEV-8), simian picornavirus 9 and avian encephalomyelitis virus (AEV). It has been reported that the IRES elements of this group are similar to HCV IRES in sequence, function and predicted secondary structure.
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Computer-assisted analysis revealed that the 626-nt-long 5′-UTR region of the DHV-1 RNA genome folds into a compact IRES-like structure with some similarities with PTV-1 and HCV-like IRESes. The prediction of the RNA structure indicates that it contains the stem-loop structures found in other type II picornaviruses but not the clover leaf structures typically found in type I picornaviruses. These data suggest the presence of an IRES element in the 5′-UTR of DHV-1 RNA that could direct viral protein synthesis. To confirm this prediction, we have examined if the 5′-UTR region of DHV-1 genome could direct translation initiation both in in vivo and in vitro assays and we have characterized the presence of accessory regulatory sequences and the requirement for eIF4F complex components.
Results
Conserved secondary structure elements in DHV-1 and HCV-like IRES RNAs
Sequence analysis of the DHV-1 5′-UTR display a secondary structure with two major domains, II and III, which contain all the structural elements that have been described as crucial for internal translation initiation. The larger domain III consists on several branching high conserved hairpin stem-loops (III abcdef) (Figure 1A), which were also found in several members of the Picornaviridae family, such as the porcine teschovirus (PTV) and the avian encephalitis virus (AEV), and in some viruses from the Flaviviridae family, such as the classical swine fever virus (CSFV) and the hepatitis C virus (HCV). DHV-1 5′-UTR is able to initiate cap-independent translation
In order to evaluate the IRES activity of the 5′-UTR region of the DHV-1 genome, a dicistronic reporter plasmid was constructed by the insertion of a cDNA corresponding to the DHV-1 5′-UTR (nts 1 to 626) between two reporter gene sequences, the first encoding CAT protein and the second encoding fLUC (DHV UTR). Dicistronic plasmid pGEM-CAT/EMCV/LUC (EMCV-IRES), which contains the EMCV IRES, was used as a positive control for internal translation initiation activity, and the plasmid pGEM-CAT/LUC (CAT/LUC), which lacks any IRES sequence, was used as a negative control. The dicistronic plasmids were in vitro transcribed as indicated in Materials and Methods and the resultant RNAs were individually added to Flexi rabbit reticulocyte lysate (RRL) system (Promega).