Duck hepatitis virus (DHV-1) is a worldwide distributed picornavirus that causes acute and fatal disease in young ducklings. Recently, the complete genome of DHV-1 has been determined and comparative sequence analysis has shown that possesses the typical picornavirus organization but exhibits several unique features. For the first time, we provide evidence that the 626-nucleotide-long 5′-UTR of the DHV-1 genome contains an internal ribosome entry site (IRES) element that functions efficiently both in vitro and in mammalian cells. The prediction of the secondary structure of the DHV-1 IRES shows significant similarity to the hepatitis C virus (HCV) IRES. Moreover, similarly to HCV IRES, DHV-1 IRES can direct translation initiation in the absence of a functional eIF4F complex. We also demonstrate that the activity of the DHV-1 IRES is modulated by a viral coding sequence located downstream of the DHV-1 5′-UTR, which enhances DHV-1 IRES activity both in vitro and in vivo. Furthermore, mutational analysis of the predicted pseudo-knot structures at the 3′-end of the putative DHV-1 IRES supported the presence of conserved domains II and III and, as it has been previously described for other picornaviruses, these structures are essential for keeping the normal internal initiation of translation of DHV-1.
Background
Duck hepatitis virus (DHV-1) is worldwide distributed and causes acute and fatal disease in young ducklings with severe economic impact in poultry industry. Although the disease was firstly reported in Long Island in 1949, the complete genome of the causing pathogen was not determined until 2006. The DHV-1 genome is 7691-nucleotide-long and encodes a polyprotein of 2250 amino acids that is proteolitically processed to the individual viral proteins. Sequence analysis has assigned DHV-1 to a new genus in the Picornaviridae family. Picornaviruses are a large family of viruses that include a number of important human and animal pathogens, such as Enterovirus, Rhinovirus, Cardiovirus, Aphthovirus, Hepatovirus, Parechovirus, Erbovirus, Kobuvirus and Teschovirus. Although these viruses have different host range and characteristics, they share common typical features, such us similar genome composition, genome structure and biological functions. The highly structured 5′-UTR of the picornavirus genome has been extensively characterized. The internal ribosome entry site (IRES) element located within this 5′-UTR directs internal initiation of viral protein synthesis in the infected cell.
The majority of host cell mRNAs are translated in a cap-dependent manner involving the recognition of their 5′-cap structure by the eIF4F complex. The eIF4F complex comprises three proteins: the eIF4E, the cellular cap-binding factor; the eIF4A, an RNA helicase responsible for the ATP-dependent elimination of secondary structures near the 5′-cap of the mRNAs; and the eIF4G, which functions as a scaffold to bind several factors such as the eIF3, the poly(A)-binding protein (PABP), the eIF4E or the eIF4A. Subsequently to the eIF4F binding to the 5′-cap structure, the 43S pre-initiation complex is recruited to the mRNA, by its interaction with the eIF3, and the selected mRNAs are efficiently translated. Initiation of translation is a major target for gene expression regulation and viruses have evolved numerous unconventional mechanisms to preferentially recruit cellular translational machinery to the viral mRNA. Often, interactions of viral proteins with the components of the eIF4F complex and with the viral mRNA allow selective viral protein translation, blocking protein synthesis of the infected cell. Translation initiation of picornaviruses proceeds by the direct interaction of the cellular machinery with the highly structured 5′ IRES elements in the viral mRNAs. Structural insights coupled with biochemical studies have revealed that the IRES substitutes for the activities of some translation initiation factors. However, internal initiation strategies used by different members of this family differ in the requirement for eIF4F complex components. For instance, EMCV IRES recruits ribosomal machinery without the contribution of the cellular cap-binding protein eIF4E but requires active eIF2, eIF3, eIF4A and the central domain of eIF4G. However, HCV IRES replaces the whole eIF4F complex and translation machinery is recruited by direct interaction of eIF3 and the viral mRNA.