Control of Translation by the 5′- and 3′-Terminal Regions of the Dengue Virus Genome

Dengue virus (DEN) is a member of the mosquito-borne group of flaviviruses that includes yellow fever, Japanese encephalitis, and West Nile (WNV) viruses. It is the causative agent of millions of human infections annually in the tropical regions of the world (14). The virus has a vertebrate host range limited to humans and monkeys, but replication and amplification also occur in mosquito vectors, principally those of the Aedes genus (4). The DEN genome is an 11-kb positive-strand RNA with a m7GpppA cap at the 5′ end but lacking a poly(A) tail at the 3′ end (29). It encodes a single long polyprotein (see Fig. ​Fig.1A)1A) that is processed through the action of viral and host proteinases to generate 10 mature structural and nonstructural proteins. The maturation pathway, the cleavage sites, and the responsible proteinases have been well described (29). However, systematic studies of the roles of the untranslated regions (UTRs) of the viral RNA in the gene expression of DEN or other flaviviruses are lacking. The UTRs are expected to play an important early role during viral infection in coordinating viral gene expression and the onset of RNA replication. FIG. 1. Luciferase reporter constructs used for assaying the roles of flaviviral UTRs in regulating translational expression. (A) The 10.7-kb DEN2 genome is diagrammed with the 5′ cap (asterisk), 5′ UTR, the single long ORF, and the 3′ ... Translation of the typical cellular mRNA relies on the recruitment of ribosomes by features in the UTRs at both ends of the RNA (11, 20, 31, 40, 41). The 5′ cap is recognized by the cytoplasmic cap binding protein eIF4E, which contacts initiation factor eIF4G. This large protein acts as a scaffold for further protein-protein interactions that support initiation. One of those interactions contacts eIF3, a complex factor that is itself bound to the small ribosomal subunit. This chain of interactions thus recruits the ribosomal subunit to the 5′ terminus of the RNA. The 3′ regions of an mRNA also enhance translation. One mechanism by which this can occur is through an interaction between eIF4G and poly(A) binding protein that is bound to the poly(A) tail (47). The translation enhancing contributions of the cap and poly(A) tail are synergistic (10, 46), and the most actively translated mRNAs are thought to exist in a cyclized or closed-loop state that is considered to enhance initiation and allow for improved ribosome recycling while ensuring that truncated, damaged mRNAs are poorly translated (11, 20, 40). After initial loading at the 5′ end of the RNA, the small ribosomal subunit scans in the 3′ direction along the RNA in search of an initiation site (27, 38, 39). This is usually the 5′-most AUG triplet, at which initiation frequency is in part determined by the immediate sequence context, GCC(A/G)CCAUGG being the optimal context in mammalian cells (27). Genomes of positive-strand RNA viruses often lack a 5′ cap and/or a poly(A) tail yet must compete with cellular mRNAs for ribosomes to support robust viral gene expression. Viral RNAs that lack a 5′ cap often possess an internal ribosome entry site in the 5′ UTR that directs interaction with the small ribosomal subunit directly or through the binding of an initiation factor, such as eIF4G (8, 17, 39, 41). This can lead to initiation at an AUG triplet that is not 5′ proximal, as occurs with the picornaviruses (19) and the members of two genera (Hepacivirus and Pestivirus) of the family Flaviviridae (29). Since the genomes of DEN and other members of the genus Flavivirus have a 5′ cap and initiate translation at the 5′-most AUG triplet, standard cap-dependent initiation rather than internal ribosome entry has been considered to be the gene expression strategy used by these viruses (29). The 3′ UTRs of some viral RNAs that possess a 5′ cap but lack a poly(A) tail promote translation to an extent similar to that of UTRs with a poly(A) tail, and through a similar synergistic interaction with the cap. This is true of rotavirus mRNA (49), whose 3′-terminal GACC sequence acts as a translational enhancer (5) by binding the viral protein NSP3, which competes with poly(A) binding protein for interaction with eIF4G (49). It is also true of some plant viral RNAs whose 3′ UTRs terminate in a tRNA-like structure, like those of Tobacco mosaic virus, Brome mosaic virus (12, 13), and Turnip yellow mosaic virus (33), although in these cases, the molecular interactions have not been identified. We report here a systematic analysis of the properties of the 5′- and 3′-terminal regions of the DEN serotype 2 genome in directing translation in Vero cells, using a luciferase (LUC) reporter system. Both 5′ and 3′ DEN sequences were shown to participate in the regulation of expression, principally by modulating translational efficiency rather than RNA stability.