Gene expression

A number of studies have provided evidence that the 5'-cap and 3'-poly(A) tail of eukaryotic mRNAs work synergistically to stimulate translation through a mechanism that involves interaction of the cap-associated eukaryotic initiation

FIG. 5. Stimulation of viral mRNA translation in infected cells. An analogue of the RRV gene 7 mRNA was made that contained the ORF for green fluorescent protein (GFP) inserted inframe at the N-terminus of the ORF for VP7. The mRNA was transfected in non-infected and rotavirus-infected MA104 cells and, at 8 h post infection, the cells were examined for the expression of GFP by UV light.

FIG. 5. Stimulation of viral mRNA translation in infected cells. An analogue of the RRV gene 7 mRNA was made that contained the ORF for green fluorescent protein (GFP) inserted inframe at the N-terminus of the ORF for VP7. The mRNA was transfected in non-infected and rotavirus-infected MA104 cells and, at 8 h post infection, the cells were examined for the expression of GFP by UV light.

factor, eIF-4G, and the poly(A)-binding protein, PABP (Gallie 1998). The interaction of these proteins with the termini of the mRNAs and with each other has been proposed to cause circularization of the mRNAs within polysomes. Circularization is believed to enhance translation by stabilizing the binding of initiation factors to the mRNA and by promoting the recycling of the ribosomes from the 3' to the 5' end of the mRNA (Wells et al 1998). Because rotavirus mRNA lack poly(A) tails, the efficient translation of these mRNAs must be accomplished via a process that differs from that of prototypic eukaryotic mRNAs.

mRNA structure and translation

The location of translation enhancement elements in rotavirus mRNA has been examined by transfection of chimeric reporter mRNAs into infected and uninfected cells (Fig. 5). In one such study, chimeric mRNAs were prepared that contained 5'-caps, the ORF for luciferase and either all or portions of the 5'- and

3'-UTRs of a rotaviral mRNA or of a non-viral mRNA (Chizhikov & Patton 2000). Based on the expression of luciferase from the transfected mRNAs, results were obtained demonstrating that the last four nucleotides of the 3'-consensus sequence of rotaviral mRNAs, 5'-GACC-3', stimulate translation in infected cells. This translation enhancer (3'TE) does not stimulate translation in uninfected cells, and its activity parallels that of the expression of viral proteins in infected cells. The activity of the 3'-TE also operates independently of the sequence of the 5'-UTR, but efficient translation is cap-dependent (Chizhikov & Patton 2000). Given that the mRNAs of most group A rotaviruses end with the same sequence, 5'-GACC-3', the 3'TE is likely to represent a common element used to promote viral gene expression in infected cells. In comparison to other enhancers, the 3'TE is remarkable in its small size. Combined with studies defining the location of cis-acting replication signals in viral mRNAs, it is apparent that the 3'-consensus sequence has dual and competing functions: to enhance translation of viral mRNAs via the activity of the 3'TE and to promote the use of the mRNAs as a template for minus-strand synthesis.

Viralproteinfunction in translation

The mechanism by which the 3'TE promotes translation is suggested by studies that have been performed on rotavirus NSP3, an RNA-binding protein which specifically recognizes the 3'TE sequence (Poncet et al 1994). NSP3 has been shown by the two hybrid system and by pull down assays to interact with eIF-4GI (Piron et al 1998). Therefore, NSP3 can be considered to be a functional homologue of PABP in that both specifically bind to the 3'-end of mRNAs and both have affinity for a cap-associated initiation factor. Based on these properties, NSP3 has been proposed to catalyse the circularization of rotavirus mRNA within polysomes, thereby increasing translation efficiency.

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