TRANSLATION OF SINDBIS-VIRUS MESSENGER-RNA - ANALYSIS OF SEQUENCES DOWNSTREAM OF THE INITIATING AUG CODON THAT ENHANCE TRANSLATION

Alphaviruses, particularly Sindbis virus and Semliki Forest virus, are proving to be useful vectors for the expression of heterologous genes , In infected cells, these self-replicating vectors (replicons) transc ribe a subgenomic mRNA that codes for a heterologous protein instead o f the structural proteins, We reported recently that translation of th e reporter gene lacZ is enhanced 10-fold when the coding sequences of this gene are fused downstream of and in frame with the 5' half of the capsid gene (I. Frolov and S, Schlesinger, J, Virol. 68:8111-8117, 19 94), The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin structure. We have mutated this region by making changes in the codon s which do not affect the protein sequence but should destabilize the putative hairpin structure, These changes caused a decrease in the acc umulation of the capsid-P-galactosidase fusion protein, When these alt erations were inserted into the capsid gene in the context of the inta ct Sindbis virus genome, they led to a decrease in the rate of virus f ormation but did not affect the final yield, We also altered the origi nal sequence to one that has 12 contiguous G . C base pairs and should form a stable hairpin, The new sequence was essentially as effective as the original had been in enhancement of translation and in the rate of virus formation. The position of the predicted hairpin structure i s important for its function; an insertion of 9 nucleotides or a delet ion of 9 nucleotides decreased the level of translation, The insertion of a hairpin structure at a particular location downstream of the ini tiating AUG appears to be a way that alphaviruses have evolved to enha nce translation of their mRNA, and, as a consequence, they produce hig h levels of the structural proteins which are needed for virus assembl y, This high level of translation requires an intracellular environmen t in which host cell protein synthesis is inhibited.