TRANSLATION OF SINDBIS VIRUS MESSENGER-RNA - EFFECTS OF SEQUENCES DOWNSTREAM OF THE INITIATING CODON

One incentive for developing the alphavirus Sindbis virus as a vector for the expression of heterologous proteins is the very high level of viral structural proteins that accumulates in infected cells. Although replacement of the structural protein genes by a heterologous gene sh ould lead to an equivalent accumulation of the heterologous protein, t he Sindbis virus capsid protein is produced at a level 10- to 20-fold higher than that of any foreign protein. Chimeric mRNAs which contain the first 275 nucleotides of the Sindbis virus 26S mRNA fused to the l acZ gene are also translated at the higher level. The enhancing sequen ces, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin-like structure; deletion s in this region destroy the activity. These sequences enhance transla tion in infected cells but have the opposite effect in uninfected cell s. Furthermore, translation of this RNA in infected cells is suppresse d by a second viral RNA lacking the hairpin-like structure, but transl ation of the latter RNA is not affected. We propose that the hairpin-l ike structure presents a barrier to the movement of the ribosomes duri ng translation of mRNA. In infected cells, under conditions in which t his mRNA is essentially the only RNA being translated, a slowdown in t he transit of the ribosomes gives factors present at low concentration s a chance to bind to the translation complex and permits a high level of functional complexes to be formed. In uninfected cells and in infe cted cells translating two different viral subgenomic mRNAs, a pause i n the movement of the ribosomes along the RNA is no longer an advantag e, because the required factors are now usurped by other translation c omplexes.