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The in vitro translation of viral RNA in a cell-free system was developed using synthetic, capped messenger RNA (mRNA) prepared by enzymatic oligo-capping. The in vitro translation produced the same structural proteins as well as the same ribonucleoprotein complex (RNP) as in vivo. However, unlike in vivo, the polyribosomes observed in the in vitro translation were truncated in number. The efficiency of translation of the uncapped mRNA was a function of its length, being optimal for a length of 15-20 bases. Studies with a variety of RNAs indicated the presence of a strong requirement for an initiating AUG codon which can function as an efficient initiation codon when either in the correct reading frame or in the 3′-proximal location relative to the 5′ end of the mRNA.Substitution of cysteine-15 with aspartic acid in the extracellular domain of human CD58 suppresses protein expression but is not sufficient for CD58-LAR.
We have previously identified and characterized a novel mycobacterial CD58-like adhesion receptor, CMRa-1 (CD58-LAR), that exhibits an unusual transmembrane topology with 12 epidermal growth factor (EGF)-like domains in its extracellular region. To investigate the role of CD58 in immune responses, we prepared several CD58-LAR-specific mutants by substituting cysteine-15 with various amino acids. We detected neither the mutant containing glutamine at position 15, nor the mutant containing asparagine or glutamic acid at position 15. In contrast, the mutant containing aspartic acid at position 15 showed