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[ILLUSTRATION OMITTED] The biotechnology industry has faced many challenges since its inception in the late 1970s. The ability to translate new discoveries into viable therapies that could be produced on [...]

We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3′ untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevisoocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3′ UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. In X. laevisoocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3′ UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3′ UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3′ UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis.

ABSTRACTHerpes simplex virus specifies two sets of transcripts from theUL24gene, short transcripts (e.g., 1.4 kb), processed at the UL24poly(A) site, and long transcripts (e.g., 5.6 kb), processed at the UL26poly(A) site. The 1.4- and 5.6-kb transcripts initiate from the same promoter but are expressed with early and late kinetics, respectively. Measurements of transcript levels following actinomycin D treatment of infected cells revealed that the 1.4- and 5.6-kb UL24transcripts have similar stabilities, consistent with UL24transcript kinetics being regulated by differential polyadenylation rather than by differential stabilities. Although the UL24poly(A) site, which gives rise to short transcripts, is encountered first during processing, long transcripts processed at the UL26site are equally or more abundant; thus, operationally, the UL24site is weak. Using a series of viral ICP27mutants, we investigated whether ICP27, which has been suggested to stimulate the usage of weak poly(A) sites, stimulates 1.4-kb transcript accumulation. We found that accumulation of 1.4-kb transcripts did not require ICP27 during viral infection. Rather, ICP27 was required for full expression of 5.6-kb transcripts, and the decrease in 5.6-kb transcripts relative to 1.4-kb transcripts was not due solely to reduced DNA synthesis. Our results indicate that temporal expression of UL24transcripts can be regulated by differential polyadenylation and that although ICP27 is not required for processing at the operationally weakUL24poly(A) site, it does modulate 5.6-kb transcript levels at a step subsequent to transcriptional initiation.