THE HUMAN ASTROVIRUS RNA-DEPENDENT RNA-POLYMERASE CODING REGION IS EXPRESSED BY RIBOSOMAL FRAMESHIFTING

The genomic RNA of human astrovirus serotype 1 (HAst-1) contains three open reading frames (ORFs), 1a, 1b, and 2. ORF 1b is located downstre am of, and overlaps, 1a, and it has been suggested on the basis of seq uence analysis that expression of ORF 1b is mediated through -1 riboso mal frameshifting. To examine this possibility, a cDNA fragment contai ning the 1a-1b overlap region was cloned within a reporter gene and pl aced under the control of the bacteriophage SP6 promoter in a recombin ant plasmid. Synthetic transcripts derived from this plasmid, when tra nslated in the rabbit reticulocyte lysate cell-free system, specified the synthesis of polypeptides whose size and antibody reactivity were consistent with an efficient -1 ribosomal frameshift event at the over lap region. The HAst-1 frameshift signal has two essential components, a heptanucleotide slippery sequence, A(6)C, and a stem-loop structure in the RNA. The presence of this structure was confirmed by complemen tary and compensatory mutation analysis and by direct structure probin g with single- and double-stranded RNA-specific reagents. The HAst-1 f rameshift signal, like that present at the overlap of the gag and pro genes of the retrovirus human T-cell lymphotrophic virus type II, does not involve the formation of an RNA pseudoknot.