Structural analysis of the-1 ribosomal frameshift elements in giardiavirusmRNA

The RNA polymerase of giardiavirus (GLV) is synthesized as a fusion protein through a -1 ribosomal frameshift in a; region where gag and pol open read ing frames (ORFs) overlap. A heptamer, CCCUUUA, and a potential pseudoknot found in the overlap were predicted to be required for the frameshift. A 68 -nuelcotide (at) cDNA fragment containing these elements was inserted betwe en the GLV 5' 631-nt cDNA and the out-of-frame luciferase gene that require d a -1 frameshift within the 68-nt fragment for expression. Giardia lamblia trophozoites transfected with the transcript of this construct showed a fr ameshift frequency at 1.7%, coinciding with the polymerase-to-capsid protei n ratio in GLV. The heptamer is required for the frameshift but can be repl aced with other sequences of the same motif, Mutations placing stop codons in the 0 or -1 frame, located directly before or after the heptamer, implic ated the latter as the site for the -1 frameshift. Shortening or destroying the putative stem decreased the frameshift efficiency threefold; the effic iency was fully recovered by mutations to restore the stem. Deleting 18 nt from the 3' end of the 68-nt fragment, which formed the second stem in the putative pseudoknot, had no effect on the frequency of the frameshift. Chem ical probing of the RNA secondary structure in the frameshift region showed that bases resistant to chemical modification were clustered in the putati ve stem structures, thus confirming the presence of the postulated stem-loo p, while all the bases in the loop, were chemically modified, thus ruling o ut their capability of forming a pseudoknot. These results confirmed the co nclusion based, on data from the mutation study that there is but a simple stem-loop downstream from the heptamer. Together, they constitute the struc tural elements for a -1 ribosomal frameshift in the GLV transcript.