Our laboratory has previously demonstrated that the tRNAs involved in ribosomal frameshifting in HIV and other retroviral infected cells are hypomodified (i.e., these tRNAs are missing a specific, highly modified base). Thus, a correlation exists in infected cells between the occurrence of hypomodified tRNAs and their utilization in ribosomal frameshifting in the corresponding retrovirus. A very important unresolved question then is a hypomodified tRNA actually required in ribosomal frameshifting? In the event such a tRNA is required, then reversal of this process (i.e., converting the undermodified tRNA to the corresponding fully modified tRNA in retroviral infected cells) provides an avenue for inhibiting retroviral expression. Our attention in the last year has therefore focused on developing an assay that will unequivocally demonstrate whether or not hypomodified tRNAs are required in frameshifting. We are in the process of changing MMTV and HIV genes encoding the respective frameshift sites to remove all methionine codons and inserting one methionine codon in the -1 reading frame immediately downstream of the frameshift site. Further, we are purifying the hypomodified tRNAs and the corresponding fully modified tRNAs which decode the frameshift signals in HIV and MMTV. Triple label experiments will be carried out employing 35S-methionine and 14C-(hypomodified)- and 3H-(fully modified)-aminoacyl-tRNA (which is required at the frameshift site) in rabbit reticulocyte lysates programmed with the corresponding mRNA generated from mutant HIV or MMTV clones. Isolation of the resulting frameshift peptide which is labeled with 35S-methionine and sequence analysis of this peptide should unequivocally demonstrate whether the hypomodified or fully modified tRNA is responsible for donating its amino acid to the frameshift site.
