The major goal of this project is to determine if host aminoacyl-tRNAs required for decoding the frameshift signal in human immunodeficient virus (HIV) and other retroviruses are altered (i.e., hypomodified) from the normal cellular aminoacyl-tRNAs. Preliminary evidence from this laboratory has suggested that these tRNAs in HIV and other retroviral infected cells are hypomodified. The mouse mammary tumor virus (MMTV) gag-pro frameshift signal (sequence A AAA AAC, where C is the site of the frameshift) is being used as a model for these studies. We obtained the wild type frameshift signal and a series of mutations at the frameshift site in MMTV as a tool for determining if the tRNA required to alter the reading frame is hypomodified. The level of frameshifting is reduced dramatically with an AAU mutant even though AAC/AAU codons are decoded by the same asparagine isoacceptor with similar efficiencies at other mRNA sites. Hypomodified asparagine tRNA which lacks the highly modified Q base is known to preferentially read AAC over AAU codons. Thus, the observation that the AAC codon at the frameshift site promotes ribosomal frameshifting more efficiently than the AAU codon suggests that the tRNA lacking Q base is required for decoding the frameshift signal. We have not been successful in utilizing in vitro assays thus far to demonstrate a requirement for hypomodified isoacceptors in ribosomal frameshifting due to the endogenous tRNA population in the assay system. However, we have developed an in vivo assay for ribosomal frameshifting utilizing microinjection techniques into Xenopus oocytes and are presently assaying the ability of purified hypomodified isoacceptors to alter the reading frame of the wild type and mutant MMTV gag-pro frameshift signals.
