Earlier studies from this laboratory suggested that tRNAs involved in frameshifting of HIV and other retroviruses lack highly modified bases in their anticodon loop (i.e., they are hypomodified). The mouse mammary tumor virus (MMTV) gag-pro frameshift signal (sequence A AAA AAC where C is the site of the frameshift) and a series of mutations at the frameshift site in MMTV are being used to determine if the tRNAs that alter the reading frame are indeed hypomodified. Asparagine tRNA, which lacks the highly modified Q base in the wobble position of its anticodon, is known to preferentially read AAC over AAU codons. Studies in this and other laboratories show that the level of frameshifting is reduced dramatically with an AAU mutant even though AAC/AAU codons are decoded by the same asparagine iso- acceptor with similar efficiencies at other mRNA sites. These observa- tions provide further evidence that asparagine tRNA, which lacks Q base, promotes ribosomal frameshifting. We developed an in vivo assay for MMTV ribosomal frameshifting by microinjecting RNA generated from the frameshift site, which was cloned in an appropriate expression vector, into Xenopus oocytes and isolating the translation product by a specific immunoassay. Preliminary studies suggest the amount of frameshifting is reduced when oocytes are also incubated in the presence of Q base. In the event a hypomodified tRNA that lacks Q base (Q is supplied in the diet of mammals) is required for ribosomal frameshifting, it provides an avenue for preventing or prolonging retrovirus expression through diet and it also provides a target site for possible inhibition of retroviral (HIV) expression. In another study, we have purified the HIV primer tRNA (tRNA[Lys-3]) to homogeniety and its interaction with HIV reverse transcriptase was examined in the laboratory of Dr. S. Wilson. The latter studies have aided in pinpointing the site of attachment of the enzyme to RNA and thus provide an additional target site for possible inhibition of HIV expression.
