RIBOSOMAL FRAMESHIFTING IN HIV AND OTHER RETROVIRAL INFECTED CELLS

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Previously we have provided strong evidence that the host tRNAs in human immunodeficiency virus (HIV)-infected and other retroviral-infected cells involved in ribosomal frameshifting of these retroviruses are hypomodified (i.e., these tRNAs lack a specific, highly modified base within the anticodon loop). A very significant unresolved question is whether a hypomodified tRNA is utilized in retroviral ribosomal frameshifting. If an undermodified tRNA is required, then alteration of this process (i.e., changing such a tRNA to the corresponding fully modified tRNA in cells infected with retrovirus) provides a possible means of inhibiting retroviral expression. To determine if hypo-modified isoacceptors are required in ribosomal frameshifting, we constructed ribosomal frameshift signals for HIV, SRV-1 and mouse mammary tumor virus (MMTV) that will unequivocally demonstrate if hypomodified tRNAs are required in this event. All methionine codons (AUG) have been mutated in the HIV and MMTV gag-pol genes to leucine codons (CUG) and a single methionine codon has been inserted into their -1 reading frames immediately downstream of the frameshift site. Sequences have been inserted into each construct corresponding to the amino terminus and carboxy terminus of GAG and GAG-POL so that the resulting proteins can be specifically isolated by immunoprecipitation. In addition, the hypo-modified tRNAs and the corresponding fully modified tRNAs which decode the frameshift signals in HIV and MMTV have been purified. We will, in the next year, carry out in vitro protein synthesis and isolate the resulting frameshift peptide that will demonstrate which tRNA is required to promote frameshifting. Further, we have begun growing HL60 cells chronically infected with HIV on chemically defined media in the presence and absence of selenium. These studies may likely (by analogy to the affect of selenium on the expression of other mammalian retroviruses) show that selenium inhibits HIV expression. We will then examine the affect of selenium on the nuclear kB-2 factor which is required for HIV expression and which preliminary studies from other laboratories have suggested that selenium inhibits its expression.