Synthetic tRNALys,3 as the replication primer for the HIV-1HXB2 and HIV-1Mal genomes.

In order to determine the contribution of modified bases on the efficiency with which tRNA(Lys,3) is used in vitro as the HIV-1 replication primer, the properties of synthetic derivatives prepared by three independent methods were compared to the natural, i.e. fully modified, tRNA. When prepared directly by in vitro run-off transcription, we show here that the predominant tRNA species is 77 nt, representing a non-templated addition of a single nucleotide. As a consequence, this aberrant tRNA inefficiently primes (-) strand strong stop DNA synthesis from the primer binding site (PBS) on the HIV-1 viral RNA genome to which it must hybridize. In contrast, correctly sized tRNA(Lys,3) can be prepared by (i) total chemical synthesis and ligation of 'half' tRNAs, (ii) transcription of a cassette whose DNA template contained strategically placed 2'-O-Methyl-containing ribonucleotides and (iii) processing from a larger precursor by means of targeted cleavage with Escherichia coli RNase H. When each of these 76 nt tRNAs was supplemented into a (-) strand strong stop DNA synthesis reaction utilizing the HXB2 strain of HIV-1, the amount of product obtained was comparable to that from the fully modified counterpart. Parallel assays monitoring early events in (-) strand strong stop DNA synthesis using either the HXB2 or Mal strain of HIV-1 RNA as the template indicated little difference in the pattern or total product amount when primed with either natural or synthetic tRNA(Lys,3). In addition, nuclease mapping of PBS-bound tRNA suggests inter-molecular base pairing between bases of the tRNA anticodon domain and the U-rich U5-IR loop of the viral 5' leader region is less stable on the HIV-1(HXB2) genome than the HIV-1(Mal) isolate.