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

AUTOR(ES)

Miller, Jennifer T.

FONTE

Oxford University Press

RESUMO

In order to determine the contribution of modified bases on the efficiency with which tRNALys,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 tRNALys,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 tRNALys,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-1HXB2 genome than the HIV-1Mal isolate.

Documentos Relacionados

• Cleavage of the HIV replication primer tRNALys,3 in human cells expressing bacterial anticodon nuclease.
• Interaction of HIV-1 reverse transcriptase with a synthetic form of its replication primer, tRNA(Lys,3).
• On the importance of the primer activation signal for initiation of tRNAlys3-primed reverse transcription of the HIV-1 RNA genome
• Phosphorothioate oligonucleotides derived from human immunodeficiency virus type 1 (HIV-1) primer tRNALys3 are strong inhibitors of HIV-1 reverse transcriptase and arrest viral replication in infected cells.
• The nucleocapsid protein specifically anneals tRNALys-3 onto a noncomplementary primer binding site within the HIV-1 RNA genome in vitro