Assembly, purification and crystallization of an active HIV-1 reverse transcriptase initiation complex

Pata, Janice D.; King, B.; Steitz, Thomas A.

doi:10.1093/nar/gkf620

Cited by 7 publications

(8 citation statements)

References 39 publications

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“…Wild-type HIV-1 RT was produced from p6HRT-PROT (38); Y181I͞Y188L and K103N mutants of HIV-1 RT were produced from derivatives of pUC12N͞ p51(His) (39,40). HIV-1 RT initiation activity was assessed by following the incorporation of 3 H-dCTP into newly synthesized DNA by using an in vitro transcribed tRNA Lys-3 primer annealed to a 36-nt viral RNA template sequence (32,33). Assays were performed in a final volume of 100 l in 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

“…CP-94,707 (Fig. 2a) is an HIV-1 RT inhibitor that was identified in a DRUG PFINDER high-throughput screening program by using a tRNA Lys-3 -primed DNA synthesis assay (32,33). This initiation event differs from elongation in several important ways: it is the only stage of replication in which both the primer and the template are RNA, the kinetics of nucleotide incorporation are slower (34)(35)(36), and more possibilities exist for contact between the primer and HIV-1 RT because the tRNA is a large asymmetric molecule, rather than a simple duplex.…”

mentioning

confidence: 99%

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Structure of HIV-1 reverse transcriptase bound to an inhibitor active against mutant reverse transcriptases resistant to other nonnucleoside inhibitors

Pata

Stirtan

Goldstein

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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We have determined the crystal structure of the HIV type 1 reverse transcriptase complexed with CP-94,707, a new nonnucleoside reverse transcriptase inhibitor (NNRTI), to 2.8-Å resolution. In addition to inhibiting the wild-type enzyme, this compound inhibits mutant enzymes that are resistant to inhibition by nevirapine, efavirenz, and delaviridine. In contrast to other NNRTI complexes where tyrosines 181 and 188 are pointing toward the enzyme active site, the binding pocket in this complex has the tyrosines pointing the opposite direction, as in the unliganded protein structure, to accommodate CP-94,707. This conformation of the pocket has not been observed previously in NNRTI complexes and substantially alters the shape and surface features that are available for interactions with the inhibitor. One ring of CP-94,707 makes extensive stacking interactions with tryptophan 229, one of the few residues in the NNRTI-binding pocket that cannot readily mutate to give rise to drug resistance. In this conformation of the pocket, mutations of tyrosines 181 and 188 are less likely to disrupt inhibitor binding. Modeling the asparagine mutation of lysine 103 shows that a hydrogen bond between it and tyrosine 188 could form as readily in the CP-94,707 complex as it does in the apoenzyme structure, providing an explanation for the activity of this inhibitor against this clinically important mutant.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Structure of HIV-1 reverse transcriptase bound to an inhibitor active against mutant reverse transcriptases resistant to other nonnucleoside inhibitors

Pata

Stirtan

Goldstein

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…The reverse transcriptase (RT) domain contains the conserved amino acid sequences characteristic of retrotransposons, and consists of subdomains RT0 to RT7. Subdomain RT5 was a completely conserved YADD motif, well known to be part of the active center of the HIV-1 retrovirus (Pata et al, 2002). The Gk.Int1 intron-encoded ORF, GK1296, with a putative length of 603 amino acid residues, is partially similar to the ORFs contained in eukaryotic (Saccharomyces cerevisiae) (Lazowska et al, 1994) and bacterial (Bacillus cereus (Rasko et al, 2004), B. anthracis (Read et al, 2003), Lactococcus lactis (Mills et al, 1996)) group II introns.…”

Section: Characterization Of Group II Intron-encoded Orfmentioning

confidence: 99%

Housekeeping recA gene interrupted by group II intron in the thermophilic Geobacillus kaustophilus

Chee

Takami

2005

Gene

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“…These data indicated that the extra nucleotide can influence the resonance of bases in the immediate vicinity and therefore lead to errors in data interpretation (46). Ribozyme cleavage has also been used to create a homogeneous RNA population in assays as diverse as virus replication and crystallographic analyses of plant and animal virus replicases (44,45,47). Pata et al (47) performed crystallization of HIV-1 RT with its cognate template/primer, and created a tRNA Lys,3 of defined length by cleavage with a hammerhead ribozyme.…”

Section: Discussionmentioning

confidence: 99%

“…Ribozyme cleavage has also been used to create a homogeneous RNA population in assays as diverse as virus replication and crystallographic analyses of plant and animal virus replicases (44,45,47). Pata et al (47) performed crystallization of HIV-1 RT with its cognate template/primer, and created a tRNA Lys,3 of defined length by cleavage with a hammerhead ribozyme. In this case, however, due to the sequence requirement of the ribozyme, the technique was limited by the necessity for a mutation at the 3 0 end of the tRNA.…”

Section: Discussionmentioning

confidence: 99%

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

Miller¹

2004

Nucleic Acids Research

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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.

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Assembly, purification and crystallization of an active HIV-1 reverse transcriptase initiation complex

Cited by 7 publications

References 39 publications

Structure of HIV-1 reverse transcriptase bound to an inhibitor active against mutant reverse transcriptases resistant to other nonnucleoside inhibitors

Structure of HIV-1 reverse transcriptase bound to an inhibitor active against mutant reverse transcriptases resistant to other nonnucleoside inhibitors

Housekeeping recA gene interrupted by group II intron in the thermophilic Geobacillus kaustophilus

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

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