Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity.

Sherman, Paula A.; Fyfe, James A.

doi:10.1073/pnas.87.13.5119

Cited by 328 publications

(281 citation statements)

References 25 publications

Supporting

Mentioning

275

Contrasting

Order By: Relevance

“…After infection, the viral reverse transcriptase synthesizes a DNA copy (vDNA) of the viral RNA. HIV integrase is responsible for inserting the viral DNA into the host genome, an event that includes a number of biochemically discrete steps (2)(3)(4)(5). Integrase acts in a multimeric form (6), binding to ends of the long terminal repeats (LTR) of viral DNA and, although still in the cytoplasm, cleaving two bases from each 3Ј terminus (4,7).…”

Section: Hiv-1mentioning

confidence: 99%

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Dicker

Terry

Lin

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

In this study, eight different HIV-1 integrase proteins containing mutations observed in strand transfer inhibitor-resistant viruses were expressed, purified, and used for detailed enzymatic analyses. All the variants examined were impaired for strand transfer activity compared with the wild type enzyme, with relative catalytic efficiencies (k p /K m ) ranging from 0.6 to 50% of wild type. The origin of the reduced strand transfer efficiencies of the variant enzymes was predominantly because of poorer catalytic turnover (k p ) values. However, smaller secondorder effects were caused by up to 4-fold increases in K m values for target DNA utilization in some of the variants. All the variants were less efficient than the wild type enzyme in assembling on the viral long terminal repeat, as each variant required more protein than wild type to attain maximal activity. In addition, the variant integrases displayed up to 8-fold reductions in their catalytic efficiencies for 3-processing. The Q148R variant was the most defective enzyme. The molecular basis for resistance of these enzymes was shown to be due to lower affinity binding of the strand transfer inhibitor to the integrase complex, a consequence of faster dissociation rates. In the case of the Q148R variant, the origin of reduced compound affinity lies in alterations to the active site that reduce the binding of a catalytically essential magnesium ion. Finally, except for T66I, variant viruses harboring the resistance-inducing substitutions were defective for viral integration.

show abstract

Section: Hiv-1mentioning

confidence: 99%

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Dicker

Terry

Lin

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…IN is the only viral protein necessary to carry out the integration reaction (15,33,54). Moreover, the enzymatic activity of recombinant IN can be studied in vitro using oligonucleotide substrates that mimic the viral LTR ends, IN, and a divalent metal cation (Mg 2ϩ or Mn 2ϩ ) (10,54).…”

mentioning

confidence: 99%

“…Moreover, the enzymatic activity of recombinant IN can be studied in vitro using oligonucleotide substrates that mimic the viral LTR ends, IN, and a divalent metal cation (Mg 2ϩ or Mn 2ϩ ) (10,54). In addition to the 3Ј-end-processing and strand transfer reactions, recombinant IN can also catalyze the reversal of the integration reaction, termed disintegration.…”

mentioning

confidence: 99%

Human Immunodeficiency Virus Type 1 (HIV-1) Integrase: Resistance to Diketo Acid Integrase Inhibitors Impairs HIV-1 Replication and Integration and Confers Cross-Resistance to l -Chicoric Acid

Lee¹,

Robinson

2004

J Virol

View full text Add to dashboard Cite

show abstract

“…Integrase then catalyzes the attachment of the recessed 3′ ends to the target DNA (strand transfer) (3)(4)(5). In vitro, purified integrase protein can carry out the terminal cleavage (6,7) and strand transfer reactions (8)(9)(10)(11). Purified integrase can also carry out an apparent reversal of strand transfer, termed "disintegration" (12).…”

mentioning

confidence: 99%

The Mobility of an HIV-1 Integrase Active Site Loop Is Correlated with Catalytic Activity^,

et al. 1999

View full text Add to dashboard Cite

Replication of HIV-1 requires the covalent integration of the viral cDNA into the host chromosomal DNA directed by the virus-encoded integrase protein. Here we explore the importance of a protein surface loop near the integrase active site using protein engineering and X-ray crystallography. We have redetermined the structure of the integrase catalytic domain (residues 50-212) using an independent phase set at 1.7 Å resolution. The structure extends helix R4 on its N-terminal side (residues 149-154), thus defining the position of the three conserved active site residues. Evident in this and in previous structures is a conformationally flexible loop composed of residues 141-148. To probe the role of flexibility in this loop, we replaced Gly 140 and Gly 149, residues that appear to act as conformational hinges, with Ala residues. X-ray structures of the catalytic domain mutants G149A and G140A/G149A show further rigidity of R4 and the adjoining loop. Activity assays in vitro revealed that these mutants are impaired in catalysis. The DNA binding affinity, however, is minimally affected by these mutants as assayed by UV cross-linking. We propose that the conformational flexibility of this active site loop is important for a postbinding catalytic step.Integration of the viral cDNA into a host chromosome is required for viral replication. Prior to integration, viral cDNA is cleaved on each strand near the 3′ end by integrase (terminal cleavage), probably to remove nontemplated extra bases occasionally added by reverse transcriptase (1, 2). Integrase then catalyzes the attachment of the recessed 3′ ends to the target DNA (strand transfer) (3-5). In vitro, purified integrase protein can carry out the terminal cleavage (6, 7) and strand transfer reactions (8-11). Purified integrase can also carry out an apparent reversal of strand transfer, termed "disintegration" (12).The structure of full-length integrase has not yet been identified. However, the structure of each of its three domains has been determined in isolation. The amino-terminal domain (amino acids 1-50) is composed of three R-helices with an embedded zinc binding site (13,14). The central domain [amino acids 50-212 (50-212 1 )] is composed of mixed R-helix and -sheet (15), forming a compact fold seen previously in several polynucleotidyl phosphotransferases (16)(17)(18)(19). This domain by itself can carry out covalent chemistry on permissive disintegration substrates, an indication of its role in catalysis (20,21). A conserved amino acid sequence motif, D,D-35-E, is found in the central domain of integrases and some related prokaryotic transposases. The carboxyl-terminal domain (amino acids 213-288) is composed of a five-stranded -barrel resembling an SH3 domain, and contributes to DNA binding (22,23). All three domains form dimers independently, and the full-length integrase is likely to act as a higher-order multimer (reviewed in ref 24).We have used X-ray crystallography and protein engineering to investigate the active site of HIV-1 integrase....

show abstract

Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity.

Cited by 328 publications

References 25 publications

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Human Immunodeficiency Virus Type 1 (HIV-1) Integrase: Resistance to Diketo Acid Integrase Inhibitors Impairs HIV-1 Replication and Integration and Confers Cross-Resistance to l -Chicoric Acid

The Mobility of an HIV-1 Integrase Active Site Loop Is Correlated with Catalytic Activity^,

Contact Info

Product

Resources

About

Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity.

Cited by 328 publications

References 25 publications

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Human Immunodeficiency Virus Type 1 (HIV-1) Integrase: Resistance to Diketo Acid Integrase Inhibitors Impairs HIV-1 Replication and Integration and Confers Cross-Resistance to l -Chicoric Acid

The Mobility of an HIV-1 Integrase Active Site Loop Is Correlated with Catalytic Activity,

Contact Info

Product

Resources

About

The Mobility of an HIV-1 Integrase Active Site Loop Is Correlated with Catalytic Activity^,