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
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            -Chicoric Acid

Lee, Deborah J; Robinson, William E.

doi:10.1128/jvi.78.11.5835-5847.2004

Cited by 35 publications

(44 citation statements)

References 67 publications

(110 reference statements)

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“…In this assay, integration of E92Q, T66I/E92Q, and N155H was reduced 1.4 -2-fold (per unit of viral DNA produced during infection) versus the WT virus. A similar reduction in integration efficiency measured by this technique was observed for several other variants selected by diketo acid inhibitors (45). The virus with the greatest integration defect (3-fold) was Q148R.…”

Section: Discussionsupporting

confidence: 52%

“…However, the attenuation reported here for T66I is greater than that reported by others. In a combined 3Ј-processing/strand transfer reaction in the presence of Mn 2ϩ the specific activity of T66I was 43% of wild type integrase (45). Others have reported that the specific activity of T66I in 3Ј-cleavage was 110% of wild type, also using Mg 2ϩ as the metal cofactor (38).…”

Section: Discussionmentioning

confidence: 95%

“…Thus, resistance substitutions, in general, give rise to defects in both catalytic activities of integrase, presumably because of the overlapping spatial arrangements and shared geometry of the active sites. For example, resistance to the integrase inhibitor L-chicoric acid generated E92K and Q148A, which were found to have greatly attenuated catalytic activities (4 and 14% of wild type, respectively) in a combined 3Ј-processing/strand transfer reaction (45).…”

Section: Discussionmentioning

confidence: 99%

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

Dicker

Terry

Lin

et al. 2008

Journal of Biological Chemistry

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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.

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Section: Discussionsupporting

confidence: 52%

Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

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

Dicker

Terry

Lin

et al. 2008

Journal of Biological Chemistry

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“…For PR, for example, the amino acids involved in specific recognition of the polyprotein cleavage sites were found to be among those that change when drug resistance was selected (65). Inhibitors of HIV-1 IN that function in the nanomolar range to inhibit the joining of the cellular and viral DNAs have been described (62)(63)(64). Macrophage-and T cell linetropic strains of HIV-1 propagated in cells in the presence of L731,988 and L708,906, two diketo acid inhibitors of strand transfer, acquire specific mutations in the integrase coding region (M154I, S153Y, N155S, L74M, S230R, and T66I) that confer anti-retroviral resistance.…”

Section: Discussionmentioning

confidence: 99%

Identification of Amino Acids in HIV-1 and Avian Sarcoma Virus Integrase Subsites Required for Specific Recognition of the Long Terminal Repeat Ends

Chen

Weber

Harrison

et al. 2006

Journal of Biological Chemistry

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A tetramer model for HIV-1 integrase (IN) with DNA representing 20 bp of the U3 and U5 long terminal repeats (LTR) termini was assembled using structural and biochemical data and molecular dynamics simulations. It predicted amino acid residues on the enzyme surface that can interact with the LTR termini. A separate structural alignment of HIV-1, simian sarcoma virus (SIV), and avian sarcoma virus (ASV) INs predicted which of these residues were unique. To determine whether these residues were responsible for specific recognition of the LTR termini, the amino acids from ASV IN were substituted into the structurally equivalent HIV-12 DNA integration is a concerted process that occurs in defined stages. After assembly of a stable complex of the viral integrase (IN) and host cell proteins with specific DNA sequences at the end of the HIV-1 LTR, the terminal dinucleotides are removed from each 3Ј end by endonucleolytic processing. The viral DNA 3Ј ends are then covalently linked to the host cell target DNA in a concerted reaction. Biochemical details concerning the processing and joining steps in the integration process have been reported for several retroviruses including avian sarcomaleukosis virus (ASV), murine leukemia virus (MuLV), and HIV-1. Specific DNA sequences at the 3Ј end of the viral LTR are required for recognition by the assembled viral integrase complex. Typically, the terminal 12-20 nucleotides are sufficient. After 3Ј processing of the CAXX sequence from the ends, viral DNA ends are joined by the viral integrase and the gapped intermediates are repaired, and unpaired viral 5Ј ends are excised by host nucleases. This results in a 4 -6-base pair duplication of host cell DNA, depending upon the virus, flanking the integrated viral DNA. Both the 3Ј processing and viral DNA-host DNA joining steps require integrase to be assembled on the specific viral DNA substrate. Integration of viral into host DNA occurs with limited sequence specificity (1). Several cell proteins have been reported to affect the integration process (2-8).Much of the information available concerning the molecular mechanism of integration comes from the use of reconstituted systems employing duplex oligodeoxyribonucleotides (oligos). The ASV IN, for example, catalyzes specific cleavage at the 3Ј end of the strands adjacent to the conserved CA dinucleotide using 15-bp substrates corresponding to the ASV U3 or U5 termini (9, 10). Similar substrates were used to demonstrate the joining reaction in which one oligo integrated into another (11-12). For HIV-1 duplex oligo substrates of comparable size, selected nucleotide substitutions in the U5 and U3 LTR regions were shown to affect one or both of the catalytic functions of HIV-1 integrase. Nucleic acid substitutions in the HIV-1 U5 LTR region, for example, can inhibit 3Ј processing (e.g. positions 1-5 and 9 -11) or not (e.g. positions 6 -8 and 12-14) (13). Other changes at specific nucleic acid positions in the HIV-1 U3 and U5 LTR regions can affect each of the catalytic reactions, althou...

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“…Published models of HIV IN (19,22) suggest that these specific STIs bind to the IN active site in a pocket flanked by the end of the LTR such that the diketoacid moiety, or its mimic, is positioned to coordinate with both of the catalytic magnesium atoms (23)(24)(25) (14,19,22,(26)(27)(28)(29). Amino acid substitutions which confer resistance to STIs cluster around the IN active site (30)(31)(32)(33)(34)(35)(36)(37), and changes to the 5′ end of the LTR reduce the affinity of a specific STI for its binding site (16). Published models do not address the issue of the precise placement of the 3′ terminus of the viral LTR in the inhibitor-bound state.…”

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confidence: 99%

The Terminal (Catalytic) Adenosine of the HIV LTR Controls the Kinetics of Binding and Dissociation of HIV Integrase Strand Transfer Inhibitors

et al. 2008

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Specific HIV integrase strand transfer inhibitors are thought to bind to the integrase active site, positioned to coordinate with two catalytic magnesium atoms in a pocket flanked by the end of the viral LTR. A structural role for the 3′ terminus of the viral LTR in the inhibitor-bound state has not previously been examined. This study describes the kinetics of binding of a specific strand transfer inhibitor to integrase variants assembled with systematic changes to the terminal 3′ adenosine. Kinetic experiments are consistent with a two-step binding model in which there are different functions for the terminal adenine base and the terminal deoxyribose sugar. Adenine seems to act as a "shield" which retards the rate of inhibitor association with the integrase active site, possibly by acting as an internal competitive inhibitor. The terminal deoxyribose is responsible for retarding the rate of inhibitor dissociation, either by sterically blocking inhibitor egress or by a direct interaction with the bound inhibitor. These findings further our understanding of the details of the inhibitor binding site of specific strand transfer inhibitors.

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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

Abstract: The diketo acids are potent inhibitors of human immunodeficiency virus (HIV) integrase (IN).

Cited by 35 publications

References 67 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

Identification of Amino Acids in HIV-1 and Avian Sarcoma Virus Integrase Subsites Required for Specific Recognition of the Long Terminal Repeat Ends

The Terminal (Catalytic) Adenosine of the HIV LTR Controls the Kinetics of Binding and Dissociation of HIV Integrase Strand Transfer Inhibitors

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