Structural Organization of Avian Retrovirus Integrase in Assembled Intasomes Mediating Full-site Integration

Vora, Ajaykumar C.; Bera, Sibes; Grandgenett, Duane P.

doi:10.1074/jbc.m314270200

Cited by 18 publications

(25 citation statements)

References 49 publications

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“…This result is consistent with the finding of others that IN binds to the 10 terminal base pairs of the viral DNA (24) that influence the efficiency of integration (11). Ten base pairs is approximately half of the DNA length protected by the full-length IN oligomer in previously-reported stable IN-DNA complexes (11,25). From a biological perspective, having IN protect the viral DNA ends from nucleases strategically ensures the integrity of the viral ends, which are essential for viral integration and, thereby, for viral propagation.…”

Section: Dna Tethering Overcomes In Requirement Of the Ntd For Strandsupporting

confidence: 81%

Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs

Alian¹,

Griner²,

Chiang³

et al. 2009

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

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IN consists of 3 functional domains: the N-terminal domain (NTD; residues 1-51) that contains a conserved ''HH-CC'' zincbinding motif, the catalytic core domain (CCD; residues 52-210) with the catalytic residues (D64, D116, and E152), and the Cterminal domain (CTD; residues 210-288) that contributes to DNA binding (2). In solution, recombinant IN exists in a dynamic equilibrium between monomers, dimers, tetramers, and higherorder oligomers (3, 4). Monomers are reportedly inactive in vitro, whereas dimers are able to catalyze 3Ј processing and integration of 1 viral end (4-9). Tetramers, which have also been isolated from human cells expressing HIV-1 IN (10), can catalyze integration of 2 viral DNA ends into target DNA (7, 11), but the exact nature of the IN complex mediating 3Ј processing and strand transfer reactions remains to be determined. The integration step is an attractive drug target given its essential role in the viral life cycle and the lack of a cellular IN homologue. Strand transfer inhibitors appear to bind significantly better to IN when it is assembled on its DNA substrate than to IN alone (12). To date there is only 1 structure of an inhibitor bound to IN (13), and that is in the absence of DNA. The compound binds at the active site; however, it dimerizes across a crystallographic 2-fold axis and therefore might not be in its bioactive configuration.Structure-based understanding of the mechanisms of the action of IN inhibitors and optimization of compounds as potential drugs targeting HIV-1 IN have been hampered by the inability to capture and crystallize IN-DNA complexes. Two key factors have contributed to this problem: first, the high salt concentration (Ϸ1 M NaCl) required to maintain full-length IN in solution interferes with DNA binding; second, IN has intrinsically low affinity for DNA. To overcome these 2 obstacles, we used disulfide cross-linking to generate soluble, catalytically-active, covalent IN-DNA complexes. A similar strategy, covalent disulfide cross-linking between HIV-1 reverse transcriptase (RT) and DNA, mediated crystallization of the .Previous cross-linking from cysteinal mutations in the CTD (6) and CCD (15) Here, we describe an IN cysteine mutant, IN Y143C , which is able to form IN-DNA complexes efficiently. The IN Y143C -DNA complexes form stable tetramers in solution, retain single-end strand transfer activity, show increased resistance to protease and nuclease digestion, and bind a strand transfer inhibitor. This IN-DNA complex can serve as an in vitro platform to identify and evolve strand transfer inhibitors of HIV integration and as a means of understanding the basis for a key part of the integration reaction.

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Section: Dna Tethering Overcomes In Requirement Of the Ntd For Strandsupporting

confidence: 81%

Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs

Alian¹,

Griner²,

Chiang³

et al. 2009

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

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“…The total yields of the HIV-1 full-site products are comparable to those observed with HIV-1 PIC at 45 to 90 min of incubation at 37°C (6, 11-13, 20, 21, 39). Similar results were also observed with avian myeloblastosis virus (AMV) and recombinant Rous sarcoma virus (RSV) IN titration experiments using 3.6-to 4.6-kbp DNA donors, although the synthesis of full-site products with avian IN at 5 to 10 nM is significantly faster (ϳ3-to 4-fold) and with slightly higher yields (15,50,51).…”

Section: Resultssupporting

confidence: 60%

“…AMV and recombinant RSV IN protect wt U3 and several gain-of-function att sites up to ϳ20 bp from the ends from DNase I digestion (15,50,51). This protection of att site sequences by IN at 14°C is correlated with full-site integration activity at 37°C.…”

Section: Discussionmentioning

confidence: 90%

Recombinant Human Immunodeficiency Virus Type 1 Integrase Exhibits a Capacity for Full-Site Integration In Vitro That Is Comparable to That of Purified Preintegration Complexes from Virus-Infected Cells

Sinha

Grandgenett

2005

J Virol

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Retrovirus preintegration complexes (PIC) in virus-Integration of the retrovirus linear DNA genome into host chromosomes is essential for replication of human immunodeficiency virus type 1 (HIV-1). Preintegration complexes (PIC) are formed in the cytoplasm after reverse transcription of the viral RNA. Integrase (IN) in the PIC catalyzes the excision of two nucleotides adjacent to the phylogenetically conserved CA dinucleotide from the 3Ј-OH blunt ends of the viral DNA (7). After nuclear transport of the PIC, the two viral DNA ends are inserted by IN into the host genome in a concerted fashion, here termed "full-site integration" (8,25,44). The unpaired dinucleotides at the 5Ј ends of the inserted viral DNA are removed, and the single-stranded gaps are repaired by a host cell DNA repair pathway (35). This process results in a short duplication of cell DNA ranging in size from 4 to 6 bp depending on the retrovirus species.Significant progress in our understanding of retrovirus fullsite integration was initiated by isolating PIC from virus-infected cells (8). Purified HIV-1 and murine leukemia virus (MLV) PIC are capable of incorporating ϳ15 to 50% of their viral DNA into an exogenously supplied DNA target as fullsite integration products after 45 to 90 min of incubation at 37°C (6,11,13,21,39,48). One report using HIV-1 PIC showed an ϳ95% incorporation of viral DNA into a target substrate where incorporation was essentially over after 45 min of incubation at 37°C (21). Cellular factors like barrier-toautointegration factor (BAF) appear to play a role in maintaining stable MLV and HIV-1 PIC structures, thus promoting full-site integration by preventing integration of the viral DNA into itself, termed "autointegration" (12,33,48,55).Simplified integration assays using model linear retrovirus DNA with terminal U5 and U3 long terminal repeat (LTR) sequences, purified recombinant IN, and a DNA target were also developed (9,16,32,45). These studies established that IN was the only viral protein necessary for both 3Ј-OH processing and DNA strand transfer activities.Further progress in reconstituting the HIV-1 full-site integration process was made using IN derived from nonionic detergent lysates of virus particles (10,22,23). The 3Ј-OH recessed ends containing attachment (att) site sequences from two different viral donors (480 bp) were integrated by IN in a concerted manner into a circular DNA target (bimolecular

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“…Avian retrovirus IN efficiently assembles the SC in the absence of catalysis at 14°C using 3Ј-OH recessed donor substrates (5,44,45). Examination of HIV-1 IN-DNA complexes on native gels at various times during incubation at 37°C revealed the presence of the SC migrating at ϳ3.4 kbp (Fig.…”

mentioning

confidence: 99%

Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate

Pandey

Bera

Zahm

et al. 2007

J Virol

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124

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The human immunodeficiency virus type 1 (HIV-1) life cycle involves the reverse transcription of the viral RNA genome into cDNA and subsequent integration of the viral DNA by integrase (IN) into the host chromosomes. Although the amino acid sequences of INs differ significantly between viruses, INs share three conserved structural domains and their associated functions (13). The N-terminal domain (ϳ50 residues) contains a zinc-binding region (7), promotes multimerization (46), and is necessary for 3Ј-OH processing and strand transfer. The catalytic core domain (CCD) (ϳ162 residues) contains the highly conserved acidic D, D-35-E motif (27) that is involved in coordinating Mg 2ϩ for 3Ј-OH processing and strand transfer activities (4, 13). The catalytic core is also involved in target binding for strand transfer (3,20,26,40). The C-terminal domain (ϳ35 residues) binds to the viral DNA ϳ6 to 9 bp from the long terminal repeat (LTR) end (15); the C-terminus and CCD are also involved in IN multimerization (1, 24, 29a).IN, along with the reverse transcriptase and protease, is an antiretroviral target (2,35,38). Highly active antiretroviral therapy, consisting of various combinations of reverse transcriptase and protease inhibitors, has significantly decreased HIV-1 replication in humans. The emergence of multidrugresistant HIV-1 mutants and undesirable side effects associated with certain drug combinations necessitates continuing efforts to develop novel and effective combinational therapies. The addition of inhibitors of HIV-1 IN function would enhance highly active antiretroviral therapy. Raltegravir (MK-0518), an analog of the strand transfer inhibitor L-870,810 used in this report, is in phase III human clinical trials (18,33).Oligonucleotide-based assays in vitro have identified a large number of compounds that inhibit HIV-1 IN activities in vitro (25), the majority of which are ineffective at preventing HIV-1 replication in cell culture. The "strand transfer inhibitors" were identified as being effective against recombinant IN, suppressed HIV-1 replication in cell culture and in vivo, and were so named because of their selectivity in both cases towards inhibiting strand transfer over 16,21,22,38). The first generation of strand transfer inhibitors possessed a 1,3-diketo acid (DKA) pharmacophore, which served as a template in the development of the naphthyridine carboxamide inhibitors. They are structurally analogous to and function identically to DKA inhibitors but exhibit improved metabolic and pharmacokinetic properties and are represented here by compounds L-870,810 and L-870,812 (21, 23). DKA-mediated inhibition is accomplished by the contact of the DKA moiety with the divalent metal ion in the CCD of IN (19,38), and efficient inhibitor binding occurs only with IN bound to the viral DNA substrate (14,22,38

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Structural Organization of Avian Retrovirus Integrase in Assembled Intasomes Mediating Full-site Integration

Cited by 18 publications

References 49 publications

Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs

Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs

Recombinant Human Immunodeficiency Virus Type 1 Integrase Exhibits a Capacity for Full-Site Integration In Vitro That Is Comparable to That of Purified Preintegration Complexes from Virus-Infected Cells

Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate

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