Structure of the termini of DNA intermediates in the integration of retroviral DNA: Dependence on IN function and terminal DNA sequence

Roth, Monica J.; Schwartzberg, Pamela L.; Goff, Stephen P.

doi:10.1016/0092-8674(89)90401-7

Cited by 261 publications

(227 citation statements)

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“…The cleavage products migrated more slowly than the corresponding fragments from chemical sequencing reactions, which are known to terminate in 3'-PO4 (19). This suggests that HIV IN produced fragments with 3'-OH termini, as was previously demonstrated in murine in vitro retroviral integration systems (20)(21)(22). No significant cleavage was observed on the minus strand of the HIV U5 substrate, even though it contained a fortuitous CA dinucleotide located two bases from the 3' end.…”

Section: Resultsmentioning

confidence: 62%

“…The oligonucleotide mimic of the ASLV U3 terminus was likewise not a substrate for HIV IN, although this same oligonucleotide was shown to be a substrate for avian IN (18). A substantially reduced level of cleavage was observed with an HIV U5 substrate in which the C in the conserved CA dinucleotide was replaced with a T. Surprisingly, a similar change in the DNA sequence of a murine leukemia virus resulted in only a slight reduction in the level of the 3'-recessed viral DNA precursor in infected cells and in the rate of replication compared with the wildtype virus (22 The role of the retroviral IN in this process has not been fully elucidated. Genetic evidence suggests that IN is required for integration (1)(2)(3).…”

Section: Resultsmentioning

confidence: 93%

“…Genetic evidence suggests that IN is required for integration (1)(2)(3). Furthermore, in cells infected with murine leukemia viruses, functional IN is required for the formation of the 3'-recessed linear DNA molecule that is the probable immediate precursor to the integrated form of the DNA (21,22). A minimal role for IN would therefore be the selective removal of bases from the termini of the viral DNA in preparation for joining to the host cell DNA.…”

Section: Resultsmentioning

confidence: 99%

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

Sherman

Fyfe

1990

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

328

274

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The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2" over Mg2".

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Section: Resultsmentioning

confidence: 62%

Section: Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

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

Sherman

Fyfe

1990

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

328

274

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“…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)(4)(5). In vitro, purified integrase protein can carry out the terminal cleavage (6,7) and strand transfer reactions (8)(9)(10)(11).…”

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confidence: 99%

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

et al. 1999

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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....

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“…The viral integrase (IN) engages the LTR ends prior to catalyzing two spatially and temporally distinct chemical reactions. Soon after their synthesis (3), IN site-specifically processes each LTR end adjacent to an invariant CA dinucleotide (4,5), yielding CA OH 3Ј-hydroxyl groups that serve as the nucleophiles for the second reaction, DNA strand transfer. Because the viral replication intermediate at this point gains the ability to catalyze DNA strand transfer activity, 3Ј-processing operationally marks the transition of the RTC to the pre-integration complex (PIC) (Fig.…”

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confidence: 99%

Retroviral Integrase Proteins and HIV-1 DNA Integration

Krishnan

Engelman

2012

Journal of Biological Chemistry

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Retroviral integrases catalyze two reactions, 3-processing of viral DNA ends, followed by integration of the processed ends into chromosomal DNA. X-ray crystal structures of integrase-DNA complexes from prototype foamy virus, a member of the Spumavirus genus of Retroviridae, have revealed the structural basis of integration and how clinically relevant integrase strand transfer inhibitors work. Underscoring the translational potential of targeting virus-host interactions, small molecules that bind at the host factor lens epithelium-derived growth factor/ p75-binding site on HIV-1 integrase promote dimerization and inhibit integrase-viral DNA assembly and catalysis. Here, we review recent advances in our knowledge of HIV-1 DNA integration, as well as future research directions.Retroviral replication proceeds through an obligate proviral or integrated DNA recombination intermediate. Integration provides a favorable environment for efficient gene expression, ensures inheritance of the virus in both daughter cells during mitosis, and forms the basis for latent HIV-1 reservoirs that persist in the face of highly active antiretroviral therapy. The early events of retroviral replication take place within the context of nucleoprotein complexes that are derived from the core of the infecting virus particle (1, 2). Within the confines of the reverse transcription complex (RTC), 2 the reverse transcriptase enzyme copies single-stranded viral RNA into a linear double-stranded DNA molecule containing a copy of the LTR sequence at each end. The viral integrase (IN) engages the LTR ends prior to catalyzing two spatially and temporally distinct chemical reactions. Soon after their synthesis (3), IN site-specifically processes each LTR end adjacent to an invariant CA dinucleotide (4, 5), yielding CA OH 3Ј-hydroxyl groups that serve as the nucleophiles for the second reaction, DNA strand transfer. Because the viral replication intermediate at this point gains the ability to catalyze DNA strand transfer activity, 3Ј-processing operationally marks the transition of the RTC to the pre-integration complex (PIC) (Fig. 1). In the strand transfer step, IN utilizes the 3Ј-oxygen atoms to cut chromosomal DNA in a staggered fashion and simultaneously join the viral DNA ends to the 5Ј-phosphates of the target DNA (4, 6, 7). The resulting DNA recombination intermediate, with unjoined viral DNA 5Ј-ends, is repaired by host cell enzymes to yield the integrated provirus flanked by the duplication of the sequence of the staggered DNA cut (Fig. 1). The spumaviruses, which compose one of seven Retroviridae genera, are an exception to this generalized scheme, as DNA synthesis is largely complete prior to target cell infection (8). Research on the functionality of the active nucleoprotein complexes that mediate HIV-1 DNA integration has led to the development of antiviral inhibitors that target the IN and block integration. IN Domain Structure and Reaction MechanismRetroviral INs belong to a superfamily of proteins known as the retroviral IN superfam...

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Structure of the termini of DNA intermediates in the integration of retroviral DNA: Dependence on IN function and terminal DNA sequence

Cited by 261 publications

References 39 publications

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

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

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

Retroviral Integrase Proteins and HIV-1 DNA Integration

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Structure of the termini of DNA intermediates in the integration of retroviral DNA: Dependence on IN function and terminal DNA sequence

Cited by 261 publications

References 39 publications

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

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

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

Retroviral Integrase Proteins and HIV-1 DNA Integration

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Product

Resources

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