Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis

Woerner, Amy M.; Marcus-Sekura, Carol J.

doi:10.1093/nar/21.15.3507

Cited by 122 publications

(100 citation statements)

References 35 publications

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“…In our UV cross-linking assays with oligonucleotides, the GP(Y/F) fragment did bind DNA with high efficiently. The finding that a region of the C-terminal domain bound DNA and that it was a nonspecific DNA binding activity paralleled the activities of other INs (31)(32)(33)(34)(35)(36). However, our study of single amino acid substitutions indicated that the GP(Y/F) residues within the GP(Y/F) fragment did not contribute to this DNA binding.…”

Section: Discussionsupporting

confidence: 56%

The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein

Ebina

Chatterjee²,

Judson³

et al. 2008

Journal of Biological Chemistry

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Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and crosslinking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining. Retroviruses and long terminal repeat (LTR)3 retrotransposons are closely related elements that depend on integrase (IN) to insert their cDNA into the genome of host cells. IN proteins are composed of three structurally distinct domains (1). The N-terminal domains contain an HHCC motif, and the catalytic core domains in the center of INs possess the DDE residues that mediate catalysis. The C-terminal domains of INs have little sequence conservation but possess nonspecific DNA binding activity.INs expressed as recombinant proteins possess a variety of catalytic activities. Oligonucleotides that model the termini of LTRs are trimmed by IN in a processing reaction that removes terminal nucleotides 3Ј of the conserved CA. Once the processing reaction is complete, strand transfer occurs. In this reaction, the 3Ј-hydroxyls of the terminal A serve as nucleophiles in transesterification reactions that cleave phosphodiester bonds in the target DNA and make covalent bonds between the 3Ј-ends of the viral DNA and the 5Ј-ends of the target DNA (2, 3). Under highly specific conditions, IN can catalyze concerted integration, the simultaneous insertion of two ends of donor DNA into the same site of target DNA (4 -7). In addition, INs also catalyze disintegration, the reverse of integration that uses model substrates that mimic one end of an LTR inserted into target DNA (8,9). This assay is a particularly sensitive method for measuring catalytic activity of INs, perhaps because it detects strand breaking and joining within a single substrate molecule, an intramolecular reaction.Currently, no molecular structures exist of an IN that possesses all three domains. The structures that do exist are of individual domains and do not include bound DNA. As a result, the function of the C-terminal domain in integration is not clear. An additional difficulty in determining the function of the C-terminal domains stems from their low levels of sequence conservation. However, close examination of C termini did identify two separate sequence modules that exist either alone or in combination in a wide variety of INs (10). One module termed the G...

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Section: Discussionsupporting

confidence: 56%

The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein

Ebina

Chatterjee²,

Judson³

et al. 2008

Journal of Biological Chemistry

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“…In line with this observation, it is worth noting that the C-terminal domain of IN, which has been implicated in both nonspecific (Engelman et al, 1994;Heuer and Brown, 1997;Lutzke et al, 1994;Vink et al, 1993;Woerner and Marcus-Sekura, 1993) and specific (Gao et al, 2001) DNA binding, is intact in the K156E/K159E mutant. Consistent with this notion, a considerable degree of nuclear accumulation was evident in transiently transfected cells at steady-state (Fig.…”

Section: Localization Of In Fused To An Nesmentioning

confidence: 61%

“…et al, unpublished) or by DNase I digestion followed by extraction under hypotonic conditions . These results highlight that HIV-1 IN tightly binds chromosomal DNA in vivo, a phenomenon that might be mediated by the nonspecific DNAbinding properties of the central core (Engelman et al, 1994;Heuer and Brown, 1997;Katzman and Sudol, 1995;Shibagaki and Chow, 1997) and/or C-terminal domains (Engelman et al, 1994;Heuer and Brown, 1997;Lutzke et al, 1994;Vink et al, 1993;Woerner and Marcus-Sekura, 1993).…”

Section: Resultsmentioning

confidence: 91%

Intracellular transport of human immunodeficiency virus type 1 integrase

Devroe

Engelman

Silver

2003

Journal of Cell Science

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The human immunodeficiency virus type 1 integrase protein has karyophilic properties; that is, it localizes to the cell nucleus according to a range of assays. As an essential component of the preintegration complex, it has been suggested that the karyophilic properties of integrase might facilitate transport of the preintegration complex through the nuclear pore complexes of nondividing cells. However, no experiments have satisfactorily identified a nuclear localization signal within integrase. In this work, we investigated the karyophilic properties of integrase in intact cells with hopes of identifying a genuine transferable nuclear localization signal. Our results confirm that integrase tightly binds chromosomal DNA in vivo. However, our analysis determined that large integrase fusion proteins are unable to access the nucleus, indicating that integrase might lack a transferable nuclear localization signal. In addition, we present several lines of evidence to indicate that DNA binding might facilitate integrase nuclear accumulation. Furthermore, our data indicate integrase is degraded in the cytoplasm by a proteasome-dependent process, an event that probably contributes to the apparent nuclear accumulation of integrase. These results provide new insight into human immunodeficiency virus type 1 integrase intracellular dynamics.

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“…The modest integration defect exhibited by the RRR mutant may be attributable to a lack of acetylation, but we believe that the lysine-to-arginine changes may degrade one of the functional activities of the integrase, perhaps by altering the ability of the IN CTD to bind either viral or target DNA (3,4,7,8,10,11,20,21,27,40,41). Although attempts to characterize integrase-DNA binding by computer modeling have been made (1,5,6,17,22,37,44), definitive structural details await description.…”

mentioning

confidence: 99%

“…Recently it has been shown that HIV-1 integrase (IN) is a substrate for histone acetyltransferase (HAT)-mediated p300 posttranslational acetylation both in vitro and in vivo (3). Interestingly, p300 modification targets lysine residues within the IN C-terminal domain (CTD), a domain known to possess intrinsic DNA binding activity in vitro (10,11,20,21,27,40,41). Since p300 acetylation affects the ability of a variety of DNA binding proteins to interact specifically with their respective target DNAs (12-14, 19, 30, 35, 42), these observations are of significant interest, especially in light of the related finding that posttranslational acetylation by p300 regulates HIV-1 integration (3).…”

mentioning

confidence: 99%

Posttranslational Acetylation of the Human Immunodeficiency Virus Type 1 Integrase Carboxyl-Terminal Domain Is Dispensable for Viral Replication

Topper

Luo

Zhadina

et al. 2007

J Virol

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Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis

Cited by 122 publications

References 35 publications

The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein

The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein

Intracellular transport of human immunodeficiency virus type 1 integrase

Posttranslational Acetylation of the Human Immunodeficiency Virus Type 1 Integrase Carboxyl-Terminal Domain Is Dispensable for Viral Replication

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