Folding of the multidomain human immunodeficiency virus type‐I integrase

Grandgenett, Duane P.; Goodarzi, Goodarz

doi:10.1002/pro.5560030604

Cited by 32 publications

(18 citation statements)

References 36 publications

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“…The TnS52-encoded protein p480 and an N-terminally histidine-tagged version, p480-His, have been over-expressed and purified, both protocols involving denaturation and renaturation. Several retroviral IN proteins have also been purified using histidine tags (Bushman et al, 1993;Jonsson et al, 1993;Katzman and Sudol, 1994) and HIV IN has been renatured by a method very similar to that described here (Grandgenett and Goodarzi, 1994). The in vitro properties of p480 and p480-His are similar, and consistent with the proposal that p480 is a transposase.…”

Section: Discussionsupporting

confidence: 61%

Tn552 transposase purification and in vitro activities.

et al. 1995

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The Staphylococcus aureus transposon Tn552 encodes a protein (p480) containing the ‘D,D(35)E’ motif common to retroviral integrases and the transposases of a number of bacterial elements, including phage Mu, the integron‐containing element Tn5090, Tn7 and IS3. p480 and a histidine‐tagged derivative were overexpressed in Escherichia coli and purified by methods involving denaturation and renaturation. DNase I footprinting and gel binding assays demonstrated that p480 binds to two adjacent, directly repeated 23 bp motifs at each end of Tn552. Although donor strand cleavage by p480 was not detected, in vitro conditions were defined for strand transfer activity with transposon end fragments having pre‐cleaved 3′ termini. Strand transfer was Mn(2+)‐dependent and appeared to join a single left or right end fragment to target DNA. The importance of the terminal dinucleotide CA‐3′ was demonstrated by mutation. The in vitro activities of p480 are consistent with its proposed function as the Tn552 transposase.

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Section: Discussionsupporting

confidence: 61%

Tn552 transposase purification and in vitro activities.

et al. 1995

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“…The membranes were blocked in 5% fat-free milk in Tris-saline buffer (0.05% Tween 20) for 1 h at room temperature. The blot was incubated overnight at 4°C with rabbit polyclonal HIV-1 IN peptide directed-antisera (1:1000 dilution)52, followed by incubating with HRP conjugated anti-rabbit antibody (1:125,000 dilution, Pierce). N-terminal (1–16 residues) and C-terminal (276–288) peptide antisera were used.…”

Section: Methodsmentioning

confidence: 99%

Molecular Interactions between HIV-1 Integrase and the Two Viral DNA Ends within the Synaptic Complex that Mediates Concerted Integration

Bera

Pandey

Vora

et al. 2009

Journal of Molecular Biology

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Summary A macromolecular nucleoprotein complex in retrovirus-infected cells, termed the preintegration complex, is responsible for the concerted integration of linear viral DNA genome into host chromosomes. Isolation of sufficient quantities of the cytoplasmic preintegration complexes for biochemical and biophysical analyses is difficult. We investigated the architecture of human immunodeficiency virus type-1 (HIV-1) nucleoprotein complexes involved in the concerted integration pathway in vitro. HIV-1 integrase (IN) non-covalently juxtaposes two viral DNA termini forming the synaptic complex, a transient intermediate in the integration pathway and shares properties associated with the preintegration complex. IN slowly processes two nucleotides from the 3′ OH ends and performs the concerted insertion of two viral DNA ends into target DNA. IN remains associated with the concerted integration product, termed the strand transfer complex. The synaptic complex and strand transfer complex can be isolated on native agarose gels for biochemical and biophysical analyses. In this report, in-gel fluorescence resonance energy transfer measurements demonstrated that the energy transfer efficiencies between the juxtaposed Cy3 and Cy5 5′-end labeled viral DNA ends in the synaptic complex (0.68 ± 0.09) was significantly different than observed in the strand transfer complex (0.07 ± 0.02). The calculated distances were 46 ± 3 Å and 83 ± 5 Å, respectively. DNaseI footprint analysis of the complexes revealed that IN protects U5 and U3 DNA sequences up to ~32 bp from the end, suggesting two IN dimers were bound per terminus. Enhanced DNaseI cleavages were observed at nucleotide positions 6 and 9 from the terminus on U3 but not on U5 suggesting independent assembly events. Protein-protein cross-linking of IN within these complexes revealed the presence of dimers, tetramers, and a larger-size multimer (>120 Kd). Our results suggest a new model where two IN dimers individually assemble on U3 and U5 ends prior to the non-covalent juxtaposition of two viral DNA ends, producing the synaptic complex.

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“…Primary antibodies for HIV-1 RT or HA-tagged RT detection were rabbit antiserum, mouse anti-RT (13, 21), or anti-HA (Sigma). Rabbit antiserum served as the primary antibody for HIV-1 IN detection (18); the secondary antibody was either a rabbit antimouse or donkey antirabbit horseradish peroxidase (HRP)-conjugated antibody. The manufacturer's protocols were followed for HRP activity detection (PerkinElmer).…”

Section: Methodsmentioning

confidence: 99%

A Single Amino Acid Substitution in HIV-1 Reverse Transcriptase Significantly Reduces Virion Release

Chiang

Wang

Pan

et al. 2010

J Virol

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HIV-1 protease (PR) mediates the proteolytic processing of virus particles during or after virus budding. PR activation is thought to be triggered by appropriate Gag-Pol/Gag-Pol interaction; factors affecting this interaction either enhance or reduce PR-mediated cleavage efficiency, resulting in markedly reduced virion production or the release of inadequately processed virions. We previously showed that a Gag-Pol deletion mutation involving the reverse transcriptase tryptophan (Trp) repeat motif markedly impairs PR-mediated virus maturation and that an alanine substitution at W401 (W401A) or at both W401 and W402 (W401A/ W402A) partially or almost completely negates the enhancement effect of efavirenz (a nonnucleoside reverse transcriptase inhibitor) on PR-mediated virus processing efficiency. These data suggest that the Trp repeat motif may contribute to the PR activation process. Here we demonstrate that due to enhanced Gag cleavage efficiency, W402 alanine or leucine substitution significantly reduces virus production. However, W402 replacement with phenylalanine does not significantly affect virus particle assembly or processing, but it does markedly impair viral infectivity in a single-cycle infection assay. Our results demonstrate that a single amino acid substitution at HIV-1 RT can radically affect virus assembly by enhancing Gag cleavage efficiency, suggesting that in addition to contributing to RT biological function during the early stages of virus replication, the HIV-1 RT tryptophan repeat motif in a Gag-Pol context may play an important role in suppressing the premature activation of PR during late-stage virus replication.

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Folding of the multidomain human immunodeficiency virus type‐I integrase

Cited by 32 publications

References 36 publications

Tn552 transposase purification and in vitro activities.

Tn552 transposase purification and in vitro activities.

Molecular Interactions between HIV-1 Integrase and the Two Viral DNA Ends within the Synaptic Complex that Mediates Concerted Integration

A Single Amino Acid Substitution in HIV-1 Reverse Transcriptase Significantly Reduces Virion Release

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