Genetic analysis of the human immunodeficiency virus type 1 integrase protein

Shin, Cha‐Gyun; Taddeo, Brunella; Haseltine, William A.; Farnet, Chris M.

doi:10.1128/jvi.68.3.1633-1642.1994

Cited by 116 publications

(83 citation statements)

References 63 publications

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“…Previous alignments of retroviral IN sequences have identified several highly conserved residues which are likely to be involved in essential aspects of the integration reaction (13,18,21,23). Mutagenesis of such residues in human immunodeficiency virus type 1 (HIV-1) IN and analyses of the resulting recombinant proteins (9,13,25,32) or virions (14,24,29,33) have largely confirmed these predictions. Our previous muta-…”

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confidence: 88%

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Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication

Cannon

Byles

Kingsman

et al. 1996

J Virol

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We have previously identified a residue in the carboxyl terminus of human immunodeficiency virus type 1 integrase (HIV-1 IN), W-235, the requirement for which is only revealed in viral assays for integrase function (P. M. Cannon, W. Wilson, E. Byles, S. M. Kingsman, and A. J. Kingsman, J. Virol. 68:4768-4775, 1994.). Our further analysis of this region of retroviral IN has now identified several sequence motifs which are conserved in all the retroviruses we examined, apart from human spumaretrovirus. We have made mutations within these motifs in HIV-1 IN and examined their phenotypes when reintroduced into an infectious proviral clone. The deleterious effects of several of these mutations demonstrate the importance of these regions for IN function in vivo. We observed a further discrepancy, at a motif that is only conserved in the lentiviruses, in the ability of mutants to function in in vitro and in vivo assays. Substitutions both in this region and at W-235 abolish HIV-1 infectivity but do not affect particle production, morphology, reverse transcription, or nuclear import in T-cell lines. Taken together with the in vitro data suggesting that neither of these residues is directly involved in the catalytic reactions of IN, it seems likely that we have identified regions of IN that are essential for interactions with other components of the integration machinery.

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confidence: 88%

“…(A and E) wild-type WI3; (B and F) mutant L2 (K186Q); (C and G) mutant C1 (W235E); (D and H) mutant W235A. (14,29). We therefore examined particle composition both by Western blot (immunoblot) analysis and by electron microscopy.…”

Section: Conserved Sequences In the Carboxyl Terminus Of Retroviral Imentioning

confidence: 99%

Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication

Cannon

Byles

Kingsman

et al. 1996

J Virol

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“…It is a relatively protease-resistant domain (18) and contains the active site for all polynucleotidyl transfer activities (16,18,30,49,53). Essential to the active site is the highly conserved D,D-35-E region [D-X (39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58) -D-X (35) -E] with invariant residues at D-64, D-116, and E-152 (18,28,30). The region of integrase necessary for homodimer formation has been mapped to this core domain by both genetic and biochemical analyses (17,25,50).…”

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confidence: 99%

Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells

Wiskerchen

Muesing

1995

J Virol

247

134

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Integrase is the only viral protein necessary for integration of retroviral DNA into chromosomal DNA of the host cell. Biochemical analysis of human immunodeficiency virus type 1 (HIV-1) integrase with purified protein and synthetic DNA substrates has revealed extensive information regarding the mechanism of action of the enzyme, as well as identification of critical residues and functional domains. Since in vitro reactions are carried out in the absence of other viral proteins and they analyze strand transfer of only one end of the donor substrate, they do not define completely the process of integration as it occurs during the course of viral infection. In an effort to further understand the role of integrase during viral infection, we initially constructed a panel of 24 HIV-1 mutants with specific alanine substitutions throughout the integrase coding region and analyzed them in a human T-cell line infection. Of these mutant viruses, 12 were capable of sustained viral replication, 11 were replication defective, and 1 was temperature sensitive for viral growth. The replication defective viruses express and correctly process the integrase and Gag proteins. Using this panel of mutants and an additional set of 18 mutant viruses, we identified nine amino acids which, when replaced with alanine, destroy integrase activity. Although none of the replication-defective mutants are able to integrate into the host genome, a subset of them with alterations in the catalytic triad are capable of Tat-mediated transactivation of an indicator gene linked to the viral long terminal repeat promoter. We present evidence that integration of the HIV-1 provirus is essential not only for productive infection of T cells but also for virus passage in both cultured peripheral blood lymphocytes and macrophage cells.

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“…Class II IN substitutions or deletion of entire IN impair proper particle assembly [11, 13–25], morphogenesis [11, 15, 21–23, 26–28] and reverse transcription in target cells [10, 11, 17, 19–21, 23, 25–44], in some cases without impacting IN catalytic function [15, 16, 19, 20, 30, 31, 34, 36, 45–47]. A hallmark morphological defect of these viruses is the formation of aberrant viral particles with viral ribonucleoprotein (vRNP) complexes mislocalized outside of the conical CA lattice [11, 15, 21–23, 26–28]. Strikingly similar morphological defects are observed in virions produced from cells treated with allosteric integrase inhibitors (ALLINIs, also known as LEDGINs, NCINIs, INLAIs or MINIs) [26, 27, 48-55].…”

Section: Introductionmentioning

confidence: 99%

“…Eccentric virions generated via class II IN substitutions or ALLINI treatment are defective for reverse transcription in target cells [10, 11, 17, 19–21, 23, 25–44, 48, 49, 51, 54, 58, 62] despite containing equivalent levels of RT and vRNA genome as wild type (WT) particles [26, 63]. In addition, neither the condensation of the viral genome by NC [26, 63] nor its priming [63] appear to be affected.…”

Section: Introductionmentioning

confidence: 99%

Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis

Elliott

Eschbach

Koneru

et al. 2019

Preprint

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27A large number of HIV-1 integrase (IN) alterations, referred to as class II substitutions, exhibit 28 pleotropic effects during virus replication. However, the underlying mechanism for the class II 29 phenotype is not known. Here we demonstrate that all tested class II IN substitutions 30 compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly 31 reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely 32 affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii) 33 directly compromising IN-RNA interactions without substantially affecting IN levels or functional 34 IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral 35 ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of 36 the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for 37 the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion 38 maturation. 39 40 42 between the HIV-1 Gag and Gag-Pol polyproteins, and the viral RNA (vRNA) genome. At the 43 plasma membrane of an infected cell, Gag and Gag-Pol molecules assemble around a vRNA 44 dimer and bud from the cell as a spherical immature virion, in which the Gag proteins are 45 radially arranged [1-3]. As the immature virion buds, the viral protease enzyme is activated and 46 cleaves Gag and Gag-Pol into their constituent domains triggering virion maturation [1, 2].47 During maturation the cleaved nucleocapsid (NC) domain of Gag condenses with the RNA 48 genome and pol-encoded viral enzymes [reverse transcriptase (RT) and integrase (IN)] inside 49 the conical capsid lattice, composed of the cleaved capsid (CA) protein, which together form the 50 core [1-3]. 51 3 After infection of a target cell, RT in the confines of the reverse transcription 52 complex (RTC) synthesizes linear double stranded DNA from vRNA [4]. The vDNA is 53 subsequently imported into the nucleus, where the IN enzyme catalyzes its insertion into the 54 host cell chromosome [5, 6]. Integration is mediated by the intasome nucleoprotein complex that 55 consists of a multimer of IN engaging both ends of linear vDNA [7]. While the number of IN 56 protomers required for intasome function varies across Retroviridae, single particle cryogenic 57 electron microscopy (cryo-EM) structures of HIV-1 and Maedi-visna virus indicate that lentivirus 58 integration proceeds via respective higher-order dodecamer and hexadecamer IN arrangements 59 [8, 9], though a lower-order intasome comprised of an HIV-1 IN tetramer was also resolvable by 60 cryo-EM [9].61 A number of IN substitutions which specifically arrest HIV-1 replication at the integration 62 step have been described [10]. These substitutions are grouped into class I to delineate them 63 from a variety of other IN substitutions, which exhibit pleiotropic effects and are collectively 64 referred to as class II substitutions [10-12]. Class ...

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Genetic analysis of the human immunodeficiency virus type 1 integrase protein

Cited by 116 publications

References 63 publications

Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication

Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication

Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells

Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis

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