Drug-Resistant HIV-1 Proteases Identify Enzyme Residues Important for Substrate Selection and Catalytic Rate

Ridky, Todd W.; Kikonyogo, Alexandra; Leis, Jonathan; Gulnik, Sergei V.; Copeland, Terry D.; Erickson, John E.; Wlodawer, Alexander; Kurinov, Igor; Harrison, Robert W.; Weber, Irene T.

doi:10.1021/bi980612k

Cited by 57 publications

(48 citation statements)

References 28 publications

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“…The amino acids that change in developing the drug resistance are predicted to be among those that are involved in specific recognition of the substrates. For PR, for example, the amino acids involved in specific recognition of the polyprotein cleavage sites were found to be among those that change when drug resistance was selected (65). Inhibitors of HIV-1 IN that function in the nanomolar range to inhibit the joining of the cellular and viral DNAs have been described (62)(63)(64).…”

Section: Discussionmentioning

confidence: 99%

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Identification of Amino Acids in HIV-1 and Avian Sarcoma Virus Integrase Subsites Required for Specific Recognition of the Long Terminal Repeat Ends

Chen

Weber

Harrison

et al. 2006

Journal of Biological Chemistry

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A tetramer model for HIV-1 integrase (IN) with DNA representing 20 bp of the U3 and U5 long terminal repeats (LTR) termini was assembled using structural and biochemical data and molecular dynamics simulations. It predicted amino acid residues on the enzyme surface that can interact with the LTR termini. A separate structural alignment of HIV-1, simian sarcoma virus (SIV), and avian sarcoma virus (ASV) INs predicted which of these residues were unique. To determine whether these residues were responsible for specific recognition of the LTR termini, the amino acids from ASV IN were substituted into the structurally equivalent HIV-12 DNA integration is a concerted process that occurs in defined stages. After assembly of a stable complex of the viral integrase (IN) and host cell proteins with specific DNA sequences at the end of the HIV-1 LTR, the terminal dinucleotides are removed from each 3Ј end by endonucleolytic processing. The viral DNA 3Ј ends are then covalently linked to the host cell target DNA in a concerted reaction. Biochemical details concerning the processing and joining steps in the integration process have been reported for several retroviruses including avian sarcomaleukosis virus (ASV), murine leukemia virus (MuLV), and HIV-1. Specific DNA sequences at the 3Ј end of the viral LTR are required for recognition by the assembled viral integrase complex. Typically, the terminal 12-20 nucleotides are sufficient. After 3Ј processing of the CAXX sequence from the ends, viral DNA ends are joined by the viral integrase and the gapped intermediates are repaired, and unpaired viral 5Ј ends are excised by host nucleases. This results in a 4 -6-base pair duplication of host cell DNA, depending upon the virus, flanking the integrated viral DNA. Both the 3Ј processing and viral DNA-host DNA joining steps require integrase to be assembled on the specific viral DNA substrate. Integration of viral into host DNA occurs with limited sequence specificity (1). Several cell proteins have been reported to affect the integration process (2-8).Much of the information available concerning the molecular mechanism of integration comes from the use of reconstituted systems employing duplex oligodeoxyribonucleotides (oligos). The ASV IN, for example, catalyzes specific cleavage at the 3Ј end of the strands adjacent to the conserved CA dinucleotide using 15-bp substrates corresponding to the ASV U3 or U5 termini (9, 10). Similar substrates were used to demonstrate the joining reaction in which one oligo integrated into another (11-12). For HIV-1 duplex oligo substrates of comparable size, selected nucleotide substitutions in the U5 and U3 LTR regions were shown to affect one or both of the catalytic functions of HIV-1 integrase. Nucleic acid substitutions in the HIV-1 U5 LTR region, for example, can inhibit 3Ј processing (e.g. positions 1-5 and 9 -11) or not (e.g. positions 6 -8 and 12-14) (13). Other changes at specific nucleic acid positions in the HIV-1 U3 and U5 LTR regions can affect each of the catalytic reactions, althou...

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Section: Discussionmentioning

confidence: 99%

“…This enzyme contains Ser substituted for Cys 56 , Cys 65 , and Cys 280 , and His substituted for Phe 185 , all of which improve the solubility properties of the enzyme. Positions 56 and 280 have different amino acids among the structurally aligned enzymes (see Fig.…”

Section: Structural Model Of Hiv-1 In With Ltr Dna-although a Crystalmentioning

confidence: 99%

Identification of Amino Acids in HIV-1 and Avian Sarcoma Virus Integrase Subsites Required for Specific Recognition of the Long Terminal Repeat Ends

Chen

Weber

Harrison

et al. 2006

Journal of Biological Chemistry

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“…Next, we consecutively chose 15 substrates predicted to be noncleavable by the CLM by HXB2 HIV-1 protease, allowing at most four amino acids to be identical at any same positions among all the substrates already selected (including the cleavable substrates already chosen above). If none of the remaining substrates met the requirements, a five-amino-acid similarity was allowed (Table 3, numbers [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33]. We then used the CLM to predict cleavability of the chosen 30 substrates by mutant HIV-1 proteases I84V, L90M, and I84V þ L90M.…”

Section: Methodsmentioning

confidence: 99%

A Look inside HIV Resistance through Retroviral Protease Interaction Maps

Kontijevskis¹,

Prūsis²,

Petrovska³

et al. 2005

PLoS Comput Biol

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Retroviruses affect a large number of species, from fish and birds to mammals and humans, with global socioeconomic negative impacts. Here the authors report and experimentally validate a novel approach for the analysis of the molecular networks that are involved in the recognition of substrates by retroviral proteases. Using multivariate analysis of the sequence-based physiochemical descriptions of 61 retroviral proteases comprising wild-type proteases, natural mutants, and drug-resistant forms of proteases from nine different viral species in relation to their ability to cleave 299 substrates, the authors mapped the physicochemical properties and cross-dependencies of the amino acids of the proteases and their substrates, which revealed a complex molecular interaction network of substrate recognition and cleavage. The approach allowed a detailed analysis of the molecular-chemical mechanisms involved in substrate cleavage by retroviral proteases.Citation: Kontijevskis A, Prusis P, Petrovska R, Yahorava S, Mutulis F, et al. (2007) A look inside HIV resistance through retroviral protease interaction maps. PLoS Comput Biol 3(3): e48.

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“…The amino acids flanking the target scissile bond are generally hydrophobic (16,33,37). A number of studies have used peptide or Gag substrates in an attempt to define the role of specific amino acids at different positions flanking the scissile bond as they impact the rate of cleavage by the viral protease (3,5,13,14,19,30,34,36,38,44,45,51,(53)(54)(55); recently reviewed in reference 27). In the present study we have examined the ability of approximately 15 different amino acids in the P1 position of the five different Gag cleavage sites to support cleavage by the HIV-1 protease (the P1 position is the amino acid immediately upstream of the scissile bond, and the P1Ј position is the amino acid immediately downstream of the scissile bond).…”

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confidence: 99%

Replacement of the P1 Amino Acid of Human Immunodeficiency Virus Type 1 Gag Processing Sites Can Inhibit or Enhance the Rate of Cleavage by the Viral Protease

Pettit

Henderson

Schiffer

et al. 2002

J Virol

105

116

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Processing of the human immunodeficiency virus type 1 (HIV-1) Gag precursor is highly regulated, with differential rates of cleavage at the five major processing sites to give characteristic processing intermediates. We examined the role of the P1 amino acid in determining the rate of cleavage at each of these five sites by using libraries of mutants generated by site-directed mutagenesis. Between 12 and 17 substitution mutants were tested at each P1 position in Gag, using recombinant HIV-1 protease (PR) in an in vitro processing reaction of radiolabeled Gag substrate. There were three sites in Gag (MA/CA, CA/p2, NC/p1) where one or more substitutions mediated enhanced rates of cleavage, with an enhancement greater than 60-fold in the case of NC/p1. For the other two sites (p2/NC, p1/p6), the wild-type amino acid conferred optimal cleavage. The order of the relative rates of cleavage with the P1 amino acids Tyr, Met, and Leu suggests that processing sites can be placed into two groups and that the two groups are defined by the size of the P1 amino acid. These results point to a trans effect between the P1 and P1 amino acids that is likely to be a major determinant of the rate of cleavage at the individual sites and therefore also a determinant of the ordered cleavage of the Gag precursor.

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Drug-Resistant HIV-1 Proteases Identify Enzyme Residues Important for Substrate Selection and Catalytic Rate

Cited by 57 publications

References 28 publications

Identification of Amino Acids in HIV-1 and Avian Sarcoma Virus Integrase Subsites Required for Specific Recognition of the Long Terminal Repeat Ends

Identification of Amino Acids in HIV-1 and Avian Sarcoma Virus Integrase Subsites Required for Specific Recognition of the Long Terminal Repeat Ends

A Look inside HIV Resistance through Retroviral Protease Interaction Maps

Replacement of the P1 Amino Acid of Human Immunodeficiency Virus Type 1 Gag Processing Sites Can Inhibit or Enhance the Rate of Cleavage by the Viral Protease

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