Comparison of the HIV‐1 and HIV‐2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag‐Pol polyproteins

Tőzsér, József; Bláha, Ivo; Copeland, Terry D.; Wondrak, Ewald M.; Oroszlan, Stephen

doi:10.1016/0014-5793(91)80362-7

Cited by 157 publications

(68 citation statements)

References 25 publications

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“…Previously, we performed extensive comparisons of the specificities of HIV-1 and HIV-2 proteinases using oligopeptides representing naturally occurring cleavage sites in their Gag and Gag-Pol polyproteins (15). These cleavage sites have been classified as type 1, which contains an aromatic amino acid and Pro at P 1 and P 1 Ј, respectively, and type 2, which has mainly hydrophobic residues but not Pro at the site of cleavage.…”

Section: Substitution Of Amino Acids Of a Type 1 Cleavage Site Peptidmentioning

confidence: 99%

“…These cleavage sites have been classified as type 1, which contains an aromatic amino acid and Pro at P 1 and P 1 Ј, respectively, and type 2, which has mainly hydrophobic residues but not Pro at the site of cleavage. We showed that an oligopeptide (peptide 1 in Table I) representing the cleavage site in p66 of HIV-1 for generating the p51 subunit of the heterodimeric reverse transcriptase of HIV-1 and another peptide (peptide 2 in Table I) representing the homologous sequence in p68 of HIV-2, and therefore proposed to be the cleavage site (22), were substrates of the HIV proteinases (15). These peptides, which match type 2 cleavage site sequences, were the starting points for our design of palindromic substrates since they are partly symmetric with aromatic amino acids at the P 1 , P 1 Ј, and they contain negatively charged residues at the P 3 and P 3 Ј positions.…”

Section: Substitution Of Amino Acids Of a Type 1 Cleavage Site Peptidmentioning

confidence: 99%

“…However, peptide 2 was found to be a much poorer substrate of the HIV proteinases than peptide 1 (see Table I and Ref. 15). Subsequently Fan et al (24) demonstrated that in fact the sequence of peptide 2 does not represent the actual cleavage site required to be cleaved to produce the smaller subunit of the HIV-2 reverse transcriptase.…”

Section: Substitution Of Amino Acids Of a Type 1 Cleavage Site Peptidmentioning

confidence: 99%

“…However, there is no obvious preference for symmetrical sequences in HIV proteinase cleavage sites (listed in Ref. 15) or in other retroviral proteinase cleavage sites (see Ref. 1).…”

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Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity

Tőzsér¹,

Bagossi²,

Weber³

et al. 1997

Journal of Biological Chemistry

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Two major types of cleavage sites with different sequence preferences have been proposed for the human immunodeficiency virus type 1 (HIV-1) proteinase. To understand the nature of these sequence preferences better, single and multiple amino acid substitutions were introduced into a type 1 cleavage site peptide, thus changing it to a naturally occurring type 2 cleavage site sequence. Our results indicated that the previous classification of the retroviral cleavage sites may not be generally valid and that the preference for a residue at a particular position in the substrate depends strongly on the neighboring residues, including both those at the same side and at the opposite side of the peptide backbone of the substrate. Based on these results, pseudosymmetric (palindromic) substrates were designed. The retroviral proteinases are symmetrical dimers of two identical subunits; however, the residues of naturally occurring cleavage sites do not show symmetrical arrangements, and no obvious symmetrical substrate preference has been observed for the specificity of HIV proteinase. To examine the role of the asymmetry created by the peptide bonds on the specificity of the respective primed and nonprimed halves of the binding site, amino acid substitutions were introduced into a palindromic sequence. In general, the results suggested that the asymmetry does not result in substantial differences in specificity of the S 3 and S 3 subsites, whereas its effect is more pronounced for the S 2 and S 2 subsites. Although it was possible to design several good palindromic substrates, asymmetrical arrangements may be preferred by the HIV proteinase.

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Section: Substitution Of Amino Acids Of a Type 1 Cleavage Site Peptidmentioning

confidence: 99%

Section: Substitution Of Amino Acids Of a Type 1 Cleavage Site Peptidmentioning

confidence: 99%

Section: Substitution Of Amino Acids Of a Type 1 Cleavage Site Peptidmentioning

confidence: 99%

“…However, there is no obvious preference for symmetrical sequences in HIV proteinase cleavage sites (listed in Ref. 15) or in other retroviral proteinase cleavage sites (see Ref. 1).…”

mentioning

confidence: 99%

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Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity

Tőzsér¹,

Bagossi²,

Weber³

et al. 1997

Journal of Biological Chemistry

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“…Within the viral context, the NC-p1 cleavage site is the slowest (50), final (37,54), and, therefore, rate-determining site in HIV-1 Gag to be processed by the viral protease (6,37). NC-p1 has a polar Asn at the P1 position, while a hydrophobic or aromatic residue is found at the same location in the other substrate sequences.…”

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confidence: 99%

Structural Basis for Coevolution of a Human Immunodeficiency Virus Type 1 Nucleocapsid-p1 Cleavage Site with a V82A Drug-Resistant Mutation in Viral Protease

Prabu-Jeyabalan

Nalivaika

King

et al. 2004

J Virol

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Maturation of human immunodeficiency virus (HIV) depends on the processing of Gag and Pol polyproteinsby the viral protease, making this enzyme a prime target for anti-HIV therapy. Among the protease substrates, the nucleocapsid-p1 (NC-p1) sequence is the least homologous, and its cleavage is the rate-determining step in viral maturation. In the other substrates of HIV-1 protease, P1 is usually either a hydrophobic or an aromatic residue, and P2 is usually a branched residue. NC-p1, however, contains Asn at P1 and Ala at P2. In response to the V82A drug-resistant protease mutation, the P2 alanine of NC-p1 mutates to valine (AP2V). To provide a structural rationale for HIV-1 protease binding to the NC-p1 cleavage site, we solved the crystal structures of inactive (D25N) WT and V82A HIV-1 proteases in complex with their respective WT and AP2V mutant NC-p1 substrates. Overall, the WT NC-p1 peptide binds HIV-1 protease less optimally than the AP2V mutant, as indicated by the presence of fewer hydrogen bonds and fewer van der Waals contacts. AlaP2 does not fill the P2 pocket completely; PheP1 makes van der Waals interactions with Val82 that are lost with the V82A protease mutation. This loss is compensated by the AP2V mutation, which reorients the peptide to a conformation more similar to that observed in other substrate-protease complexes. Thus, the mutant substrate not only binds the mutant protease more optimally but also reveals the interdependency between the P1 and P2 substrate sites. This structural interdependency results from coevolution of the substrate with the viral protease.Human immunodeficiency virus type 1 (HIV-1) matures after the viral protease processes (35) the Gag and Pol polyproteins at 10 substrate locations (3, 15). Therefore, inhibition of HIV-1 protease represents an important avenue for antiviral therapy (13, 48). The substrate sequences cleaved by the protease are nonhomologous (3), with the sequence of the nucleocapsid-p1 (NC-p1) substrate being the most different. In spite of the poor sequence homology among the substrate sites, a series of substrate-protease crystal structures led us to hypothesize that substrate specificity in HIV-1 protease results from the enzyme's recognizing an asymmetric shape (or envelope) rather than a particular amino acid sequence (40). This shape results from the conformation that a particular substrate sequence can adopt, implying that an interdependency necessarily exists among the different substrate residue sites.All of the protease inhibitors whose designs were structure based bind competitively (8,18,28,52,53) at the active site. Since these inhibitors bind at the same site as the substrates, many protease residues contact both substrates and inhibitors. Drug resistance, which often develops in the presence of therapeutic protease inhibitors, results from high viral turnover, the infidelity of the viral reverse transcriptase (16,42,43), and selective pressure on the virus. With drug resistance, the protease no longer binds as tightly to inhibitors but r...

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Targets of a protease inhibitor, KNI-272, in HIV-1-infected cells

et al. 2001

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The targets of a protease inhibitor, KNI-272, in the HIV-1 life cycle were investigated in this study. Neither expression of HIV-1 Gag proteins nor production of virus particles was detected in cells infected acutely with HIV-1 cultured in the presence of KNI-272. Although HIV-1 proviral DNA was detected in the cells by PCR, the inhibitor depressed the amount of the proviral DNA in a concentration dependent manner. These results indicate that one of the targets of KNI-272 occurs in the stage before the expression of viral structural proteins. No direct inhibition of reverse transcription was found with the inhibitor. To confirm the inhibition of viral protease, persistently HIV-1-infected cells were cultured in the presence of the inhibitor and examined by electron microscopy for the morphology of HIV-1 particles. Doughnut-shaped immature particles were observed in the extracellular space of the cells, and disrupted semicircular shaped particles were also seen at the higher concentration of KNI-272. A bioassay for infectivity showed that the virus particles were not infectious, and immunofluorescent assay using anti-p17 antibody, that does not react with the precursor of Gag protein, revealed that Gag precursor p55 protein in the cells was not processed. Thus, KNI-272 blocked the maturation of viral particles. Consequently, KNI-272 has at least two inhibition targets in the stages of the HIV-1 life cycle.

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Comparison of the HIV‐1 and HIV‐2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag‐Pol polyproteins

Cited by 157 publications

References 25 publications

Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity

Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity

Structural Basis for Coevolution of a Human Immunodeficiency Virus Type 1 Nucleocapsid-p1 Cleavage Site with a V82A Drug-Resistant Mutation in Viral Protease

Targets of a protease inhibitor, KNI-272, in HIV-1-infected cells

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