scholarly journals HIV-2 protease is inactivated after oxidation at the dimer interface and activity can be partly restored with methionine sulphoxide reductase

2000 ◽  
Vol 346 (2) ◽  
pp. 305-311 ◽  
Author(s):  
David A. DAVIS ◽  
Fonda M. NEWCOMB ◽  
Jackob MOSKOVITZ ◽  
Paul T. WINGFIELD ◽  
Stephen J. STAHL ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Peptide Mapping ◽  
Hiv Protease ◽  
Reversible Inactivation ◽  
Dimer Interface ◽  
Oxidative Inactivation ◽  
Human Immunodeficiency Viruses ◽  
Methionine Residues ◽  
Hiv 1

Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically. Here we demonstrate a related yet distinct mechanism of reversible inactivation of the HIV-2 protease. Exposure of the HIV-2 protease to H2O2 resulted in conversion of the two methionine residues (Met-76 and Met-95) to methionine sulphoxide as determined by amino acid analysis and mass spectrometry. This oxidation completely inactivated protease activity. However, the activity could be restored (up to 40%) after exposure of the oxidized protease to methionine sulphoxide reductase. This treatment resulted in the reduction of methionine sulphoxide 95 but not methionine sulphoxide 76 to methionine, as determined by peptide mapping/mass spectrometry. We also found that exposure of immature HIV-2 particles to H2O2 led to the inhibition of polyprotein processing in maturing virus particles comparable to that demonstrated for HIV-1 particles. Thus oxidative inactivation of the HIV protease in vitro and in maturing viral particles is not restricted to the type 1 proteases. These studies indicate that two distinct retroviral proteases are susceptible to inactivation after a very minor modification at residue 95 of the dimer interface and suggest that the dimer interface might be a viable target for the development of novel protease inhibitors.

In vitro susceptibility of clinical isolates of HIV-1 to XM323, a non-peptidyl HIV protease inhibitor

AIDS ◽  
1994 ◽  
Vol 8 (6) ◽  
pp. 753-756 ◽  
Author(s):  
Dean L. Winslow ◽  
Douglas Mayers ◽  
Helen Scarnati ◽  
James Lane ◽  
Arlene Bincsik ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Clinical Isolates ◽  
Hiv Protease ◽  
Hiv Protease Inhibitor ◽  
In Vitro Susceptibility ◽  
Hiv 1

A C2 Symmetry-Based HIV Protease Inhibitor, A77003, Irreversibly Inhibits Infectivity of HIV-1 In Vitro

1994 ◽  
Vol 10 (6) ◽  
pp. 735-743 ◽  
Author(s):  
SEIJI KAGEYAMA ◽  
DAVID T. HOEKZEMA ◽  
YOHKO MURAKAWA ◽  
EIJI KOJIMA ◽  
TAKUMA SHIRASAKA ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Hiv Protease ◽  
Hiv Protease Inhibitor ◽  
Hiv 1

Oxidation-resistant variants of recombinant anti-leucoprotease are better inhibitors of human-neutrophil-elastase-induced emphysema in hamsters than natural recombinant antileucoprotease

1991 ◽  
Vol 81 (6) ◽  
pp. 777-784 ◽  
Author(s):  
A. Rudolphus ◽  
R. Heinzel-Wieland ◽  
V. A. M. M. Vincent ◽  
D. Saunders ◽  
G. J. Steffens ◽  
...  
Keyword(s):  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Site Directed Mutagenesis ◽  
Human Neutrophil Elastase ◽  
Polymorphonuclear Leucocytes ◽  
Inhibitory Effects ◽  
Oxidative Inactivation ◽  
Methionine Residues

1. Antileucoprotease, being sensitive to oxidative inactivation, can be produced by recombinant techniques. Via site-directed mutagenesis, two mutants of recombinant antileucoprotease were produced in which one or more of the oxidation-sensitive methionine residues were replaced by leucine: in rALP242, methionine-73 was replaced by leucine, and in rALP231, leucine was substituted for four methionine residues. In vitro, native antileucoprotease and the recombinant antileucoprotease preparations have similar inhibitory characteristics towards human neutrophil elastase. We hypothesized that replacement of methionine residues in the antileucoprotease molecule would result in a reduced oxidation sensitivity of the mutants. 2. After incubation of recombinant antileucoprotease and its mutants with increasing dosages of cis-platinum(II)diammine dichloride, we observed that native antileucoprotease and recombinant antileucoprotease were inactivated by this reagent to the same extent. Compared with this, rALP242 was less inactivated, whereas the inhibitory capacity of rALP231 was not influenced by cis-platinum(II)diammine dichloride at all. 3. After incubation of recombinant antileucoprotease, rALP242 and rALP231 with triggered polymorphonuclear leucocytes, which are thought to produce an excess of oxidants, we measured residual inhibitory activities towards human neutrophil elastase of 10%, 55% and 87%, respectively. 4. In vivo, the inhibitory effects of intratracheally administered rALP242 and rALP231 towards human-neutrophil-elastase-induced emphysema were significantly greater than that of recombinant antileucoprotease. There were no significant differences between the mutants. With respect to secretory cell metaplasia and haemorrhage, rALP231 tended to be a better inhibitor than recombinant antileucoprotease and rALP242. 5. We conclude that the recombinant antileucoprotease mutants are less sensitive to oxidation and consequently inhibit human-neutrophil-elastase-induced emphysema to a greater extent than recombinant antileucoprotease.

scholarly journals The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

2003 ◽  
Vol 77 (1) ◽  
pp. 366-374 ◽  
Author(s):  
Steven C. Pettit ◽  
Sergei Gulnik ◽  
Lori Everitt ◽  
Andrew H. Kaplan
Keyword(s):  
Mutational Analysis ◽  
Expression System ◽  
Enzyme Activation ◽  
Nucleocapsid Proteins ◽  
Dimer Interface ◽  
Late Event ◽  
In The Wild ◽  
Retroviral Proteases ◽  
Hiv 1

ABSTRACT Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

scholarly journals 4′-Ethynyl Nucleoside Analogs: Potent Inhibitors of Multidrug-Resistant Human Immunodeficiency Virus Variants In Vitro

2001 ◽  
Vol 45 (5) ◽  
pp. 1539-1546 ◽  
Author(s):  
Ei-Ichi Kodama ◽  
Satoru Kohgo ◽  
Kenji Kitano ◽  
Haruhiko Machida ◽  
Hiroyuki Gatanaga ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Reverse Transcriptase ◽  
Wide Spectrum ◽  
Nucleoside Analogs ◽  
Transcriptase Inhibitor ◽  
Multidrug Resistant ◽  
Human Immunodeficiency Viruses ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACT A series of 4′-ethynyl (4′-E) nucleoside analogs were designed, synthesized, and identified as being active against a wide spectrum of human immunodeficiency viruses (HIV), including a variety of laboratory strains of HIV-1, HIV-2, and primary clinical HIV-1 isolates. Among such analogs examined, 4′-E-2′-deoxycytidine (4′-E-dC), 4′-E-2′-deoxyadenosine (4′-E-dA), 4′-E-2′-deoxyribofuranosyl-2,6-diaminopurine, and 4′-E-2′-deoxyguanosine were the most potent and blocked HIV-1 replication with 50% effective concentrations ranging from 0.0003 to 0.01 μM in vitro with favorable cellular toxicity profiles (selectivity indices ranging 458 to 2,600). These 4′-E analogs also suppressed replication of various drug-resistant HIV-1 clones, including HIV-1M41L/T215Y, HIV-1K65R, HIV-1L74V, HIV-1M41L/T69S-S-G/T215Y, and HIV-1A62V/V75I/F77L/F116Y/Q151M. Moreover, these analogs inhibited the replication of multidrug-resistant clinical HIV-1 strains carrying a variety of drug resistance-related amino acid substitutions isolated from HIV-1-infected individuals for whom 10 or 11 different anti-HIV-1 agents had failed. The 4′-E analogs also blocked the replication of a non-nucleoside reverse transcriptase inhibitor-resistant clone, HIV-1Y181C, and showed an HIV-1 inhibition profile similar to that of zidovudine in time-of-drug-addition assays. The antiviral activity of 4′-E-thymidine and 4′-E-dC was blocked by the addition of thymidine and 2′-deoxycytidine, respectively, while that of 4′-E-dA was not affected by 2′-deoxyadenosine, similar to the antiviral activity reversion feature of 2′,3′-dideoxynucleosides, strongly suggesting that 4′-Eanalogs belong to the family of nucleoside reverse transcriptase inhibitors. Further development of 4′-E analogs as potential therapeutics for infection with multidrug-resistant HIV-1 is warranted.

In vitro anti-HIV-1 activity of HIV protease inhibitor KNI-272 in resting and activated cells: implications for its combined use with AZT or ddI

1995 ◽  
Vol 28 (1) ◽  
pp. 25-38 ◽  
Author(s):  
Sudhichai Chokekijchai ◽  
Takuma Shirasaka ◽  
John N. Weinstein ◽  
Hiroaki Mitsuya
Keyword(s):  
Protease Inhibitor ◽  
Hiv Protease ◽  
Hiv Protease Inhibitor ◽  
Combined Use ◽  
Anti Hiv ◽  
Hiv 1

Double trouble? Gag in conjunction with double insert in HIV protease contributes to reduced DRV susceptibility

2019 ◽  
Vol 476 (2) ◽  
pp. 375-384 ◽  
Author(s):  
Alison Williams ◽  
Adriaan Basson ◽  
Ikechukwu Achilonu ◽  
Heini W. Dirr ◽  
Lynn Morris ◽  
...  
Keyword(s):  
Specific Activity ◽  
Catalytic Efficiency ◽  
Drug Susceptibility ◽  
Hiv Protease ◽  
Titration Calorimetry ◽  
Replication Capacity ◽  
Subtype C ◽  
Viral Replication Capacity ◽  
Hiv 1

AbstractHIV protease is essential for processing the Gag polyprotein to produce infectious virions and is a major target in antiretroviral therapy. We have identified an unusual HIV-1 subtype C variant that contains insertions of leucine and asparagine (L38↑N↑L) in the hinge region of protease at position 38. This was isolated from a protease inhibitor naïve infant. Isothermal titration calorimetry showed that 10% less of L38↑N↑L protease was in the active conformation as compared with a reference strain. L38↑N↑L protease displayed a ±50% reduction in KM and kcat. The catalytic efficiency (kcat/KM) of L38↑N↑L protease was not significantly different from that of wild type although there was a 42% reduction in specific activity for the variant. An in vitro phenotypic assay showed the L38↑N↑L protease to be susceptible to lopinavir (LPV), atazanavir (ATV) and darunavir in the context of an unrelated Gag. However, in the presence of the related Gag, L38↑N↑L showed reduced susceptibility to darunavir while remaining susceptible to LPV and ATV. Furthermore, a reduction in viral replication capacity (RC) was observed in combination with the related Gag. The reduced susceptibility to darunavir and decrease in RC may be due to PTAPP duplication in the related Gag. The present study shows the importance of considering the Gag region when looking at drug susceptibility of HIV-1 protease variants.

scholarly journals Envelope residue 375 substitutions in simian–human immunodeficiency viruses enhance CD4 binding and replication in rhesus macaques

2016 ◽  
Vol 113 (24) ◽  
pp. E3413-E3422 ◽  
Author(s):  
Hui Li ◽  
Shuyi Wang ◽  
Rui Kong ◽  
Wenge Ding ◽  
Fang-Hua Lee ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Germ Line ◽  
Virus Entry ◽  
Rhesus Macaques ◽  
Critical Determinant ◽  
Human Immunodeficiency Viruses ◽  
Subtype A ◽  
Hiv 1

Most simian–human immunodeficiency viruses (SHIVs) bearing envelope (Env) glycoproteins from primary HIV-1 strains fail to infect rhesus macaques (RMs). We hypothesized that inefficient Env binding to rhesus CD4 (rhCD4) limits virus entry and replication and could be enhanced by substituting naturally occurring simian immunodeficiency virus Env residues at position 375, which resides at a critical location in the CD4-binding pocket and is under strong positive evolutionary pressure across the broad spectrum of primate lentiviruses. SHIVs containing primary or transmitted/founder HIV-1 subtype A, B, C, or D Envs with genotypic variants at residue 375 were constructed and analyzed in vitro and in vivo. Bulky hydrophobic or basic amino acids substituted for serine-375 enhanced Env affinity for rhCD4, virus entry into cells bearing rhCD4, and virus replication in primary rhCD4 T cells without appreciably affecting antigenicity or antibody-mediated neutralization sensitivity. Twenty-four RMs inoculated with subtype A, B, C, or D SHIVs all became productively infected with different Env375 variants—S, M, Y, H, W, or F—that were differentially selected in different Env backbones. Notably, SHIVs replicated persistently at titers comparable to HIV-1 in humans and elicited autologous neutralizing antibody responses typical of HIV-1. Seven animals succumbed to AIDS. These findings identify Env–rhCD4 binding as a critical determinant for productive SHIV infection in RMs and validate a novel and generalizable strategy for constructing SHIVs with Env glycoproteins of interest, including those that in humans elicit broadly neutralizing antibodies or bind particular Ig germ-line B-cell receptors.

Computational Prospecting for the Pharmacological Mechanism of Activity: HIV-1 Inhibition by Ixoratannin A-2

2020 ◽  
Vol 16 (4) ◽  
pp. 376-388 ◽  
Author(s):  
Olujide O. Olubiyi ◽  
Thomas O. Idowu ◽  
Abiodun O. Ogundaini ◽  
Goodness Orhuah
Keyword(s):  
Antiviral Activity ◽  
Molecular Size ◽  
Multidrug Resistant ◽  
The Other ◽  
Hiv Protease ◽  
Strong Interactions ◽  
Replication Assay ◽  
Protease Enzyme ◽  
Hiv 1

Background: Ixora coccinea is a tropical ornamental shrub employed in ethnomedicine for the treatment of a number of diseases none of which include the Human Immunodeficiency Virus (HIV) infection. Ixoratannin A-2, one of the constituents, was previously identified via virtual-screening and experimentally confirmed to possess significant anti-HIV-1 activity in an in vitro CD4+ replication assay. This activity was observed to be significantly reduced in degree in viruses lacking the protein Vpu. This suggests the involvement of Vpu as well as other extra-Vpu macromolecules in its antiviral activity. Methods: In the present computational search for the identity of the other macromolecules that could possibly explain the observed activity, a panel of fourteen established HIV-1 macromolecular targets was assembled against which ixoratannin A-2 and other major phytoconstituents of I. coccinea were virtually screened. Results: Structural analyses of the computed ligand-bound complexes, as well as the careful investigation of the thermodynamic attributes of the predicted binding, revealed subtle selectivity patterns at the atomistic level that suggest the likely involvement of multiple macromolecular processes. Some of the binding interactions were found to be thermodynamically favourable, including the multidrug-resistant HIV protease enzyme, CXCR4 and the human elongin C protein all of which formed reasonably strong interactions with ixoratannin A-2 and other constituents of I. coccinea. Conclusion: Ixoratannin A-2’s ability to favourably interact with multiple HIV-1 and human targets could explain its observed extra-Vpu antiviral activity. This, however, does not imply uncontrolled binding with all available targets; on the other hand, molecular size of ixoratannin A-2 and combination of functional groups confer on it a decent level of selectivity against many of the investigated HIV/AIDS targets.

Inhibition of the integrases of human immunodeficiency viruses type 1 and type 2 by reverse transcriptases

2002 ◽  
Vol 361 (3) ◽  
pp. 557-566 ◽  
Author(s):  
Iris OZ ◽  
Orna AVIDAN ◽  
Amnon HIZI
Keyword(s):  
Protein Structures ◽  
Three Dimensional ◽  
Rnase H ◽  
Strand Transfer ◽  
Leukaemia Virus ◽  
Reverse Transcriptases ◽  
Show Evidence ◽  
Human Immunodeficiency Viruses ◽  
Hiv 1

We present evidence that the integrases (INs) of HIV types 1 and 2 are inhibited in vitro by the reverse transcriptases (RTs) of HIV-1, HIV-2 and murine leukaemia virus. Both 3′-end processing and 3′-end joining (strand transfer) activities of IN were affected by the RTs. Full inhibitions were accomplished with most RT and IN combinations tested at around equimolar RT/IN ratios. The disintegration activity of IN was also inhibited by RTs. Neither DNA synthesis nor the ribonuclease H (RNase H) domain of RT were involved in IN inhibition, since specific DNA polymerase inhibitors did not affect the level of IN inhibition, and the p51 isoform of HIV-1 RT (which lacks the RNase H domain) is as effective in inhibiting IN as the heterodimeric p66/p51 isoform. On the other hand, the catalytic activities of HIV RTs were not affected by the INs, showing that RTs can inhibit IN activities, whereas INs do not inhibit RTs. We postulate that sequences and/or three-dimensional protein structures common to RTs interact with INs and inhibit their activities. We show evidence for this hypothesis and discuss the possible sites of IN involved in this interaction.

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