Human Immunodeficiency Virus Type 1 Protease Activity Research Articles

Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model

Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR’s specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes.

HIV-1 protease and reverse transcriptase inhibition by tiger milk mushroom (Lignosus rhinocerus) sclerotium extracts: In vitro and in silico studies

Background and aimLignosus rhinocerus (LR) is an edible mushroom with a variety of medicinal properties such as neurostimulation, immunomodulation, anti-inflammation, anti-oxidation, anti-proliferation, anti-diabetes and especially antiviral activity. Human immunodeficiency virus type-1 (HIV-1) needs the HIV-1 protease (PR) and reverse transcriptase (RT) for its replication. Therefore, both HIV-1 PR and RT are important targets for antiretroviral drug development. Experimental procedureThe crude hexane (LRH), ethanol (LRE) and water (LRW) extracts of LR were in vitro screened for inhibitory activity against HIV-1 PR and RT, then anti-HIV-1 activity on the infected MOLT-4 cells were determined. Chemical constituents of the extracts were identified by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC)-MS. The identified compounds were in silico analysed for drug-likeness property and molecular modelling. Results and conclusionAccording to our screening assays, LRE and LRW significantly inhibited both enzymes (25–55%), while LRH suppressed only the HIV-1 PR activity (88.97%). At 0.5 mg/ml of LRW showed significant inhibition of HIV-1 induced syncytial formation and p24 production in the infected MOLT-4 cells. Investigation of chemical analysis revealed that major groups of identified constituents found in the extracts were fatty acids, peptides and terpenoids. In silico analysis showed that heliantriol F and 6 alpha-fluoroprogesterone displayed great binding energies with HIV-1 PR and HIV-1 RT, respectively. These findings suggest that LR could be a potential source of compounds to inhibit HIV-1 PR and/or RT activities in vitro. Furthermore, our results provide beneficial data for the development of novel HIV-1 PR and RT inhibitors.

A Direct Interaction with RNA Dramatically Enhances the Catalytic Activity of the HIV-1 Protease In Vitro

Though the steps of human immunodeficiency virus type 1 (HIV-1) virion maturation are well documented, the mechanisms regulating the proteolysis of the Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) remain obscure. One proposed mechanism argues that the maturation intermediate p15NC must interact with RNA for efficient cleavage by the PR. We investigated this phenomenon and found that processing of multiple substrates by the HIV-1 PR was enhanced in the presence of RNA. The acceleration of proteolysis occurred independently from the substrate's ability to interact with nucleic acid, indicating that a direct interaction between substrate and RNA is not necessary for enhancement. Gel-shift assays demonstrated the HIV-1 PR is capable of interacting with nucleic acids, suggesting that RNA accelerates processing reactions by interacting with the PR rather than the substrate. All HIV-1 PRs examined have this ability; however, the HIV-2 PR does not interact with RNA and does not exhibit enhanced catalytic activity in the presence of RNA. No specific sequence or structure was required in the RNA for a productive interaction with the HIV-1 PR, which appears to be principally, though not exclusively, driven by electrostatic forces. For a peptide substrate, RNA increased the kinetic efficiency of the HIV-1 PR by an order of magnitude, affecting both turnover rate (kcat) and substrate affinity (Km). These results suggest that an allosteric binding site exists on the HIV-1 PR and that HIV-1 PR activity during maturation could be regulated in part by the juxtaposition of the enzyme with virion-packaged RNA.

Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

Establishment of a new cell line inducibly expressing HIV-1 protease for performing safe and highly sensitive screening of HIV protease inhibitors

The human immunodeficiency virus type 1 (HIV-1) protease (PR) plays an essential role in processing viral polyproteins into mature proteins. As a result, it is a major target for the development of drugs against AIDS. However, due to the rapid emergence of drug-resistant HIV, the development of novel HIV PR inhibitors is urgently needed. We recently established a new cell line E-PR293 which can be used as a safe, convenient and highly efficient assay system to screen HIV-1 PR inhibitors. In the cells, the HIV-1 PR is expressed in a chimeric protein with the green fluorescence protein (GFP). This assay measures the PR activity as a function of either the fluorescence of GFP or the cytotoxic activity of HIV-1 PR which is expressed in the cell. E-PR293 cells were maintained in the presence of doxycycline, which suppresses the expression of HIV-1 PR. The removal of doxycycline induces the expression of HIV-1 PR, which is used to screen HIV-1 PR inhibitors. In E-PR293 cells, the 50% inhibitory concentration of the cytotoxic effects by nelfinavir and saquinavir were as low as nanomolar levels, almost equal to those found in the HIV-infection assay.

Inhibition of HIV-1 replication by a peptide dimerization inhibitor of HIV-1 protease

Peptides based on the amino (N) and carboxy (C)-terminal regions of human immunodeficiency virus type-1 (HIV-1) protease and on the C-terminus of p6 * can inhibit HIV-1 protease activity by preventing dimerization. We developed a peptide dimerization inhibitor, P27, that included these domains and a cell permeable domain derived from HIV-1 Tat. P27 inhibited wild type (WT) and protease inhibitor (PI)-resistant HIV-1 protease (IC 50: 0.23–0.32 μM). Kinetic and biochemical assays confirmed that P27 inhibits protease dimerization. Fluorescein-labeled peptide accumulated in MT-2 cells and protected acutely infected MT-2 cells from HIV-1-induced cytotoxicity (IC 50: 5.1 μM). P27 also inhibited p24 accumulation from H9 and U937 cells chronically infected with WT or PI-resistant HIV-1. Immunoblot analysis on the supernatants and infected cells revealed a block in virus release by P27 rather than an inhibition of polyprotein processing. However, inhibition of p55 Gag processing by active-site inhibitors was enhanced when combined with P27, suggesting that P27 can affect protease function in maturing virions. Although P27 was rationally designed to block dimerization of the mature HIV-1 protease, the effects of P27 on HIV-1 replication may be related to partial inhibition of Gag-Pol processing leading to a disruption in virus release.

Inhibition of Cellular HIV-1 Protease Activity by Lysyl-tRNA Synthetase

During early assembly of human immunodeficiency virus type 1 (HIV-1), an assembly complex is formed, the components of which include genomic RNA, Gag, GagPol, tRNA(Lys), and lysyl tRNA synthetase (LysRS). Directly increasing or decreasing cellular expression of LysRS results in corresponding changes in viral infectivity and in the viral concentrations of LysRS, tRNA(Lys), and, surprisingly, reverse transcriptase (RT). Since altering the cellular expression of LysRS does not lead to a change in the incorporation of the RT precursor protein, GagPol, in protease-negative HIV-1, we propose that the altered viral content of RT resulting from alterations in cellular LysRS concentration results from the ability of LysRS to inhibit premature activation of Gag-Pol viral protease within the complex. Supporting this hypothesis, we find that increases and decreases in cellular LysRS expression are accompanied by 5-8-fold increases and 5-fold decreases, respectively, in the cytoplasmic proteolysis of Gag and GagPol to mature viral proteins. Using a novel bioluminescence resonance energy transfer assay to directly measure HIV-1 protease activity in vivo also indicates that the overexpression of LysRS in the cell reduces viral protease activity.

Blocking of human immunodeficiency virus type-1 virion autolysis by autologous p2(gag) peptide.

Our previous study suggested that the p2(gag) peptide, AEAMSQVTNTATIM, inhibits human immunodeficiency virus type 1 (HIV-1) protease (PR) activity in vitro. In this study, Ala substitutions (Met4Ala and Thr8Ala) and deletion of amino acid Asn9 within the nona p2(gag) peptide (AEAMSQVTN) were found to decrease the inhibitory effect on HIV-1 PR activity. Furthermore, treatment of PMA-activated latently infected T lymphocytes, ACH-2 cells, with the p2(gag) peptide (100 and 250 micro M) resulted in a decrease in the amount of p24(gag )in the resultant viral lysates derived from the cell-free supernatant. In addition, the HIV-1-Tat-p2(gag) fusion peptide was synthesized to effectively deliver the p2(gag) peptide into the cells. The fusion peptide was incorporated into chronically infected T lymphocytes, CEM/LAV-1 cells, as detected on indirect immunofluorescence analysis using anti-p2(gag) peptide monoclonal antibodies, which recognize the nona peptide (AEAMSQVTN) derived from the N-terminus of the p2(gag) peptide, and cleaved by HIV-1 PR in vitro. Treatment of CEM/LAV-1 cells with the fusion peptide also resulted in a decrease in the amount of p24(gag )in the resultant viral lysate derived from the cell-free supernatant. Taken together, these data suggest that the p2(gag) peptide consequently blocks the autolysis of HIV-1 virions for the conservation of viral species.

Oxidative modifications of kynostatin-272, a potent human immunodeficiency virus type 1 protease inhibitor: potential mechanism for altered activity in monocytes/macrophages.

Previous studies have indicated that human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) are less active at blocking viral replication in HIV-1 infected peripheral blood monocytes/macrophages (M/M) than in HIV-1-infected T cells. We explored the hypothesis that oxidative modification and/or metabolism of the PIs in M/M might account for this reduced potency. We first tested the susceptibility of several PIs (kynostatin-272 [KNI-272], saquinavir, indinavir, ritonavir, or JE-2147) to oxidation after exposure to hydrogen peroxide (H(2)O(2)): only KNI-272 was highly susceptible to oxidation. Treatment of KNI-272 with low millimolar concentrations of H(2)O(2) resulted in mono-oxidation of the sulfur in the S-methyl cysteine (methioalanine) moiety, as determined by reversed-phase high-performance liquid chromatography and mass spectrometry (RP-HPLC/MS). Higher concentrations of H(2)O(2) led to an additional oxidation of the sulfur in the thioproline moiety of KNI-272. None of the PIs were metabolized or oxidized when added to T cells and cultured for up to 12 days. However, when KNI-272 was added to M/M, the concentration of the original KNI-272 steadily decreased with a corresponding increase in the production of three KNI-272 metabolites as identified by RP-HPLC/MS. The structures of these metabolites were different from those produced by H(2)O(2) treatment. The two major products of M/M metabolism of KNI-272 were identified as isomeric forms of KNI-272 oxidized solely on the thioproline ring. Both metabolites had reduced capacities to inhibit HIV-1 protease activity when tested in a standard HIV-1 protease assay. These studies demonstrate that antiviral compounds can be susceptible to oxidative modification in M/M and that this can affect their antiviral potency.

Involvement of matrix metalloproteinases in human immunodeficiency virus type 1-induced replication by clinical Mycobacterium avium isolates.

The role of Mycobacterium avium isolates in modulating human immunodeficiency virus type 1 (HIV-1) replication was examined by use of an in vitro, resting T cell system. Two human clinical isolates (serotypes 1 and 4) but not an environmental M. avium isolate (serotype 2) enhanced HIV-1 replication. The M. avium-induced HIV-1 replication was not associated with cell activation or differential cytokine production or utilization. Addition of matrix metalloproteinase (MMP) inhibitors and their in vivo regulators, tissue inhibitors of metalloproteinases-1 and -2, abrogated M. avium-induced HIV-1 replication 80%-95%. The MMP inhibitors did not have any effect on the HIV-1 protease activity, suggesting that they may affect cellular processes. Furthermore, MMP-9 protein was differentially expressed after infection with clinical M. avium isolates and paralleled HIV-1 p24 production. Collectively, these data suggest that M. avium-induced HIV-1 replication is mediated, in part, through the induction of MMP-9.

Competitive Inhibition of Human Immunodeficiency Virus Type-1 Protease by the Gag-Pol Transframe Protein

The human immunodeficiency virus type-1 (HIV-1) transframe protein p6* is located between the structural and enzymatic domains of the Gag-Pol polyprotein, flanked by the nucleocapsid (NC) and the protease (PR) domain at its amino and carboxyl termini, respectively. Here, we report that recombinant highly purified HIV-1 p6* specifically inhibits mature HIV-1 PR activity. Kinetic analyses and cross-linking experiments revealed a competitive mechanism for PR inhibition by p6*. We further demonstrate that the four carboxyl-terminal residues of p6* are essential but not sufficient for p6*-mediated inhibition of PR activity. Based on these results, we suggest a role of the transframe protein p6* in regulating HIV-1 PR activity during viral replication.

Peptides derived from HIV-1 vif: a non-substrate based novel type of HIV-1 protease inhibitors

The retroviral protease (PR) is absolutely essential for completion of human immunodeficiency virus multiplication cycle, and cannot be replaced by any cellular function. Thus PR, like reverse transcriptase, is an ideal target for the development of anti-AIDS therapy. A large number of human immunodeficiency virus type-1 (HIV-1) PR inhibitors have been developed, and several are currently used as anti-AIDS drugs. These inhibitors are mainly based on the natural PR cleavage sites within the viral Gag and Gag-Pol precursors. The major difficulty encountered while using anti-HIV therapeutic agents in patients has been the rapid emergence of drug-resistant viral strains. Most of the mutations which convert the PR into inhibitor-resistant are located within the substrate binding subsites of the enzyme. Recently, it has been shown that the HIV-1 auxiliary protein Vif, and especially the N-terminal half of Vif (N′-Vif) specifically interacts with the viral PR and inhibits its activity. We now show that efficient inhibition of HIV-1 PR activity can be achieved using Vif-derived peptides. Based on the above model we have performed peptide mapping of N′-Vif in order to find a small peptidic lead compound which inhibits PR activity. The screening revealed that peptides derived from two regions in Vif spanning from residues 30–65 and 78–98 inhibit PR activity in vitro, specifically bind HIV-PR and inhibit HIV-1 production in vivo. Further mapping of these regions revealed the lead compounds Vif81–88 and Vif88–98. These peptides specifically inhibit and bind HIV-1 PR, but do not affect pepsin and rous sarcoma virus protease. In contrast to other known PR inhibitors, these peptides are not substrate-based and their sequences do not resemble the sequences of the natural PR substrates (cleavage sites). Moreover, the Vif-derived peptides themselves are not cleaved by HIV-1 PR. Conversion of the lead peptides into small backbone cyclic peptidomimetics is taking place nowadays in order to turn these lead compounds into metabolically stable selective novel type of HIV-PR non-substrate-based inhibitors.

Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.

Development of HIV-1 protease expression methods using the T7 phage promoter system.

New and simple human immunodeficiency virus type 1 (HIV-1) protease expression methods in Escherichia coli were developed using the T7 phage promoter system. In order to suppress leaky HIV-1 protease expression under the control of the T7 polymerase, two new methods were tested. One involved the introduction of supplementary T7 promoter regions into host cells [E. coli BL-21 (DE3)] containing the HIV-1 protease gene under the control of the T7 promoter. It was expected that the supplementary T7 promoter regions would compete with the HIV-1 protease expression vector for the T7 polymerase binding. The other involved the infection of late-log-phase cultures of E. coli JM109 harboring the same HIV-1 protease expression vector with the M13 phage expressing T7 polymerase. Both methods were effective, and transformants with the mature HIV-1 protease expression vector showed ten times higher HIV-1 protease activity than activities obtained with the autoprocessing vector. The expression systems described here are convenient and are also easily applicable for the expression of other proteins toxic for E. coli.

Antiviral properties of simple difunctionalized enols targeted to the HIV-1 protease

The human immunodeficiency virus type 1 (HIV-1) protease catalyses the specific cleavage of the virion structural polyproteins p55gag and p160gag-pol and is, therefore, essential for viral maturation. We have previously reported a series of low molecular weight non-peptidic enol-based compounds that inhibit the HIV-1 protease activity in a competitive fashion (Vaillancourt et al., Bioorg. Med. Chem., 2 (1994) 343–355). Here we demonstrate that VS-215 and VS-261, two of these non-peptidic inhibitors, impair viral polyprotein maturation and exhibit antiviral activity in infected MT4 cells. The ID 50 for these two compounds ranged between 24 and 50 μM whereas their TD 50 ranged between 60 and 200 μM depending on the cell lines used. The calculated therapeutic index of these two inhibitors both had values of 2.5 even though they were shown to be non cytotoxic at their ID 50. Their calculated permeability index ranged between 0.09 and 0.79 suggesting that these enol-based inhibitors efficiently reach the site of protease activity. These results provide new information on the therapeutic potential of this new class of protease inhibitors and emphasize the usefulness of enol chemistry in the development of anti-HIV-1 protease inhibitors.

Intrinsic activity of human immunodeficiency virus type 1 protease heterologous fusion proteins in mammalian cells.

We have generated various mammalian expression constructs that produce fusion proteins of human immunodeficiency virus type 1 (HIV-1) protease (PR) with the HIV-1 Nef protein. The expression of these proteins is inducible by the HIV-1 Tat protein. High-level expression of proteolytically active PR was produced from PR imbedded into Nef coding sequences, flanked by PR cleavage sites. The fusion protein was cleaved nearly to completion and did not exhibit the regulated processing that is seen with the virally encoded PR. No cytotoxic effect of PR expression was detected. The self-cleavage of PR could be inhibited by a specific inhibitor of HIV-1 PR (U75875). Elimination of the aminoterminal PR cleavage site did not have a measurable effect on cleavage of the precursor fusion protein. The cleaved fusion proteins appeared to be extremely unstable in the transfected cells. These findings demonstrate the intrinsic activity of HIV-1 PR in mammalian cells, in the context of a heterologous fusion protein.