Activity Of HIV-1 Reverse Transcriptase Research Articles

Thermostable Kunitz trypsin inhibitor with cytokine inducing, antitumor and HIV-1 reverse transcriptase inhibitory activities from Korean large black soybeans

A large number of trypsin inhibitors belonging to various types have been purified from different kinds of legumes. In this study, by using liquid chromatography, a Kunitz type trypsin inhibitor (KBTI) with a molecular weight of 20107.645 Da was purified from Korean large black soybeans. KBTI reduced the proteolytic activities of trypsin and alpha-chymotrypsin with the activity of approximately 8520 BAEE units/mg and approximately 24 BTEE units/mg, respectively. It showed high thermal stability (0-100 degrees C) as well as stability over a large range of pH values (pH 3-11). Furthermore, KBTI inhibited HIV-1 reverse transcriptase activity with an IC(50) value of 0.71 microM and induced the release of pro-inflammatory cytokines such as TNF-alpha, IL-1beta, IL-2 and interferon-gamma at the mRNA level. KBTI exerted weak antiproliferative activity toward CNE-2 and HNE-2 nasopharyngeal cancer cells, MCF-7 breast cancer cells, and Hep G2 hepatoma cells. KBTI was destitute of mitogenic, ribonuclease and antifungal activities.

Campesin, a thermostable antifungal peptide with highly potent antipathogenic activities.

An 9.4-kDa antifungal peptide designated as campesin was isolated from seeds of the cabbage Brassica campestris. The isolation procedure involved affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Q-Sepharose and Mono S, and gel filtration on Superdex 75 and Superdex Peptide. The peptide was adsorbed on the first three chromatographic media. It exerted an inhibitory action on mycelial growth including Fusarium oxysporum and Mycosphaerella arachidicola, with an IC50 of 5.1 μM and 4.4 μM, respectively. The peptide was characterized by remarkable thermostability and pH stability. It inhibited proliferation of HepG2 and MCF cancer cells with an IC50 of 6.4 μM and 1.8 μM, and the activity of HIV-1 reverse transcriptase with an IC50 of 3.2 μM. It demonstrated lysolecithin binding activity.

Dipyridodiazepinone derivatives; synthesis and anti HIV-1 activity

Ten dipyridodiazepinone derivatives were synthesized and evaluated for their anti HIV-1 reverse transcriptase activity against wild-type and mutant type enzymes, K103N and Y181C. Two of them were found to be promising inhibitors for HIV-1 RT.

Establishment of a real-time PCR assay for detection of the activity of HIV-1 reverse transcriptase

Objective To reconstruct the initiative procedure of HIV-1 reverse transcription in vitro and establish a methodology of assessing activity of HIV-1 reverse transcriptase (RT) with real-time PCR Methods The tRNALys-3 gene was amplified from genome in healthy individuals through polymerase chain reaction (PCR), and then T7 transcription promoter was added in 5'-terminal of the tRNALys-3. The tRNA[Lys-3 cRNA product was obtained by applying T7 RNA polymerase through a transcription reaction. The 5'-LTR-PBS DNA was also obtained by transcription reaction from the HIV-1 infectious clone and inserted into pGEM-T easy vectors. 5'-LTR-PBS cRNA was obtained by applying SP6 RNA polymerase whose combining site was located in pGEM-T easy vectors. Then the two RNA samples was catalyzed by two kinds of standard reverse transcriptases (SuperScript Ⅲ and HIV-1 standard reverse transcriptase, respectively) and the cDNA was synthesised. The relative activity of RT was determined with the real-time PCR. Results The tRNALys-3 primer and the SP6-5'-LTR-PBS RNA were procured accurately, whose length were 93bp and 872 bp, respectively. After the following serial dilution of Super Script Ⅲ and HIV-1 standard reverse transeriptase:1 : 10, 1: 100, 1:1 000, 1:10 000, each step of reverse transcription process worked successfully. Real-time PCR results showed that Ct values of the two groups were 13.9, 18. 3, 20. 9, 24. 9 and 20. 4, 25. 5, 28. 7, 32. 5 respectively. Conclusion A novel real-time PCR method is developed to assay the RT activity directly through reconstructing the initiation of HIV reproduction, which may be helpful for clinical management, screening of new antiretroviral drugs, and drug resistance test. Key words: HIV-1; HIV reverse transcriptase; Polymerase chain reaction

Isolation and Characterization of Juncin, an Antifungal Protein from Seeds of Japanese Takana (Brassica juncea Var. integrifolia)

An 18.9 kDa antifungal protein designated juncin was isolated from seeds of the Japanese takana (Brassica juncea var. integrifolia). The purification protocol employed comprised anion-exchange chromatography on Q-Sepharose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration on Superdex 75. Juncin was adsorbed on Affi-gel blue gel and SP-Sepharose but unadsorbed on Q-Sepharose. The protein exhibited antifungal activity toward the phytopathogens Fusarium oxysporum, Helminthosporium maydis, and Mycosphaerella arachidicola with IC(50) values of 13.5, 27, and 10 μM, respectively. It was devoid of mitogenic activity toward splenocytes and nitric oxide inducing activity toward macrophages. It inhibited the proliferation of hepatoma (HepG2) and breast cancer (MCF7) cells with IC(50) values of 5.6 and 6.4 μM, respecitvely, and the activity of HIV-1 reverse transcriptase with an IC(50) of 4.5 μM. Its N-terminal sequence differed from those of antifungal proteins that have been reported to date. Compared with Brassica campestris and Brassica alboglabra antifungal peptides, juncin exhibits a different molecular mass and N-terminal amino acid sequence but similar biological activities.

Trypsin-Chymotrypsin Inhibitors from Vigna mungo Seeds

Three trypsin-chymotrypsin inhibitors were isolated from seeds of the black gram (Vigna mungo) with a procedure that entailed cation exchange chromatography on SP-Sepharose, anion exchange chromatography on Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q and Mono S, and gel filtration by FPLC on Superdex 75. Two of the trypsin-chymotrypsin inhibitors were adsorbed on the first four types of chromatographic media. All three inhibitors have a molecular mass of 16 kDa as judged by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The trypsin inhibitory activity of the inhibitors was attenuated in the presence of the reducing agent dithiothreitol. The remaining inhibitor was unadsorbed on SP-Sepharose but adsorbed on Q-Sepharose, Mono Q and Mono S. The protease inhibitors did not exert any inhibitory effect on hepatoma (Hep G2) and breast cancer (MCF 7) cells or antifungal action toward Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. Two of the inhibitors slightly inhibited the activity of HIV-1 reverse transcriptase, with an IC50 in the millimolar range.

Musa acuminata (Del Monte banana) lectin is a fructose-binding lectin with cytokine-inducing activity

A homodimeric, fructose-binding lectin was isolated from Del Monte bananas by using a protocol that involved ion-exchange chromatography on DEAE-cellulose and SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. Not only fructose, but also glucose, mannose, rhamnose and glucosamine could inhibit the lectin. The N-terminal amino acid sequence of its identical 15-kDa subunits was similar to lectins from other Musa species except for the deletion of the N-terminal glycine residue in Del Monte banana lectin. The hemagglutinating activity was stable up to 80 degrees C and also stable in the range pH 1-13. However, the hemagglutinating activity dwindled to an undetectable level at 90 degrees C. The lectin was capable of eliciting a mitogenic response in murine splenocytes and inducing the expression of the cytokines interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 in splenocytes. The lectin also inhibited proliferation of leukemia (L1210) cells and hepatoma (HepG2) cells and the activity of HIV-1 reverse transcriptase. The additional information obtained in the present study includes demonstration of fructose-binding activity and cytokine-inducing activity of Del Monte banana lectin. Fructose binding is an unusual characteristic of plant lectins. It is possible that the banana lectin can be developed into a useful anti-HIV, immunopotentiating and antitumor agent in view of its trypsin stability and thermostability.

Broad Spectrum Antiviral Fractions from the Lichen Ramalina farinacea (L.) Ach.

Background: An ethylacetate-soluble fraction (ET4) from the lichen Ramalina farinacea has previously been shown to inhibit the infectivity of lentiviral and adenoviral vectors, as well as wild-type HIV-1. We now determined the antiviral activity of ET4 against other wild-type viruses, including the herpes simplex virus type 1 (HSV-1) and the respiratory syncytial virus (RSV). Methods: Wild-type HIV-1, HSV-1 or RSV were pre-incubated with various concentrations of ET4 for 30 min at 37°C before adding to P4CCR5 indicator cell line (HIV-1), ELVIS TM indicator cell line (HSV-1) or HEp2 cell line (RSV) in 96-well microtitre plates. Controls contain virus alone without ET4. The anti-HIV and anti-HSV activities were quantified by estimating beta-galactosidase expression of the respective indicator cell lines while the anti-RSV activity was determined via an immunofluorescent technique, employing monoclonal mouse antibody against the P-protein of RSV. Toxicity of ET4 to cell lines was evaluated in parallel using either the BrdU incorporation method or the MTT method. The effect of ET4 on the enzymatic activity of HIV-1 reverse transcriptase was also evaluated using a chemiluminescent reverse transcriptase assay. Bioassay-guided fractionation of the whole methanol extract of R. farinacea involved sequential screening of HPLC fractions using a vector-based assay technique. Results:ET4 inhibited HSV-1 and RSV potently (IC₅₀ = 6.09 and 3.65 μg/ml, respectively). Time-of-addition studies suggest that both entry and post-entry steps of the HIV-1 replication cycle and the entry step of the RSV replication cycle are targeted. Furthermore, ET4 inhibited HIV-1 reverse transcriptase with an IC₅₀ of 0.022μg/ml. Bioassay-guided fractionation of ET4 led to the identification sub-fraction rfO, with activity against lentiviral vector and HIV-1 (RNA viruses) but not against HSV-1 (DNA virus) and sub-fraction rfM, with activity against HSV-1 but not against the lentiviral vector. Conclusions:ET4 represents a novel fraction from the lichen R. farinacea with broad spectrum antiviral activity against DNA viruses (adenovirus and HSV-1) and RNA viruses (HIV-1 and RSV). The effect against DNA and RNA viruses is mediated by different sub-fractions within R. farinacea.

Brassiparin, an antifungal peptide fromBrassica parachinensisseeds

Aims: To isolate and characterize an antifungal peptide from the seeds of Brassica parachinensis L.H.Bailey. Methods and Results: An antifungal peptide designated as brassiparin was isolated. It exhibited a molecular mass of 5716 Da. It potently inhibited mycelial growth in a number of fungal species including Fusarium oxysporum, Helminthosporium maydis, Mycosphaerella arachidicola and Valsa mali. The antifungal activity of brassiparin toward M. arachidicola exhibited pronounced thermostability and pH stability. It inhibited proliferation of hepatoma (HepG2) and breast cancer (MCF7) cells and the activity of HIV‐1 reverse transcriptase. Its N‐terminal sequence differed from those of antifungal proteins which have been reported to date. Conclusions: Brassiparin can be purified by using a protocol involving ion exchange chromatography, affinity chromatography and gel filtration. It manifests potent, thermostable and pH‐stable antifungal activity. It demonstrates antiproliferative activity toward tumour cells, and inhibitory activity toward HIV‐1 reverse transcriptase. Thus, brassiparin is a defense protein. Significance and Impact of the Study: Brassiparin represents one of the few antifungal proteins reported to date from Brassica species. Its antifungal activity has pronounced pH stability and thermostability. Brassiparin exhibits other exploitable activities such as antiproliferative activity toward hepatoma and breast cancer cells and inhibitory activity toward HIV‐reverse transcriptase.

Purification and Characterization of a Lectin from Phaseolus vulgaris cv. (Anasazi Beans)

A lectin has been isolated from seeds of the Phaseolus vulgaris cv. “Anasazi beans” using a procedure that involved affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The lectin was comprised of two 30-kDa subunits with substantial N-terminal sequence similarity to other Phaseolus lectins. The hemagglutinating activity of the lectin was stable within the pH range of 1–14 and the temperature range of 0–80°C. The lectin potently suppressed proliferation of MCF-7 (breast cancer) cells with an IC50 of 1.3 μM, and inhibited the activity of HIV-1 reverse transcriptase with an IC50 of 7.6 μM. The lectin evoked a mitogenic response from murine splenocytes as evidenced by an increase in [3H-methyl]-thymidine incorporation. The lectin had no antifungal activity. It did not stimulate nitric oxide production by murine peritoneal macrophages. Chemical modification results indicated that tryptophan was crucial for the hemagglutinating activity of the lectin.

A New Generation of Peptide-based Inhibitors Targeting HIV-1 Reverse Transcriptase Conformational Flexibility

The biologically active form of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) is a heterodimer. The formation of RT is a two-step mechanism, including a rapid protein-protein interaction "the dimerization step," followed by conformational changes "the maturation step," yielding the biologically active form of the enzyme. We have previously proposed that the heterodimeric organization of RT constitutes an interesting target for the design of new inhibitors. Here, we propose a new class of RT inhibitors that targets protein-protein interactions and conformational changes involved in the maturation of heterodimeric reverse transcriptase. Based on a screen of peptides derived from the thumb domain of this enzyme, we have identified a short peptide P(AW) that inhibits the maturation step and blocks viral replication at subnanomolar concentrations. P(AW) only binds dimeric RT and stabilizes it in an inactive/non-processive conformation. From a mechanistic point of view, P(AW) prevents proper binding of primer/template by affecting the structural dynamics of the thumb/fingers of p66 subunit. Taken together, these results demonstrate that HIV-1 RT maturation constitutes an attractive target for AIDS chemotherapeutics.

Purification of melibiose-binding lectins from two cultivars of Chinese black soybeans

A dimeric 50 kDa melibiose‐binding lectin was isolated from the seeds of the cultivar of soybean (Glycine max), called the small glossy black soybean. The isolation procedure comprised ion exchange chromatography on Q Sepharose, SP Sepharose and Mono Q followed by gel filtration on Superdex 75. The lectin was adsorbed on all three ion exchangers, and it exhibited an N‐terminal sequence identical to that of soybean lectin. Of all the sugars tested, melibiose most potently inhibited the hemagglutinating activity of the lectin, which was stable between pH 3‐12 and 0‐70 °C. The lectin evoked maximal mitogenic response at about the same molar concentration as Con A. However, the response was much weaker. The soybean lectin inhibited the activity of HIV‐1 reverse transcriptase as well as the proliferation of breast cancer MCF7 cells and hepatoma HepG2 cells with an IC50 of 2.82 μM, 2.6 μM and 4.1 μM, respectively. There was no antifungal activity. Another lectin was isolated from a different cultivar of soybean called little black soybean. The lectin was essentially similar to small glossy black soybean lectin except for a larger subunit molecular mass (31 kDa), a more potent mitogenic activity and lower thermostability. The results indicate that different cultivars of soybean produce lectins that are not identical in every aspect.

Marmorin, a new ribosome inactivating protein with antiproliferative and HIV-1 reverse transcriptase inhibitory activities from the mushroom Hypsizigus marmoreus

Ribosome inactivating proteins (RIPs) are enzymes that inactivate ribosomes by eliminating one or more adenosine residues from rRNA, a 9,567-Da RIP with a novel N-terminal sequence was isolated from fresh fruiting bodies of the mushroom Hypsizigus marmoreus. The protein was unadsorbed on DEAE-cellulose, adsorbed on Affi-gel blue gel, and appeared as a single peak upon gel filtration on Superdex 75. The protein, designated as marmorin, inhibited proliferation of hepatoma Hep G2 cells and breast cancer MCF-7 cells, HIV-1 reverse transcriptase activity, and translation in the rabbit reticulocyte lysate system with an IC50 of 0.15 microM, 5 microM, 30 microM, and 0.7 nM, respectively. Compared to RIPs from hairy gourd, bitter gourd, ridge gourd, garden pea, and the mushroom Flammulina velutipes, marmorin was more potent in its antiproliferative activity toward hepatoma (HepG2) and breast cancer (MCF-7) cells, similar in inhibitory potency toward HIV-1 reverse transcriptase (with the exception that it was more potent than ridge gourd RIP and bitter gourd RIP), and less potent in translation-inhibitory potency. Marmorin was devoid of antifungal, protease, RNase, mitogenic, anti-mitogenic, nitric oxide-inducing, hemagglutinating, and trypsin inhibitory activities.

An antifungal protein fromBacillus amyloliquefaciens

To isolate and characterize an antifungal protein from the culture broth of the bacterium Bacillus amyloliquefaciens. The antifungal protein designated as baciamin was isolated and exhibited a molecular mass around 50 kDa. Baciamin manifested a broad spectrum of antifungal activity. Baciamin could induce membrane permeabilization of tested fungi. Its antifungal activity was retained after incubation with trypsin and EDTA. Various ions tested did not affect its antifungal activity. Baciamin reduced the activity of HIV-1 reverse transcriptase (RT). It also inhibited proliferation of hepatoma, breast cancer and colon cancer cell lines. Baciamin augmented nitric oxide production by mouse macrophages. Bacillus amyloliquefaciens produces a broad-spectrum antifungal protein, baciamin. It induces membrane permeabilization in fungi but not in rabbit erythrocytes. Its antifungal activity is relatively thermostable, pH- and trypsin-stable. It demonstrates antiproliferative activity towards various tumour cells, nitric oxide-inducing activity towards macrophages, and inhibitory activity towards HIV-1 RT. Baciamin represents one of the few bacterial antifungal proteins reported to date. Most of the previously isolated antifungal molecules of bacterial origin are either peptides or ring compounds. Baciamin also exhibits other exploitable activities such as antitumour and immuno-enhancing activities.

Preparation and Biological Properties of a Melibiose Binding Lectin from Bauhinia variegata Seeds

A dimeric 64-kDa melibiose-binding lectin was isolated from the seeds of Bauhinia variegata. The isolation procedure comprised affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Mono Q, and gel filtration on Superdex 75. The lectin was adsorbed on the first two chromatographic media. Its hemagglutinating activity was stable after 30-min exposure to temperatures up to 70 degrees C. Since lectins may demonstrate biological activities such as antiproliferative, immunomodulatory, antifungal, antiviral, and HIV-1 reverse transcriptase inhibitory activities, the isolated lectin was tested for these activities. It was found that the lectin inhibited proliferation in hepatoma HepG2 cells and breast cancer MCF7 cells with an IC(50) of 1.4 microM and 0.18 microM, respectively. HIV-1 reverse transcriptase activity was inhibited with an IC(50) of 1.02 microM. The lectin and concanavalin A (Con A) evoked maximal mitogenic response from mouse splenocytes at similar concentrations, but the maximal response to B. variegata lectin was only 1/5 of that induced by Con A in magnitude. B. variegata lectin was devoid of antifungal activity.

Vinylogous Ureas as a Novel Class of Inhibitors of Reverse Transcriptase-Associated Ribonuclease H Activity

High-throughput screening of National Cancer Institute libraries of synthetic and natural compounds identified the vinylogous ureas 2-amino-5,6,7,8-tetrahydro-4 H-cyclohepta[ b]thiophene-3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as inhibitors of the ribonuclease H (RNase H) activity of HIV-1 and HIV-2 reverse transcriptase (RT). A Yonetani-Theorell analysis demonstrated that NSC727447, and the active-site hydroxytropolone RNase H inhibitor beta-thujaplicinol were mutually exclusive in their interaction with the RNase H domain. Mass spectrometric protein footprinting of the NSC727447 binding site indicated that residues Cys280 and Lys281 in helix I of the thumb subdomain of p51 were affected by ligand binding. Although DNA polymerase and pyrophosphorolysis activities of HIV-1 RT were less sensitive to inhibition by NSC727447, protein footprinting indicated that NSC727447 occupied the equivalent region of the p66 thumb. Site-directed mutagenesis using reconstituted p66/p51 heterodimers substituted with natural or non-natural amino acids indicates that altering the p66 RNase H primer grip significantly affects inhibitor sensitivity. NSC727447 thus represents a novel class of RNase H antagonists with a mechanism of action differing from active site, divalent metal-chelating inhibitors that have been reported.

Inhibition of HIV-1 Protease and RNase H of HIV-1 Reverse Transcriptase Activities by Long Chain Phenols from the Sarcotestas ofGinkgo biloba

Correction to: Inhibition of HIV-1 Protease and RNase H of HIV-1 Reverse Transcriptase Activities by Long Chain Phenols from the Sarcotestas of Ginkgo bilobaPlanta Med 2008; 74(05): 532-534DOI: 10.1055/s-2008-1074497

Inhibition of HIV-1 protease and RNase H of HIV-1 reverse transcriptase activities by long chain phenols from the Sarcotestas of Ginkgo biloba

Nine long chain phenols: four cardanols, two bilobols and three alkylsalicylic acids were isolated from the CH2Cl2 extracts of the sarcotestas of Ginkgo biloba for HIV-1 protease (PR) inhibitors. From these phenols, the bilobols (IC50, 2.6–5.8µM) and alkylsalicylic acids (IC50, 10.2–24.9µM) exhibited dose-dependent potent inhibitory activities on HIV-1 PR, while the cardanols did not. On the other hand, only the alkylsalicylic acids (IC50, 33.7–170.3µM) inhibited the activities of RNase H of HIV-1 reverse transcriptase (RT) while all of the compounds failed to affect the RNA dependent DNA polymerase (RDDP) of HIV-1 RT. Therefore, we regard bilobols as a new class and selective inhibitors of HIV-1 PR; in addition, alkylsalicylic acids are elucidated as new class inhibitors of HIV-1 PR and RNase H of HIV-1 RT.

Methamphetamine Enhances HIV Infection of Macrophages

Epidemiological studies have demonstrated that the use of methamphetamine (meth), a sympathomimetic stimulant, is particularly common among patients infected with HIV. However, there is a lack of direct evidence that meth promotes HIV infection of target cells. This study examined whether meth is able to enhance HIV infection of macrophages, the primary target site for the virus. Meth treatment resulted in a significant and dose-dependent increase of HIV reverse transcriptase activity in human blood monocyte-derived macrophages. Dopamine D1 receptor antagonists (SCH23390 and SKF83566) blocked this meth-mediated increase in the HIV infectivity of macrophages. Investigation of the underlying mechanisms of meth action showed that meth up-regulated the expression of the HIV entry co-receptor CCR5 on macrophages. Additionally, meth inhibited the expression of endogenous interferon-alpha and signal transducer and activator of transcription-1 in macrophages. These findings provide direct in vitro evidence to support the possibility that meth may function as a cofactor in the immunopathogenesis of HIV infection and may lead to the future development of innate immunity-based intervention for meth users with HIV infection.

A novel and exploitable antifungal peptide from kale ( Brassica alboglabra) seeds

The aim of this study was to purify and characterize antifungal peptides from kale seeds in view of the paucity of information on antifungal peptides from the family Brassicaceae, and to compare its characteristics with those of published Brassica antifungal peptides. A 5907-Da antifungal peptide was isolated from kale seeds. The isolation procedure comprised affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose and Mono S, and gel filtration on Superdex Peptide. The peptide was adsorbed on the first three chromatographic media. It inhibited mycelial growth in a number of fungal species including Fusarium oxysporum, Helminthosporium maydis, Mycosphaerella arachidicola and Valsa mali, with an IC 50 of 4.3 μM, 2.1 μM, 2.4 μM, and 0.15 μM, respectively and exhibited pronounced thermostability and pH stability. It inhibited proliferation of hepatoma (HepG2) and breast cancer (MCF7) cells with an IC 50 of 2.7 μM and 3.4 μM, and the activity of HIV-1 reverse transcriptase with an IC 50 of 4.9 μM. Its N-terminal sequence differed from those of antifungal proteins which have been reported to date.