Human Immunodeficiency Virus Type 1 Reverse Transcriptase Activity Research Articles

Internal RNA 2'-O-methylation on the HIV-1 genome impairs reverse transcription.

Viral RNA genomes are modified by epitranscriptomic marks, including 2'-O-methylation that is added by cellular or viral methyltransferases. 2'-O-Methylation modulates RNA structure, function and discrimination between self- and non-self-RNA by innate immune sensors such as RIG-I-like receptors. This is illustrated by human immunodeficiency virus type-1 (HIV-1) that decorates its RNA genome through hijacking the cellular FTSJ3 2'-O-methyltransferase, thereby limiting immune sensing and interferon production. However, the impact of such an RNA modification during viral genome replication is poorly understood. Here we show by performing endogenous reverse transcription on methylated or hypomethylated HIV-1 particles, that 2'-O-methylation negatively affects HIV-1 reverse transcriptase activity. Biochemical assays confirm that RNA 2'-O-methylation impedes reverse transcriptase activity, especially at low dNTP concentrations reflecting those in quiescent cells, by reducing nucleotide incorporation efficiency and impairing translocation. Mutagenesis highlights K70 as a critical amino acid for the reverse transcriptase to bypass 2'-O-methylation. Hence, the observed antiviral effect due to viral RNA 2'-O-methylation antagonizes the FTSJ3-mediated proviral effects, suggesting the fine-tuning of RNA methylation during viral replication.

Sticklac-Derived Natural Compounds Inhibiting RNase H Activity of HIV-1 Reverse Transcriptase.

The emergence of drug-resistant viruses is a serious concern in current chemotherapy for human immunodeficiency virus type-1 (HIV-1) infectious diseases. Hence, antiviral drugs aiming at targets that are different from those of approved drugs are still required, and the RNase H activity of HIV-1 reverse transcriptase is a suitable target. In this study, a search of a series of natural compounds was performed to identify the RNase H inhibitors. Three compounds were found to block the RNase H enzymatic activity. A laccaic acid skeleton was observed in all three natural compounds. A hydroxy phenyl group is connected to an anthraquinone backbone in the skeleton. An acetamido-ethyl, amino-carboxy-ethyl, and amino-ethyl are bound to the phenyl in laccaic acids A, C, and E, respectively. Laccaic acid C showed a 50% inhibitory concentration at 8.1 μM. Laccaic acid C also showed inhibitory activity in a cell-based viral proliferation assay. Binding structures of these three laccaic acids were determined by X-ray crystallographic analysis using a recombinant protein composed of the HIV-1 RNase H domain. Two divalent metal ions were located at the catalytic center in which one carbonyl and two hydroxy groups on the anthraquinone backbone chelated two metal ions. Molecular dynamics simulations were performed to examine the stabilities of the binding structures. Laccaic acid C showed the strongest binding to the catalytic site. These findings will be helpful for the design of potent inhibitors with modification of laccaic acids to enhance the binding affinity.

Computational and Crystallographic Analysis of Binding Structures of Inhibitory Compounds for HIV-1 RNase H Activity.

Chemotherapy of human immunodeficiency virus type-1 (HIV-1) has significantly developed over the last three decades. The emergence of drug-resistant variants is, however, still a severe problem. The RNase H activity of HIV-1 reverse transcriptase is an attractive target for a new class of antiviral drugs because there is no approved inhibitor. The nitro-furan-carbonyl and nitro-thiophene-carbonyl groups are potent scaffolds for the HIV-1 RNase H inhibitor. In this work, the binding structures of six inhibitory compounds were obtained by X-ray crystal analysis in a complex with a recombinant protein of HIV-1 RNase H domain. Every inhibitory compound was found to be bound to the catalytic site with the furan- or thiophene-ring coordinated to two divalent metal ions at the binding pocket. All the atoms in nitro, furan, carbonyl, and two metals were aligned in the nitro-furan derivatives. The straight line connecting nitro and carboxyl groups was parallel to the plane made by two metal ions and a furan O atom. The binding modes of the nitro-thiophene derivatives were slightly different from those of the nitro-furan ones. The nitro and carbonyl groups deviated from the plane made by two metals and a thiophene S atom. Molecular dynamics simulations suggested that the furan O or thiophene S atom and carbonyl O atom were firmly coordinated to the metal ions. The simulations made the planar nitro-furan moiety well aligned to the line connecting the two metal ions. In contrast, the nitro-thiophene derivatives were displaced from the initial positions after the simulations. The computational findings will be a sound basis for developing potent inhibitors for HIV-1 RNase H activity.

Inhibition of the DNA polymerase and RNase H activities of HIV-1 reverse transcriptase and HIV-1 replication by Brasenia schreberi (Junsai) and Petasites japonicus (Fuki) components

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) possesses two distinct enzymatic activities: those of RNA- and DNA-dependent DNA polymerases and RNase H. In the current HIV-1 therapy, all HIV-1 RT inhibitors inhibit the activity of DNA polymerase, but not that of RNase H. We previously reported that ethanol and water extracts of Brasenia schreberi (Junsai) inhibited the DNA polymerase activity of HIV-1 RT [Hisayoshi et al. (2014) J Biol Macromol 14:59-65]. In this study, we screened 43 edible plants and found that ethanol and water extracts of Brasenia schreberi and water extract of Petasites japonicus strongly inhibit not only the activity of DNA polymerase to incorporate dTTP into poly(rA)-p(dT)15 but also the activity of RNase H to hydrolyze the RNA strand of an RNA/DNA hybrid. In addition, these three extracts inhibit HIV-1 replication in human cells, with EC50 values of 1-2µg/ml. These results suggest that Brasenia schreberi and Petasites japonicus contain substances that block HIV-1 replication by inhibiting the DNA polymerase activity and/or RNase H activity of HIV-1 RT.

Inhibition of the Ribonuclease H Activity of HIV-1 Reverse Transcriptase by GSK5750 Correlates with Slow Enzyme-Inhibitor Dissociation

Compounds that efficiently inhibit the ribonuclease (RNase) H activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have yet to be developed. Here, we demonstrate that GSK5750, a 1-hydroxy-pyridopyrimidinone analog, binds to the enzyme with an equilibrium dissociation constant (K(d)) of ~400 nM. Inhibition of HIV-1 RNase H is specific, as DNA synthesis is not affected. Moreover, GSK5750 does not inhibit the activity of Escherichia coli RNase H. Order-of-addition experiments show that GSK5750 binds to the free enzyme in an Mg(2+)-dependent fashion. However, as reported for other active site inhibitors, binding of GSK5750 to a preformed enzyme-substrate complex is severely compromised. The bound nucleic acid prevents access to the RNase H active site, which represents a possible biochemical hurdle in the development of potent RNase H inhibitors. Previous studies suggested that formation of a complex with the prototypic RNase H inhibitor β-thujaplicinol is slow, and, once formed, it dissociates rapidly. This unfavorable kinetic behavior can limit the potency of RNase H active site inhibitors. Although the association kinetics of GSK5750 remains slow, our data show that this compound forms a long lasting complex with HIV-1 RT. We conclude that slow dissociation of the inhibitor and HIV-1 RT improves RNase H active site inhibitors and may circumvent the obstacle posed by the inability of these compounds to bind to a preformed enzyme-substrate complex.

Inhibition of HIV-1 Reverse Transcriptase Activity by Brasenia schreberi (Junsai) Components

We screened for inhibitory activities of 25 wild vegetables and fruits for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Among them, ethanol- and water-extracts of Brasenia schreberi (Junsai) and water-extract of Petasites japonicus (Fuki) strongly inhibited the HIV-1 RT activity to incorporate dTTP into poly(rA)-p(dT)15. We tested HIV-1 RT inhibitory activities of 15 polyphenols, isolated from Brasenia schreberi. Among them, gossypetin and hypolaetin 7-O-glucoside inhibited the activity.

Theoretical studies of HIV-1 reverse transcriptase inhibition

Computational methods for accurately calculating the binding affinity of a ligand for a protein play a pivotal role in rational drug design. We herein present a theoretical study of the binding of five different ligands to one of the proteins responsible for the human immunodeficiency virus type 1 (HIV-1) cycle replication; the HIV-1 reverse transcriptase (RT). Two types of approaches are used based on molecular dynamics (MD) simulations within hybrid QM/MM potentials: the alchemical free energy perturbation method, FEP, and the pathway method, in which the ligand is physically pulled away from the binding site, thus rendering a potential of mean force (PMF) for the binding process. Our comparative analysis stresses their advantages and disadvantages and, although the results are not in quantitative agreement, both methods are capable of distinguishing the most and the less potent inhibitors of HIV-1 RT activity on an RNase H site. The methods can then be used to select the proper scaffold to design new drugs. A deeper analysis of these inhibitors through molecular electrostatic potential (MEP) and calculation of the binding contribution of the individual residues shows that, in a rational design, apart from the strong interactions established with the two magnesium cations present in the RNase H site, it is important to take into account interactions with His539 and with those residues that are anchoring the metals; Asp443, Glu478, Asp498 and Asp549. The MEPs of the active site of the protein and the different ligands show a better complementarity in those inhibitors that present higher binding energies, but there are still possibilities of improving the favourable interactions and decreasing those that are repulsive in order to design compounds with higher inhibitory activity.

Inhibitory activity of 9-phenylcyclohepta[d]pyrimidinedione derivatives against different strains of HIV-1 as non-nucleoside reverse transcriptase inhibitors

BackgroundThe non-nucleoside reverse transcriptase inhibitor (NNRTI), as a major component of the highly active antiretroviral therapy (HAART) to HIV-1 (human immunodeficiency virus type 1) infected patients, required the development of new NNRTIs with improved resistance profile and decreased toxicity. Therefore, a series of novel compounds, 9-phenylcyclohepta[d]pyrimidinedione derivatives (PCPs), were designed based on the chemical structure of TNK-651, to detect anti-HIV-1 activity.Results1-[(benzyloxy)methyl]-9-phenyl-cyclohepta[d] pyrimidinedione (BmPCP) among four PCPs has antiviral activity on laboratory-adapted HIV strains (HIV-1 SF33). The results showed 50% inhibition concentrations (IC50s) of BmPCP were 0.34 μM, 1.72 μM and 1.96 μM on TZM-bl, peripheral blood mononuclear cells (PBMCs) and MT4, respectively. It was also effective against infection by the predominant HIV-1 isolates in China, with IC50s at low μM levels. Its selectivity index (SI) ranged from 67 to 266 in different cells. The results of time-of-addition assay demonstrated that BmPCP inhibited HIV-1 infection by targeting the post entry of the HIV-1 replication cycle. For inhibition of HIV-1 reverse transcriptase activity, the IC50 values of BmPCP and NVP were 1.51 and 3.67 μM, respectively.ConclusionsBmPCP with a novel structure acts as a NNRTI to inhibit HIV-1 replication and can serve as a lead compound for further development of new anti-HIV-1 drugs.

Anti-HIV-1 activity of phloroglucinol derivative, 6,6′-bieckol, from Ecklonia cava

Ecklonia cava (EC), which is an edible marine brown alga with a broad range of bioactivities, belongs to the family of Laminariaceae. The bioactive 6,6’-bieckol, one of the main phloroglucinol derivatives naturally occurred from this genus, was isolated and characterized by NMR techniques. For the first time, human immunodeficiency virus type-1 (HIV-1) inhibitory activity of 6,6’-bieckol showed wild inhibition against HIV-1 induced syncytia formation (EC 50 1.72 μM), lytic effects (EC 50 1.23 μM), and viral p24 antigen production (EC 50 1.26 μM), respectively. This result was strongly and clearly supported by the further investigation also, which 6,6’-bieckol selectively inhibited the activity of HIV-1 reverse transcriptase (RT) enzyme with EC 50 of 1.07 μM, as well as HIV-1 entry. Moreover, unlike most of other tannins, 6,6’-bieckol exhibited no cytotoxicity at concentrations which inhibited HIV-1 replication almost completely. Thus, it can be suggested that the potentially effective 6,6’-bieckol might be employed as a drug candidate for development of new generation therapeutic agents against HIV.

Broad spectrum antiviral fraction from the lichen Ramalina farinacea

Extracts from the lichen Ramalina farinacea (RF) have previously been shown to be antibacterial, antifungal and most recently, inhibitory to lentiviral and adenoviral vectors. We now determined the antiviral activity of subfractions of the ethylacetate-soluble fraction of RF against a broad spectrum of wild-type viruses, including human immunodeficiency virus type1 (HIV-1), herpes simplex type1 (HSV-1) and respiratory syncytial virus (RSV). Wild-type HIV-1, HSV-1 or RSV were pre-incubated with various concentrations of the ET4 subfraction for 30 minutes 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 the beta-galactosidase expression of the respective indicator cell lines in single round infection assays while the anti-RSV activity was determined via an immunoflourescent technique, employing monoclonal mouse antibody against the P-protein of RSV (as 1° antibody) and a peroxidase-conjugated rabbit antimouse IgG (as 2° antibody). 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 RT assay. ET4 has previously been shown to strongly inhibit the infectivity of lentiviral, retroviral and adenoviral vectors. The present result also shows very strong inhibition of HIV-1, HSV-1 and RSV, with the 50% antiviral concentrations (IC50) of 0.33, 6.09 and 3.65µg/ml respectively. Time-of-addition studies suggest that both entry and post-entry steps of the HIV-1 replication cycle are targeted. Furthermore, ET4 inhibited HIV-1 RT with an IC50 of 0.022µg/ml. ET4 may represent a novel source for lead phyto-antiviral agents. Further fractionation will reveal whether the broad spectrum antiviral activity results from a single compound or from different compounds present in ET4. Acknowledgements: The support of the Alexander von Humboldt Foundation to ECO is gratefully acknowledged. State Committee for Scientific Research, Grant No PBZ-KBN 112/P06/2005

High Sequence Conservation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase under Drug Pressure despite the Continuous Appearance of Mutations

To define the extent of sequence conservation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in vivo, the first 320 amino acids of RT obtained from 2,236 plasma-derived samples from a well-defined cohort of 1,704 HIV-1-infected individuals (457 drug naïve and 1,247 drug treated) were analyzed and examined in structural terms. In naïve patients, 233 out of these 320 residues (73%) were conserved (<1% variability). The majority of invariant amino acids clustered into defined regions comprising between 5 and 29 consecutive residues. Of the nine longest invariant regions identified, some contained residues and domains critical for enzyme stability and function. In patients treated with RT inhibitors, despite profound drug pressure and the appearance of mutations primarily associated with resistance, 202 amino acids (63%) remained highly conserved and appeared mostly distributed in regions of variable length. This finding suggests that participation of consecutive residues in structural domains is strictly required for cooperative functions and sustainability of HIV-1 RT activity. Besides confirming the conservation of amino acids that are already known to be important for catalytic activity, stability of the heterodimer interface, and/or primer/template binding, the other 62 new invariable residues are now identified and mapped onto the three-dimensional structure of the enzyme. This new knowledge could be of help in the structure-based design of novel resistance-evading drugs.

The Amino Acid Asn136 in HIV-1 Reverse Transcriptase (RT) Maintains Efficient Association of Both RT Subunits and Enables the Rational Design of Novel RT Inhibitors

The highly conserved Asn136 is in close proximity to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type 1 (HIV-1) RT. Site-directed mutagenesis has revealed that the catalytic activity of HIV-1 RT mutated at position Asn136 is heavily compromised. Only 0.07 to 2.1% of wild-type activity is retained, depending on the nature of the amino acid change at position 136. The detrimental effect of the mutations at position 136 occurred when the mutated amino acid was present in the p51 subunit but not in the p66 subunit of the p51/p66 RT heterodimer. All mutant enzymes could be inhibited by second-generation NNRTIs such as efavirenz. They were also markedly more sensitive to the inactivating (denaturating) effect of urea than wild-type RT, and the degree of increased urea sensitivity was highly correlated with the degree of (lower) catalytic activity of the mutant enzymes. Replacing wild-type Asn136 in HIV-1 RT with other amino acids resulted in notably increased amounts of free p51 and p66 monomers. Our findings identify a structural/functional role for Asn136 in stabilization of the RT p66/p51 dimer and provide hints for the rational design of novel NNRTIs or drugs targeting either Asn136 in the beta7-beta8 loop of p51 or its anchoring point on p66 (the peptide backbone of His96) so as to interfere with the RT dimerization process and/or with the structural support that the p51 subunit provides to the p66 subunit and which is essential for the catalytic enzyme activity.

Novel method of inactivation of human immunodeficiency virus type 1 by the freeze pressure generation method

It has been reported that high-pressure (over 600 MPa) treatment at room temperature inactivates human immunodeficiency virus type 1 (HIV-1), and it has recently been shown that the high pressure generated by the expansion of water due to freezing (freeze pressure generation method, or FPGM) has an inactivating effect on bacteria and fungi. In this study, we examined the effects of treatment by FPGM on HIV-1. A sturdy vessel filled with water and securely closed with a lid was kept at 0 degrees C to -30 degrees C. High pressures of 200 MPa and 250 MPa were generated at -20 degrees C and -30 degrees C, respectively. When T-cell-tropic and macrophage-tropic laboratory strains of HIV-1 were kept at -10 degrees C, the virus infectivity decreased to approximately 1/100, and was completely lost at -20 degrees C and -30 degrees C. Four T-cell-tropic and four macrophage-tropic laboratory strains and clinical isolates of HIV-1 became completely inactivated at -30 degrees C. Treatment by FPGM at -20 degrees C to -30 degrees C reduced HIV-1 reverse transcriptase activity to approximately one tenth. In addition, treatment by FPGM at -20 degrees C was found to destroy the ability of HIV-1 to bind to CD4+ cells. In conclusion, this study showed that treatment by FPGM at -20 degrees C to -30 degrees C destroyed the infectivity of a wide range of HIV-1 strains, and suggested that the mechanisms of HIV-1 inactivation were the reduction in viral enzyme activity and the loss of the cell-binding ability of a viral envelope protein.

Inhibition of Human Immunodeficiency Virus Type-1 (HIV-1) Replication at a Reverse Transcription Step by Human Cell Factor(s)

Infection of human cell with human immunodeficiency virus type-1 (HIV-1) was suppressed by cellular genetic factor(s) at reverse transcription step. Although same amount of virus adsorbed on both cells, small amount of HIV-1 (IIIB strain) infected HeLa (MAGI/CCR5) cell, while large amount of HIV-1 infected HOS (GHOST/CXCR4) cell. Regulation of virus replication at postentry level by cellular factor(s) had an important role for low efficiency of HIV-1 infection to MAGI/CCR5 cell. Provirus DNA formation in MAGI/CCR5 cell was less efficient than in GHOST/CXCR4 cell. Once GHOST/CXCR4 cell was fused with MAGI/CCR5 cell, susceptibility against HIV-1 decreased. Further, HIV-1 reverse transcriptase (RT) activity was strongly inhibited by cytosolic protein, derived from MAGI/CCR5 cell, in vitro. This research cleared a certain human cell genetically carries some factor(s) which inhibits the activity of HIV-1 RT.

Antiviral activity of diterpenes isolated from the Brazilian marine alga Dictyota menstrualis against human immunodeficiency virus type 1 (HIV-1)

The antiviral effect of the CH2Cl2/MeOH-soluble fraction from the alga Dictyota menstrualis on HIV-1 replication was evaluated in vitro. The antiretroviral activity was attributed to two diterpenes: (6R)-6-hydroxydichotoma-3,14-diene-1,17-dial, named Da-1, and (6R)-6-acetoxi-dichotoma-3,14-diene-1,17-dial, named AcDa-1. Da-1 or AcDa-1 were added to the culture medium of HIV-1-infected PM-1 cells at different times post-infection or during virus adsorption/penetration. The results indicated that the compounds affected an early step of the virus replicative cycle. Virus binding and entry into the host cells were evaluated in the presence of each diterpene, but no inhibitory effect was observed. To evaluate provirus DNA synthesis/integration into the host genome, the viral protease coding sequence was amplified from total cellular DNA. Proviral DNA was not detected in infected cells incubated with the diterpenes. To investigate the effect of the diterpenes on the reverse transcription of the viral genomic RNA, the recombinant HIV-1 reverse transcriptase (RT) was assayed in vitro in the presence of each diterpene. Da-1 and AcDa-1 inhibited the RNA-dependent DNA-polymerase activity of HIV-1 RT in a dose-dependent manner. Taken together, our results demonstrate that both diterpenes inhibit HIV-1 RT and consequently virus replication.

Antiviral activity of diterpenes isolated from the Brazilian marine alga Dictyota menstrualis against human immunodeficiency virus type 1 (HIV-1)

The antiviral effect of the CH 2Cl 2/MeOH-soluble fraction from the alga Dictyota menstrualis on HIV-1 replication was evaluated in vitro. The antiretroviral activity was attributed to two diterpenes: (6R)-6-hydroxydichotoma-3,14-diene-1,17-dial, named Da-1, and (6R)-6-acetoxi-dichotoma-3,14-diene-1,17-dial, named AcDa-1. Da-1 or AcDa-1 were added to the culture medium of HIV-1-infected PM-1 cells at different times post-infection or during virus adsorption/penetration. The results indicated that the compounds affected an early step of the virus replicative cycle. Virus binding and entry into the host cells were evaluated in the presence of each diterpene, but no inhibitory effect was observed. To evaluate provirus DNA synthesis/integration into the host genome, the viral protease coding sequence was amplified from total cellular DNA. Proviral DNA was not detected in infected cells incubated with the diterpenes. To investigate the effect of the diterpenes on the reverse transcription of the viral genomic RNA, the recombinant HIV-1 reverse transcriptase (RT) was assayed in vitro in the presence of each diterpene. Da-1 and AcDa-1 inhibited the RNA-dependent DNA-polymerase activity of HIV-1 RT in a dose-dependent manner. Taken together, our results demonstrate that both diterpenes inhibit HIV-1 RT and consequently virus replication.

Inhibition of HIV-1 reverse transcriptase and protease by phlorotannins from the brown alga Ecklonia cava.

The bioassay-directed isolation of a marine brown alga, Ecklonia cava, afforded four phlorotannin derivatives, eckol (1), 8,8'-bieckol (2), 8,4"'-dieckol (3), and phlorofucofuroeckol A (4). Among these compounds, 2 and 3 exhibited an inhibitory effect on human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease. Specifically, they inhibited the RT more potently than the protease. The inhibitory activity of compound 2 (IC(50), 0.51 microM) against HIV-1 RT was comparable to that of nevirapine (IC(50), 0.28 microM), a reference compound. An enzyme kinetic assay showed that this compound inhibited the RNA-dependent DNA synthesis activity of HIV-1 RT noncompetitively against dUTP/dTTP with a K(i) value of 0.78 microM. With respect to the homopolymeric template/primer, (rA)n(dT)15, 8,8'-bieckol (2) displayed an uncompetitive type of inhibition (K(i), 0.23 microM).

Inhibition of human immunodeficiency virus type 1 reverse transcriptase, protease and integrase by bovine milk proteins

Different proteins have been isolated from bovine milk including lactoferrin, lactoperoxidase, glycolactin, angiogenin-1, lactogenin, α-lactalbumin, lactoglobulin and casein. These proteins have been assayed for inhibitory activity against human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, protease and integrase, enzymes crucial to the HIV-1 life cycle. It was found that different milk proteins inhibited the three aforementioned HIV enzymes to different extents. Lactoferrin strongly inhibited HIV-1 reverse transcriptase but only slightly inhibited HIV-1 protease and integrase. On the other hand, α-lactalbumin, β-lactoglobulin and casein inhibited HIV-1 protease and integrase to an appreciable extent but did not inhibit HIV-1 reverse transcriptase. Glycolactin and angiogenin-1 suppressed the activity of HIV-1 reverse transcriptase by a moderate extent but more powerfully inhibited HIV-1 protease and integrase. In comparison with the other milk proteins glycolactin was a strong inhibitor of HIV-1 protease and integrase and a moderate inhibitor of HIV-1 reverse transcriptase. Lactogenin was a strong inhibitor of HIV-1 integrase, a moderate inhibitor of HIV-1 reverse transcriptase and a weak inhibitor of HIV-1 protease.

An immunodominant neutralization epitope on the 'thumb' subdomain of human immunodeficiency virus type 1 reverse transcriptase revealed by phage display antibodies.

An antibody phage display library was produced from the splenocytes of mice immunized with an infectious vaccinia virus recombinant (WRRT) expressing the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The library was panned against HIV-1 RT. Two clones, 5F and 5G, which produced Fab fragments specific for RT, were isolated. Surprisingly, both 5F and 5G Fab fragments were capable of strongly inhibiting the RNA-dependent DNA polymerase activity of HIV-1 RT. A hybridoma cell line that produces the monoclonal antibody 7C4, which strongly inhibits RT activity, was established previously using splenocytes from mice immunized with WRRT by the same immunization protocol. The epitope recognized by 7C4 exists in the region of the template primer-binding sites (or the 'helix clump') of RT. By epitope mapping and competitive ELISA analysis, it was shown that the 5F and 5G Fab fragments were directed against the same, or a very closely related, epitope that is recognized by 7C4. The neutralizing activities of the 5F, 5G and 7C4 Fab fragments correlated with their affinities for HIV-1 RT. DNA sequencing indicated that the immunoglobulin genes of the heavy chains of 5G and 7C4, as well as those of the light chains of 5F and 5G, had the same origin. These results suggest that the neutralizing epitope, which is recognized by these antibodies, becomes immunodominant after repeated immunization of mice with WRRT. This unique epitope, HIV-1 RT-specific and immunodominant neutralizing epitope (HRSINE), is a logical target for new types of HIV-1 RT inhibitors and gene therapy.

Reduction of HIV-1 Infectivity and Reverse Transcriptase Activity by High Hydrostatic Pressure Treatment.

It has been reported that high hydrostatic pressure (HHP) treatment at 400 MPa or higher inactivated a laboratory strain of human immunodeficiency virus type 1 (HIV-1). We investigated the effects of HHP treatment on the infectivity and reverse transcriptase (RT) activity of HIV-1. The sensitivity to HHP treatment differed among the HIV-1 strains. The pressure required for the inactivation of two clinical isolates was over 100 MPa higher than that of the laboratory strain. The RT activity of HIV-1 treated by HHP closely correlated with the decrease in the infectivity of each of the three strains tested. The decrease in the activity of RT may be related to the reducing the infectivity of HIV.