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PubMed1998

Optimal conditions of immune complex transfer enzyme immunoassays for antibody IgGs to HIV-1 using recombinant p17, p24, and reverse transcriptase as antigens.

The immune complex transfer enzyme immunoassays for antibody IgGs to p17, p24, and reverse transcriptase (RT) of HIV-1 were tested under various conditions. Antibody IgGs to HIV-1 were reacted for up to 20 hr with 2,4-dinitrophenyl-bovine serum albumin-recombinant HIV-1 protein conjugates and recombinant HIV-1 protein-beta-D-galactosidase conjugates, and the immune complexes formed, comprising the three components, were trapped onto polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG by incubation at 4-30 degrees C for up to 2 hr with shaking and were transferred onto polystyrene beads coated with (antihuman IgG gamma-chain) IgG in the presence of excess of epsilon N-2,4-dinitrophenyl-L-lysine by incubation at 4-30 degrees C for up to 2 hr with shaking. When serum randomly collected from an HIV-1 seropositive subject and serum included in an Western blot kit were tested, the formation of the immune complex was almost completed within 1 hr for antibody IgG to p17, within 1-2 hr for antibody IgG to p24 and within 4 hr for antibody IgG to RT. Even for antibody IgG to p17, however, the immune complex continued to be formed for at least 2 hr, when serum samples at early stages of HIV-1 infection were tested. Trapping and transferring of the immune complexes were faster at higher temperatures and were almost completed within 0.5-1.5 hr, although the amount of the immune complexes trapped and transferred at 25 and/or 30 degrees C increased for 0.5-1 hr, but subsequently tended to decline. When the formation, trapping, and transferring of the immune complexes were performed for 0.5, 1, and 1 hr, respectively, with shaking followed by 1 hr assay of bound beta-D-galactosidase activity, the sensitivities for antibody IgGs to p17, p24, and RT using 10 microliters of serum samples were similar to or significantly higher than those of the corresponding previous immune complex transfer enzyme immunoassays using 10 microliters of serum samples, in which the formation, trapping, and transferring of the immune complexes were performed for 3, 16, and 3 hr, respectively, without shaking, followed by 2.5 hr assay of bound beta-D-galactosidase activity, and the sensitivities for antibody IgGs to p17, p24, and RT using 100 microliters of serum samples were 21-22-fold, 5.5-6.3-fold, and 5.3-6.0-fold, respectively, higher. When each period of time for the formation, trapping, and transferring of the immune complexes was prolonged to up to 4 hr, the sensitivities for antibody IgGs to p17, p24, and RT using 100 microliters of serum samples were improved 88-93-fold, 15-17 fold and 20-24-fold, respectively, as compared with those of the previous ones.

Antigen-Antibody Complex↗
PubMed1986

Lack of detectable reverse transcriptase activity in human and chimpanzee sera with a high infectivity for non-A, non-B hepatitis.

A serum sample from a patient with hepatitis and samples from two experimentally infected chimpanzees, all with a high infectivity for non-A, non-B hepatitis, were tested for reverse transcriptase. Biopsy confirmed that the hepatocytes of the chimpanzees that received these sera contained the characteristic tubular structures associated with non-A, non-B hepatitis. None of these three sera revealed detectable enzyme activity. We have not been able to confirm the association of reverse transcriptase activity with non-A, non-B hepatitis reported recently.

Animals↗
PubMed2004

Roles of conformational and positional adaptability in structure-based design of TMC125-R165335 (etravirine) and related non-nucleoside reverse transcriptase inhibitors that are highly potent and effective against wild-type and drug-resistant HIV-1 variants.

Anti-AIDS drug candidate and non-nucleoside reverse transcriptase inhibitor (NNRTI) TMC125-R165335 (etravirine) caused an initial drop in viral load similar to that observed with a five-drug combination in naïve patients and retains potency in patients infected with NNRTI-resistant HIV-1 variants. TMC125-R165335 and related anti-AIDS drug candidates can bind the enzyme RT in multiple conformations and thereby escape the effects of drug-resistance mutations. Structural studies showed that this inhibitor and other diarylpyrimidine (DAPY) analogues can adapt to changes in the NNRTI-binding pocket in several ways: (1). DAPY analogues can bind in at least two conformationally distinct modes; (2). within a given binding mode, torsional flexibility ("wiggling") of DAPY analogues permits access to numerous conformational variants; and (3). the compact design of the DAPY analogues permits significant repositioning and reorientation (translation and rotation) within the pocket ("jiggling"). Such adaptations appear to be critical for potency against wild-type and a wide range of drug-resistant mutant HIV-1 RTs. Exploitation of favorable components of inhibitor conformational flexibility (such as torsional flexibility about strategically located chemical bonds) can be a powerful drug design concept, especially for designing drugs that will be effective against rapidly mutating targets.

Anti-HIV Agents↗
PubMed1994

Natural occurrence of drug resistance mutations in the reverse transcriptase of human immunodeficiency virus type 1 isolates.

Reverse transcriptase-associated amino acid substitutions related to ddC, d4T, and nevirapine resistance have been found in isolates of human immunodeficiency virus type 1 (HIV-1) from patients treated with AZT only. Sequence analysis of 23 isolates documented the presence of 4 unexpected mutations at amino acid residues related to drug resistance. Two isolates contained an aspartic residue in codon 69 associated with ddC resistance, and another a change in codon 75 associated with resistance to d4T. The Y-to-C alteration in codon 181 associated with nevirapine resistance was observed in another isolate after serial passage in cell culture in the absence of drug. Changes in substitution patterns were also noted after serial passage of four AZT resistant isolates in cell culture without inhibitors. One of the strains showed changes in codons 67 and 70 to wild-type residues. Clonal analysis showed that this alteration occurred by the selection during cell culture passage of the wild-type genotype, which was present as a minority subpopulation in the initially resistant virus stock, rather than to genetic reversion. In summary, we present evidence documenting the presence of mutations associated with drug resistance in the absence of drug treatment and supporting the role played by gentic variability in the emergence of HIV-1 antiviral resistance.

Amino Acid Sequence↗
PubMed2006

FEP-guided selection of bicyclic heterocycles in lead optimization for non-nucleoside inhibitors of HIV-1 reverse transcriptase.

Monte Carlo simulations using free energy perturbation theory have been used to guide the selection of bicyclic heterocycles in the lead optimization of non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). Good correlation is found between predicted and observed activities. Six compounds are reported with EC50 values below 20 nM for protection of human MT-2 cells against the cytopathogenicity of HIV-1. Striking variation in activity is found and analyzed for an isomeric pyrrolopyrimidine and pyrrolopyrazine pair.

Anti-HIV Agents↗
PubMed2006

Mad1 suppresses bladder cancer cell proliferation by inhibiting human telomerase reverse transcriptase transcription and telomerase activity.

OBJECTIVES: To study the effects and possible mechanisms of mitosis arrest deficiency 1 (Mad1), the heterodimerizer of Max and a transcriptional repressor, on cell proliferation of bladder cancer in vitro and in vivo. METHODS: Combining methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, luciferase assay, telomeric repeat amplification protocol-enzyme-linked immunosorbent assay, real-time reverse transcriptase polymerase chain reaction, experimental animal models, and other assays, we detected the alterations of cell proliferation, cell cycle, promoter activity and expression of human telomerase reverse transcriptase (hTERT), and telomerase activity in different treated bladder cells and xenograft tissues. RESULTS: Mad1 inhibited cell proliferation, increased G0/G1 accumulation in cell cycle distribution, decreased the transcription and expression of hTERT, and reduced telomerase activity compared with controls in T24 and EJ cells. Mad1 also arrested tumor growth and downregulated hTERT expression and telomerase activity in bladder cancer xenograft BALB/c nude mice. CONCLUSIONS: Mad1 inhibited the proliferation of human bladder cancer cells by inhibiting hTERT transcription and telomerase activity. Mad1 could be a potentially useful candidate for inhibition of bladder cancer growth.

Animals↗
PubMed1992

The observed inhibitory potency of 3'-azido-3'-deoxythymidine 5'-triphosphate for HIV-1 reverse transcriptase depends on the length of the poly(rA) region of the template.

The inhibitory potency of 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) against HIV-1 reverse transcriptase (HIV-1 RT) has been further evaluated. The results indicate that the previously reported low Ki values for AZTTP against HIV-1 RT (2.35 nM) are due neither to the to the direct tight binding of AZTTP to HIV-1 RT nor to the interaction of the enzyme with AZTMP moiety terminated primer-templates, but instead they are an artifact of the use of a homotemplate-primer [poly(rA).oligo(dT)]. With a set of RNAs of defined sequence as templates, we demonstrate that the observed Ki value for AZTTP depends on the length of the poly(rA) region following the primer in the RNA template. The more adenosyl residues in the RNA template that are available for processive incorporation of TMP moieties, the lower is the observed Ki value for AZTTP. Since the potencies of new inhibitors of HIV-1 RT are usually compared with that for AZTTP, these results have important consequences for the process of discovery of new HIV inhibitors that are of potential use in AIDS therapy.

Base Sequence↗
PubMed2004

[Antisense human telomerase reverse transcriptase inhibits leukemia cell proliferation in vitro].

OBJECTIVE: To study the inhibitory effect of antisense human telomerase reverse transcriptase (hTERT) on leukemia cell proliferation in vitro. METHODS: Sense and antisense hTERT eukaryotic expression vectors previously constructed were transfected into leukemia cell line HL(60) using SuperFect transfection reagent (Qiagen) to obtain HL(60)-s and HL(60)-as, and the G418-resistant colonies were identified for the presence of hTERT insert by PCR with T7 and pcDNA3.1/BGH reverse primers. Endogenous hTERT mRNA expression and telomerase activity were then detected by quantitative real-time RT-PCR and telomerase associated protein -silver staining in each cell line. MTT cellular proliferation assay, soft agar colony formation assay and flow cytometry were also employed to analyze the changes in proliferation capacity of leukemia cell in vitro and apoptosis of the tumor cells induced by antisense hTERT. RESULTS: Antisense hTERT remarkably reduced endogenous hTERT mRNA expression (P<0.01) and down-regulated telomerase activity in HL(60), as compared with the blank control and sense hTERT. After 25 passages of the 3 cell lines, a 7-day cell growth curve and the numbers (size) of soft agar colony formation showed that the proliferation rates and the anchorage-independent growth ability of HL(60)-as cells were significantly decreased in comparison with HL(60) and HL(60)-s cells, but a significant increase in apoptosis of HL(60)-as cells occurred as determined by flow cytometry. CONCLUSIONS: Antisense hTERT can obviously inhibit leukemia cell growth and proliferation in vitro, and this telomerase-targeted molecular biotherapy may be achieved by apoptosis pathway through down-regulation of hTERT mRNA and telomerase activity.

Antisense Elements (Genetics)↗
PubMed2005

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.

Asparagine↗
PubMed2004

High-level expression and purification of untagged and histidine-tagged HIV-1 reverse transcriptase.

We have devised simplified protocols to purify large quantities of histidine-tagged (His-tagged) and untagged heterodimeric forms of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT). Here, we report the optimization of overexpression and purification of heterodimeric RT expressed in Escherichia coli. The coding sequences of p66 and p51 subunits of RT were amplified using PCR from HXB2 HIV-1 and cloned into a bacterial expression system. The resulting expression plasmids for the RT subunits, pET-RT66 and pET-RT51, were under a strong T7/lac promoter that is induced by isopropyl-beta-d-thiogalactopyranoside. Purification of heterodimeric forms of RT was facilitated by high-level expression of these subunits that represented approximately 30-40% of total cell protein. For purification of the His-tagged heterodimeric RT, cell pellet from cells expressing the untagged p66 subunit was mixed in excess with a cell pellet expressing tagged p51. For untagged heterodimeric RT, the pellet from cells expressing p51 was mixed in excess with pellet expressing p66. Subunit dimerization occurred during cell lysis. During the subsequent chromatography steps, stable p66/p51 heterodimer was purified to homogeneity. The heterodimeric nature of the final preparations of RT was confirmed by analytical gel filtration, mass spectrometry, and denaturing gel electrophoresis. Further, the sensitivity of these enzyme preparations to AZTTP indicated that the histidine tag had no effect on nucleoside inhibitor binding, nucleotide binding or insertion, or DNA binding. The application of these expression/purification methodologies represents a useful method to purify large quantities of heterodimeric RT for structural investigations and provides an efficient protocol to produce subunit-specific amino acid alterations necessary for unambiguous structure/function investigations.

Anti-HIV Agents↗
PubMed1991

Authentic reverse transcriptase is coded by jockey, a mobile Drosophila element related to mammalian LINEs.

The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms. It is transcribed at different stages of Drosophila ontogenesis. The Drosophila LINE family includes active transposable elements. Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. As demonstrated here, a 2.23 kb DNA fragment from the region of jockey encoding the putative reverse transcriptase was stably introduced into an expression system under inducible control of the Escherichia coli lac regulatory elements. We describe the expression of the 92 kDa protein and identify this polypeptide alone as the authentic jockey reverse transcriptase based on some of its physical and enzymic properties. The jockey polymerase demonstrates RNA and DNA-directed DNA polymerase activities but lacks detectable RNase H, has a temperature optimum at 26 degrees C, requires Mg2+ or Mn2+ as a cofactor and is inactivated by sulphydryl reagent. The enzyme prefers poly(rC) and poly(rA) as template and 'activated' DNA is not effective.

Animals↗
PubMed1999

Nonlinear pharmacokinetics of efavirenz (DMP-266), a potent HIV-1 reverse transcriptase inhibitor, in rats and monkeys.

Efavirenz (EFV, Sustiva, Stocrin, DMP-266, L-743,726) is a potent and selective non-nucleoside inhibitor of HIV-1 reverse transcriptase. Pharmacokinetics of EFV was studied in rats and monkeys, the safety assessment species. In rats, after 2 and 5 mg/kg i.v. administrations, the mean CLp, Vdss, and T1/2 were 67 ml/min/kg, 5.0 liters/kg, and 1 h, respectively. EFV was metabolized completely, and the products were excreted almost exclusively via bile. At the higher dose of 15 mg/kg, the CLp was reduced by 36%, implying saturation of metabolism processes. A similar phenomenon occurred in monkeys, where the CLp declined by 60% as the i.v. dose was increased from 5 to 15 mg/kg. After oral dosing, the bioavailability of EFV in rats (10 mg/kg) and monkeys (2 mg/kg) was 16% and 42%, respectively. Higher doses in both species led to disproportionate increases in the AUC and higher Tmax values, suggesting saturation of metabolism and/or prolongation of absorption. The delay in Tmax was more pronounced in monkeys where the plasma concentrations reached plateaus and were sustained for 4 to 20 h. In rats, the prolongation of absorption was due to delayed gastric emptying as demonstrated by >10-fold slower transit of [14C]polyethylene glycol through the stomach of EFV-pretreated animals. The delayed gastric emptying in monkeys also was observed when the animals dosed at 160 mg/kg exhibited emesis, 8 h postdose, which was found to contain a substantial portion of the dose. These results demonstrated that in rats and monkeys, both delayed gastric emptying and saturation of metabolic processes played significant roles in the nonlinear pharmacokinetics of EFV.

Alkynes↗
PubMed1996

Detection of herpes simplex virus type 1 latency-associated transcript expression in trigeminal ganglia by in situ reverse transcriptase PCR.

One of the defining characteristics of herpes simplex virus type 1 (HSV-1) infection is the ability of the virus to establish a lifelong latent state in neurons. We previously demonstrated (R. Ramakrishnan, A.J. Fink, G. Jiang, P. Desai, J. C. Glorioso, and M. Levine, J. Virol. 68:1864-1873, 1994) by in situ PCR that many more neurons contain viral genomes than are detected by in situ hybridization for HSV latency-associated transcripts (LATs). To determine whether all cells which contain genomes express LATs, we examined trigeminal ganglia for LATs 1 and 8 weeks after corneal scarification with ribonucleotide reductase-deficient HSV-1 by in situ reverse transcriptase PCR. The number of LAT-positive cells detected by in situ reverse transcriptase was substantially greater than the number of cells positive by in situ hybridization and appeared to be similar to the number of cells containing HSV genomes by in situ PCR and the number of ganglionic neurons that project to the cornea as detected by retrograde labeling with Fluorogold. These results demonstrate LAT expression in many neurons containing HSV-1 genomes.

Animals↗
PubMed1996

Use of chimeric human immunodeficiency virus types 1 and 2 reverse transcriptases for structure-function analysis and for mapping susceptibility to nonnucleoside inhibitors.

The human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2) reverse transcriptases (RTs) are evolutionary related. To study the effect of homologous sequence replacements on polymerase function and to map the determinants of the lack of susceptibility of HIV-2 RT to nonnucleoside drugs, a series of chimeric HIV-1/HIV-2 RTs were constructed. Analysis of the chimeric RTs showed that wild-type levels of RNA-dependent DNA polymerase activity were retained when both finger and palm subdomains were exchanged as a unit between the two parental RTs. Analysis of enzymatically active chimeras for inhibition by the thiobenzimidazolone derivative TIBO R82150 showed that a segment of HIV-2 RT at 212-250, when placed in the HIV-1 RT context, conferred a 40-fold decrease in susceptibility to TIBO R82150. Site-directed mutagenesis of this segment found Tyr227 to be a key residue in this segment for the natural resistance of HIV-2 RT to TIBO R82150.

Amino Acids↗
PubMed2002

Amplification of the telomerase reverse transcriptase (hTERT) gene in cervical carcinomas.

The expression of telomerase reverse transcriptase (hTERT), the catalytic component of the telomerase complex, is required for activation of telomerase during immortalization and transformation of human cells. However, the biochemical and genetic mechanisms governing hTERT expression remain to be elucidated. In the present study, we examined hTERT amplification as a potential genetic event contributing to telomerase activation in cervical carcinomas. An amplification of the hTERT gene was found in 1/4 cervical cancer cell lines and 21/88 primary tumor samples derived from the patients with cervical carcinomas. An increase in the hTERT copy number was significantly correlated with higher levels of hTERT protein expression. Moreover, the hTERT alterations with the enhanced hTERT expression were exclusively observed in those tumors with high-risk human papillomavirus infection. Taken together, the hTERT gene amplification, directly or indirectly targeted by human papillomavirus, may be one of the driving forces responsible for upregulation of hTERT expression and activation of telomerase in cervical cancers.

Carcinoma↗
PubMed2000

RNA degradation and primer selection by Moloney murine leukemia virus reverse transcriptase contribute to the accuracy of plus strand initiation.

During reverse transcription, plus strand DNA synthesis is initiated at a purine-rich RNA primer generated by the RNase H activity of reverse transcriptase (RT). Specific initiation of plus strand synthesis from this polypurine tract (PPT) RNA is essential for the subsequent integration of the linear viral DNA product. Based on current models, it is predicted that priming from sites upstream of the PPT may be tolerated by the virus, whereas efficient extension from RNA primers located downstream from the PPT is predicted to generate dead-end products. By using hybrid duplex substrates derived from the Moloney murine leukemia virus long terminal repeat, we investigated the extent to which RNase H degrades the viral RNA during time course cleavage assays, and we tested the capacity of the polymerase activity of RT to use the resulting cleavage products as primers. We find that the majority of the RNA fragments generated by RNase H are 2-25 nucleotides in length, and only following extensive degradation are most fragments reduced to 10 nucleotides or smaller. Although extensive RNA degradation by RNase H likely eliminates many potential RNA primers, based on thermostability predictions it appears that some RNA fragments remain stably annealed to the DNA template. RNA primers generated by RNase H within the long terminal repeat sequence are found to have the capacity to initiate DNA synthesis by RT; however, the priming efficiency is significantly less than that observed with the PPT primer. We find that Moloney murine leukemia virus nucleocapsid protein reduces RNase H degradation and slightly alters the cleavage specificity of RT; however, nucleocapsid protein does not appear to enhance PPT primer utilization or suppress extension from non-PPT RNA primers.

Escherichia coli↗