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PubMed2006

Cytotoxic T cell responses to human telomerase reverse transcriptase in patients with hepatocellular carcinoma.

Human telomerase reverse transcriptase, hTERT, has been identified as the catalytic enzyme required for telomere elongation. hTERT is expressed in most tumor cells but seldom expressed in most human adult cells. It has been reported that 80% to 90% of hepatocellular carcinomas (HCCs) express hTERT, making the enzyme a potential target in immunotherapy for HCC. In the current study, we identified hTERT-derived, HLA-A*2402-restricted cytotoxic T cell (CTL) epitopes and analyzed hTERT-specific CTL responses in patients with HCC. Peptides containing the epitopes showed high affinity to bind HLA-A*2402 in a major histocompatibility complex binding assay and were able to induce hTERT-specific CTLs in both hTERT cDNA-immunized HLA-A*2402/Kb transgenic mice and patients with HCC. The CTLs were able to kill hepatoma cell lines depending on hTERT expression levels in an HLA-A*2402-restricted manner and induced irrespective of hepatitis viral infection. The number of single hTERT epitope-specific T cells detected by ELISPOT assay was 10 to 100 specific cells per 3 x 10(5) PBMCs, and positive T cell responses were observed in 6.9% to 12.5% of HCC patients. hTERT-specific T cell responses were observed even in the patients with early stages of HCC. The frequency of hTERT/tetramer+ CD8+ T cells in the tumor tissue of patients with HCC was quite high, and they were functional. In conclusion, these results suggest that hTERT is an attractive target for T-cell-based immunotherapy for HCC, and the identified hTERT epitopes may be valuable both for immunotherapy and for analyzing host immune responses to HCC.

Animals↗
PubMed1990

Origin and evolution of retroelements based upon their reverse transcriptase sequences.

To study the evolutionary relationship of reverse transcriptase (RT) containing genetic elements, a phylogenetic tree of 82 retroelements from animals, plants, protozoans and bacteria was constructed. The tree was based on seven amino acid domains totalling 178 residues identified in all RTs. We have also identified these seven domains in the RNA-directed RNA polymerases from various plus-strand RNA viruses. The sequence similarity of these RNA polymerases to RT suggests that these two enzymes evolved from a common ancestor, and thus RNA polymerase can be used as an outgroup to root the RT tree. A comparison of the genetic organization of the various RT containing elements and their position on the tree allows several inferences concerning the origin and evolution of these elements. The most probable ancestor of current retroelements was a retrotransposable element with both gag-like and pol-like genes. On one major branch of the tree, organelle and bacterial sequences (e.g. group II introns and bacterial msDNA) appear to have captured the RT sequences from retrotransposons which lack long terminal repeats (LTRs). On the other major branch, acquisition of LTRs gave rise to two distinct groups of LTR retrotransposons and three groups of viruses: retroviruses, hepadnaviruses and caulimoviruses.

Amino Acid Sequence↗
PubMed1996

Detection of contamination of vaccines with the reticuloendotheliosis virus by reverse transcriptase polymerase chain reaction (RT-PCR).

The reverse transcriptase polymerase chain reaction (RT-PCR) was applied to detect contamination of Marek's disease (MD) vaccine with reticuloendotheliosis virus (REV). The env primers were used for the 1st RT-PCR to amplify the DNA fragments of REV-A and -T. The rel and env primers were used for nested-PCR to confirm the sites deleted from REV-T and REV-A. Specific amplification products were detected in the 1st RT-PCR with these primers. By nested PCR with the env and the rel primer pairs, the products originating from REV-A and -T were identified. This system, using the env primer pairs, showed a specific amplification with several REV strains (REV-T, DE, CE, KI and 0202), but no amplified product was detected with MDV, NDV, IBV or ILTV. The 1st RT-PCR detected the virus in a concentration of 10(3) in 50% fluorescent antibody infectious dose per ml (FAID50/ml) and the nested PCR detected 10(1) FAID50/ml virus. The sensitivity of the RT-PCR system was found to be higher than that of the FA assay. This system provides a rapid, sensitive and specific method for detection of contamination of MD vaccines with REV-RNA, and it may be applied for quality control of live vaccines.

Animals↗
PubMed1995

The formation of the 2',5'-phosphodiester linkage in the cDNA priming reaction by bacterial reverse transcriptase in a cell-free system.

Bacterial reverse transcriptase (RT) is responsible for synthesis of multicopy single-stranded DNA (msDNA) consisting of single-stranded DNA linked to an internal guanosine residue of RNA by an unusual 2',5'-phosphodiester linkage. Here we purified a bacterial RT to homogeneity from Escherichia coli harboring the RT gene from retron-Ec73. The purified RT-Ec73 was able to synthesize msDNA in a cell-free system using an RNA template produced in vitro by T7 RNA polymerase. The in vitro synthesized msDNA was released from the template RNA only when treated with yeast debranching enzyme DBR1, a specific nuclease for a 2',5'-phosphodiester linkage. The position of the branching G residue in the template RNA and the DNA sequence of the cell-free product were identical to those of msDNA-Ec73 synthesized in vivo. These results clearly demonstrate that the formation of the 2',5'-phosphodiester linkage in msDNA synthesis is carried out by RT itself.

Base Sequence↗
PubMed1977

Production of antiserum to the reverse transcriptase of Mason-Pfizer monkey virus.

The reverse transcriptase of Mason-Pfizer monkey virus (M-PMV) has been isolated and partially purified by ion exchange chromatography. Sera from rabbits immunized with the partially purified enzyme have been shown by microimmunodiffusion analysis to be immunologically specific for the M-PMV polymerase. The immune serum also specifically inhibits M-PMV polymerase activity and this inhibitory activity has been shown to reside in the IgG fraction of the serum. The application of these reagents to examining virus identity and investigating the possible viral aetiology of human breast cancer is discussed.

Animals↗
PubMed1992

Requirements for strand transfer between internal regions of heteropolymer templates by human immunodeficiency virus reverse transcriptase.

We have examined the ability of the reverse transcriptase (RT) from human immunodeficiency virus (HIV) to carry out strand transfer synthesis (i.e., switching of the primer to a new template) from internal regions of natural-sequence RNA. A 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide was used to initiate synthesis. DNA oligonucleotides with homology to internal regions of the donor were used as acceptors. In this system, HIV RT produced strand transfer products. An HIV RT having RNase H depleted to 3% of normal (HIV RTRD) catalyzed the transfer reaction inefficiently. An RNase H-minus deletion mutant of murine leukemia virus RT was unable to catalyze strand transfer. HIV RTRD, however, efficiently catalyzed transfer when Escherichia coli RNase H was included in the reactions, while the mutant murine leukemia virus RT was not efficiently complemented by the E. coli enzyme. Evidently, RNase H activity enhances, or is required for, internal strand transfer. Two acceptors homologous to 27-nucleotide regions of the donor, one offset from the other by 6 nucleotides, were tested. The offset eliminated a sequence homologous to a prevalent DNA synthesis pause site in the donor. Strand transfer to this acceptor was about 25% less efficient, suggesting that RT pausing can enhance strand transfer. When the deoxynucleoside triphosphates in the reactions were reduced from 50 to 0.2 microM, increasing RT pausing, the efficiency of strand transfer also increased. A model for RT-catalyzed strand transfer consistent with our results is presented.

Base Sequence↗
PubMed1996

Identification of carcinoembryonic antigen-producing cells circulating in the blood of patients with colorectal carcinoma by reverse transcriptase polymerase chain reaction.

BACKGROUND: Application of the reverse transcriptase polymerase chain reaction (RT-PCR) to identification of circulating tumour cells in colorectal cancer. AIMS: To assess whether circulating malignant cells in patients with colorectal liver metastasis could be identified by RT-PCR recognition of mRNA coding for the tumour marker carcinoembryonic antigen (CEA). PATIENTS: A total of 31 with colorectal liver metastases and 22 no-cancer controls. METHODS: Specific cDNA primers for CEA transcripts were used to apply RT-PCR to tissue biopsy specimens, colon carcinoma cell lines, and peripheral blood samples from patients with colorectal liver metastases. A strongly CEA-expressive HT115 colorectal carcinoma cell line was used to spike blood samples from no-cancer control subjects. RESULTS: The limit for detection of CEA cDNA by Southern blotting using HT115 cells was 50 cells per 14 ml of spiked blood. There was a significant difference (p = 0.007) in RT-PCR positive expression between patients with liver metastasis (26/31) compared with controls (5/22). There was no significant relation between the prevalence of CEA cDNA amplification and serum CEA level or metastasis volume in patients with liver metastasis. CONCLUSIONS: This is the first study to suggest that identification of circulating colorectal cancer cells using RT-PCR for detection of CEA cDNA is feasible.

Biomarkers, Tumor↗
PubMed2000

Quantitative measurement of telomerase reverse transcriptase (hTERT) mRNA in urothelial cell carcinomas.

Telomerase reverse transcriptase (hTERT) messenger RNA has been detected in 95% of bladder tumors using RT-PCR. In this study, we quantified the expression of hTERT in 35 bladder urothelial cell carcinomas and in 6 normal bladder epithelia using a real-time quantitative PCR assay. hTERT expression was detected in all 35 urothelial cell carcinomas of varying grade and stage, but not in normal tissue samples. An increase in both pathological grade and clinical stage as prognostic parameters correlated with increased hTERT expression. Using different cutoff values for grades and stages, normalized hTERT expression values could discriminate among low, medium, and high grade tumors and between superficial and muscle-invasive tumors. We conclude that standardized real-time measurement of hTERT expression can be used for early tumor detection and may be used for determination of prognosis in urothelial cell carcinomas of the bladder.

Carcinoma, Transitional Cell↗
PubMed1996

[Detection of breast cancer micrometastases in lymph nodes by amplification of keratin 19 mRNA with reverse-transcriptase polymerase chain reaction].

Keratin 19 mRNA reverse-transcriptase polymerase chain reaction (K 19 RT-PCR) was compared with histological examination in the detection of breast cancer micrometastases in axillary lymph nodes. Sixty-three axillary lymph nodes, which were obtained from 23 breast cancer patients, were bisected. One half was studied by conventional histological analysis of hematoxylin and eosin sections. Total RNA was extracted from the other half and subjected to K 19 RT-PCR. In all the ten lymph nodes, which were histologically metastasis-positive, K 19 mRNA was detected by RT-PCR. Of the 53 histologically negative lymph nodes, five (9%) lymph nodes were found to express K 19 mRNA, indicating the presence of micrometastases which could be detected by RT-PCR but not by a histological examination. These results demonstrate the usefulness of K 19 RT-PCR in the detection of breast cancer micrometastases in lymph nodes.

Axilla↗
PubMed1992

Rifamycins as inhibitors of retroviral reverse transcriptase from M-MuLV, RAV-2, and HIV-1.

29 Rifamycins were tested for inhibition of Reverse Transcriptase (RT) as potential anti HIV drugs. Two purified commercial enzymes from M-MuLV and RAV-2 were used. Anti-RT activity was also measured on a crude lysate of HIV-1. The results show that some derivatives have interesting levels of activity on isolated M-MuLV and RAV-2 RTs, while they are less active on the RT in the crude HIV-1 lysate. The active derivatives include oximes and hydrazones, alkylaminoderivatives, open ansa-chain derivatives and derivatives carrying a modified nucleoside.

HIV Reverse Transcriptase↗
PubMed1998

DNA synthesis exhibited by the reverse transcriptase of mouse mammary tumor virus: processivity and fidelity of misinsertion and mispair extension.

We have recently expressed in bacteria an enzymatically active reverse transcriptase (RT) of mouse mammary tumor virus (MMTV), a mammalian retrovirus with a typical B-type morphology [Taube, R., Loya, S., Avidan, O., Perach, M. & Hizi, A. (1998) Biochemical J. 329, 579-587]. The purified recombinant protein was shown to possess the catalytic activities characteristic of retroviral reverse transcriptases. In the present study, we have analyzed two basic parameters characteristic of the DNA polymerase activity of the novel MMTV RT, namely the processivity and the fidelity of DNA synthesis. Two features related to fidelity were studied, the capacity to misinsert wrong nucleotides at the 3' end of the nascent DNA strand and the ability to extend 3' mispairs. The studied properties of MMTV RT were compared with those of the RT purified from virions of avian myeloblastosis virus (AMV), since AMV RT shows a relatively high sequence similarity to MMTV RT. MMTV RT shows a relative processivity of DNA synthesis which is as high as the reference AMV RT. Regarding fidelity of DNA synthesis, MMTV RT shows a fidelity of misinsertion lower than that of AMV RT, whereas its capacity to elongate mispaired DNA is lower than that of AMV RT indicating a somewhat higher fidelity. These fidelity properties are discussed also in the context of the RTs of lentiviruses, especially those of HIV, which were reported to exhibit an exceptionally low fidelity of DNA synthesis. It is clear that MMTV RT has a fidelity higher than that of lentiviral RTs.

3' Untranslated Regions↗
PubMed1993

RNase H domain mutations affect the interaction between Moloney murine leukemia virus reverse transcriptase and its primer-template.

The active sites for the polymerase and nuclease activities of Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) reside in separate domains of a single polypeptide. We have studied the effects of RNase H domain mutations on DNA polymerase activity. These mutant RTs displayed decreased processivity of DNA synthesis. We also compared complexes formed between primer-templates and mutant and wild-type reverse transcriptase (RT). Although M-MuLV RT is monomeric in solution, two molecules of RT bound DNA cooperatively, suggesting that M-MuLV RT binds primer-template as a dimer. Some mutant RTs with decreased processivity failed to form the putative dimer.

Base Sequence↗
PubMed1998

A randomized, controlled, double-blind study comparing the survival benefit of four different reverse transcriptase inhibitor therapies (three-drug, two-drug, and alternating drug) for the treatment of advanced AIDS. AIDS Clinical Trial Group 193A Study Team.

OBJECTIVE: The primary objective was to compare the effects of dual or triple combinations of HIV-1 reverse transcriptase inhibitors with respect to survival. The time to new HIV disease progression or death, toxicities, the change in CD4 cells, and plasma HIV-1 RNA concentrations in a subset of study subjects were evaluated. DESIGN: This was a multicenter randomized, double-blind, placebo-controlled study. SETTING: The study was conducted among 42 adult AIDS Clinical Trials Group sites and 7 National Hemophilia Foundation centers. PATIENTS: 1313 HIV-infected patients with CD4 counts < or = 50 cells/mm3 participated in this study, which was conducted from June 1993 to June 1996. INTERVENTION: Patients were randomized to one of four daily regimens containing 600 mg of zidovudine: zidovudine alternating monthly with 400 mg didanosine; zidovudine plus 2.25 mg of zalcitabine; zidovudine plus 400 mg of didanosine; or zidovudine plus 400 mg of didanosine plus 400 mg of nevirapine (triple therapy). MAIN OUTCOME MEASURES: The main outcome was survival (i.e., time to death). RESULTS: A significant difference in survival time was found between the four treatment groups, favoring those assigned to triple therapy (p = .02). A significant difference was also found in the delay of disease progression or death among the four treatment arms favoring the group assigned to triple therapy (p = .002). Baseline CD4 cell counts and plasma HIV-1 RNA concentrations as well as changes of CD4 counts at week 8 predicted survival for subjects in the virology substudy. CONCLUSIONS: In the pre-protease inhibitor era, a combination of triple reverse transcriptase inhibitors prolonged life and delayed disease progression in AIDS patients with advanced immune suppression.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Structure of unliganded HIV-1 reverse transcriptase at 2.7 A resolution: implications of conformational changes for polymerization and inhibition mechanisms.

BACKGROUND: HIV-1 reverse transcriptase (RT) is a major target for anti-HIV drugs. A considerable amount of information about the structure of RT is available, both unliganded and in complex with template-primer or non-nucleoside RT inhibitors (NNRTIs). But significant conformational differences in the p66 polymerase domain among the unliganded structures have complicated the interpretation of these data, leading to different proposals for the mechanisms of polymerization and inhibition. RESULTS: We report the structure of an unliganded RT at 2.7 A resolution, crystallized in space group C2 with a crystal packing similar to that of the RT-NNRTI complexes. The p66 thumb subdomain is folded into the DNA-binding cleft. Comparison of the unliganded RT structures with the DNA-bound RT and the NNRTI-bound RT structures reveals that the p66 thumb subdomain can exhibit two different upright conformations. In the DNA-bound RT, the p66 thumb subdomain adopts an upright position that can be described as resulting from a rigid-body rotation of the p66 thumb along the "thumb's knuckle' located near residues Trp239 (in strand beta 14) and Val317 (in beta 15) compared with the thumb position in the unliganded RT structure. NNRTI binding induces an additional hinge movement of the p66 thumb near the thumb's knuckle, causing the p66 thumb to adopt a configuration that is even more extended than in the DNA-bound RT structure. CONCLUSIONS: The p66 thumb subdomain is extremely flexible. NNRTI binding induces both short-range and long-range structural distortions in several domains of RT, which are expected to alter the position and conformation of the template-primer. These changes may account for the inhibition of polymerization and the alteration of the cleavage specificity of RNase H by NNRTI binding.

Anti-HIV Agents↗
PubMed2003

Validation of a model for the complex of HIV-1 reverse transcriptase with nonnucleoside inhibitor TMC125.

The structure for the complex of nonnucleoside inhibitor TMC125 and HIV-1 reverse transcriptase has been determined and validated through computation of resistance profiles using Monte Carlo/free-energy perturbation calculations. The good quantitative agreement between the computed and experimental anti-HIV activities for TMC125, nevirapine, and efavirenz with wild-type RT and four common mutants (L100I, K103N, Y181C, and Y188L) confirms the correctness of the predicted structure and provides insights into the improved potency of this novel NNRTI. The blue shading in the figure indicates basic residues.

Anti-HIV Agents↗
PubMed1996

An unusual bacterial reverse transcriptase having LVDD in the YXDD box from Escherichia coli.

A minor population of wild strains of Escherichia coli contains a gene for reverse transcriptase (RT) which is responsible for the synthesis of multicopy single-stranded DNA (msDNA), a branched DNA-RNA complex. A DNA fragment capable of synthesizing msDNA was cloned from strain ECOR-58, one of the 72 wild strains in the ECOR collection. The complete open reading frame of a novel reverse transcriptase, designated ECOR-58 RT, was identified. ECOR-58 RT consisted of 408 amino acid residues, and its 227-residue polymerase domain from residue 43 to 269 showed significant homologies to all the other bacterial RTs so far identified. Most significantly, its YXDD box, the most highly conserved sequence in all RTs from prokaryotes to eukaryotes, was found to be replaced with LVDD. ECOR-58 RT was found to be most distantly related in a phylogenetic tree to all 9 other bacterial RTs so far identified.

Amino Acid Sequence↗