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PubMed2005

High virological failure rate in HIV patients after switching to a regimen with two nucleoside reverse transcriptase inhibitors plus tenofovir.

BACKGROUND: Regimens with two nucleoside analogue reverse transcriptase inhibitors (NRTI) plus tenofovir DF have been associated with a high failure rate when administered as first line therapy. Little is known about patients with undetectable viral loads who are switched to these regimens. METHODS: A post-hoc review of the virological outcomes at 24 weeks of patients who switched from a successful (< 50 copies/ml) highly active antiretroviral therapy regimen to a tenofovir plus two NRTI combination. RESULTS: Fifty-five patients started a two NRTI plus tenofovir regimen mostly because of previous toxicity/intolerance of the original drugs (74%). After 24 weeks, only 17 patients (31%) remained virologically suppressed. Patients with a regimen including a didanosine plus tenofovir-based regimen had significantly poorer outcomes than those on other combinations (success rate 5 versus 47.1%, P = 0.001). In contrast, patients on a regimen including zidovudine plus tenofovir showed a trend towards a better outcome (75 versus 27%, P = 0.083). Multivariate analysis confirmed the combination of didanosine plus tenofovir as the only variable associated with a higher rate of failure (odds ratio 17.7; 95% confidence interval 2.1-147; P = 0.007). Patients with previous reverse transcriptase mutations presented virological failure in all cases. At failure a new pattern, including the K65R mutation with M184V or thymidine analogue mutations, was observed. CONCLUSIONS: Even in patients with suppressed viraemia, a two NRTI plus tenofovir regimen is associated with a high virological failure rate, but significant variations are found depending on the nucleosides included.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Mechanistic studies comparing the incorporation of (+) and (-) isomers of 3TCTP by HIV-1 reverse transcriptase.

Among the nucleoside inhibitors used clinically as anti-HIV drugs which target HIV-1 reverse transcriptase (RT), (-)-2', 3'-dideoxy-3'-thiacytidine [(-)SddC or 3TC] is the only analogue with the unnatural L(-) nucleoside configuration. 3TC has been shown to be more potent and less toxic than the D(+) isomer, (+)SddC, which has the natural nucleoside configuration. The mechanistic basis for the stereochemical selectivity and differential toxicity of the isomeric SddC compounds is not completely understood although a number of factors may clearly come into play including differences in uptake, metabolic activation, degradation, and transport. We used a pre-steady-state kinetic analysis to determine the maximum rate of incorporation, kpol, nucleotide-binding affinity, Kd, and efficiency of incorporation, kpol/Kd, for the (-) and (+) isomeric SddCTP compounds as well as the corresponding dideoxy and natural nucleoside triphosphates into a primer-template complex using HIV-1 reverse transcriptase. The affinity (Kd) of the dNTP was much tighter and the efficiency (kpol/Kd) of incorporation by enzyme into the primer-template complex was much higher for the DNA/RNA primer-template compared to DNA/DNA. The maximum rate of incorporation, kpol, followed the trend of dCTP > ddCTP > (+)SddCTP > (-)SddCTP while the Kd values determined for the DNA/RNA primer-template followed the order (-)SddCTP congruent with (+)SddCTP congruent with ddCTP > dCTP. The corresponding efficiency of incorporation followed the trend dCTP > ddCTP > (+)SddCTP > (-)SddCTP. These data suggest that perturbations on the ribose ring of cytidine analogues (C --> S) decrease the rate and efficiency of incorporation but enhance the binding affinity. These results are discussed in the context of a computer modeled structure of the ternary complexes of RT, DNA/RNA primer-template, and SddCTP analogues as well as implications for structure-activity relationships and further drug design. This information provides a mechanistic basis for understanding the inhibition of HIV-1 reverse transcriptase by 3TC.

Anti-HIV Agents↗
PubMed1997

Pre-steady-state kinetic characterization of wild type and 3'-azido-3'-deoxythymidine (AZT) resistant human immunodeficiency virus type 1 reverse transcriptase: implication of RNA directed DNA polymerization in the mechanism of AZT resistance.

There is lack of a correlation between biochemical studies and the observed clinical resistance of AIDS patients on long term AZT therapy. Mutant HIV-1 reverse transcriptase in the viral isolates from these patients shows a 100-fold decrease in sensitivity whereas little or no difference is observed in kinetic parameters in vitro using steady-state kinetic analysis. A detailed pre-steady-state kinetic analysis of wild type and the clinically important AZT resistant mutant (D67N, K70R, T215Y, K219Q) HIV-1 reverse transcriptase was conducted to understand the mechanistic basis of drug resistance. In contrast to steady-state techniques, a pre-steady-state kinetic analysis allows for the direct observation of catalytic events occurring at the active site of the enzyme, including subtle conformational changes enabling a greater degree of mechanistic detail. In this investigation the rate of incorporation of dTMP and AZTMP by wild type and mutant HIV-1 RT was determined using an RNA and the corresponding DNA template. The present study has shown a 1.5-fold decrease in the rate constant for polymerization (kpol) and a 2.5-fold decrease in the equilibrium dissociation constant (Kd) for AZTTP for the mutant reverse transcriptase as compared to the wild type, for RNA dependent DNA replication. These values translate into a 4-fold decrease in selectivity (kpol/Kd) for AZTMP incorporation by mutant reverse transcriptase as compared to wild type for RNA dependent DNA replication. No such decrease in selectivity was detected for DNA dependent replication. These results suggest that the basis of AZT resistance is related to RNA dependent replication rather than DNA dependent replication.

Anti-HIV Agents↗
PubMed2002

Characterization of a binding site for template competitive inhibitors of HIV-1 reverse transcriptase using photolabeling derivatives.

Analogues of a novel class of template-competitive reverse transcriptase inhibitors (Li, K.; Lin, W.; Chong, K. H.; Moore, B. M.; Doughty, M. B. Bioorg. Med. Chem. 2002, 10, 507) were analyzed as photoprobes of HIV-1 reverse transcriptase (RT) heterodimer. The two photoprobes, 2-(4-azidophenacyl)thio-1,N(6)-etheno-2'-deoxyadenosine 5'-triphosphate 2 and the tetrafluoro analogue 2-(4-azido-2,3,5,6-tetrafluorophenacyl)thio-1,N(6)-etheno-2'-deoxyadenosine 5'-triphosphate 3, photodecomposed at 3500 A with half-lives of 4.0 and 2.5 min, respectively. Analysis of the photoproducts of 2m demonstrated that the etheno group is stable but the azido decomposes primarily to the 2-(S-[3H-diazepinon-4-yl]thio)-1,N(6)-etheno-dAMP. Photolysis of both 2 and 3 with RT resulted in a time-dependent loss of activity, with maximum inactivation of 83 and 60%, respectively. Both 2 and 3 showed concentration-dependent photoinactivation of RT in the concentration range from 0 to 100 microM, with EC(50)s of 20 and 25 microM and maximum inactivation of 80 and 60%, respectively. Both the time and concentration dependent photoinactivation were strongly protected by template-primer, but only poorly inhibited by even high concentrations of TTP. Radiolabeled analogues [beta,gamma-(32)P]-2 and [beta,gamma-(32)P]-3 photoincorporated into the p66 subunit, an incorporation also protected by template primer. Identification of the site of incorporation was problematic for both photoprobes, but evidence presented is consistent with labeling sites for the phenacyl side chains of both 2 and 3 in the template grip. Nevertheless, the photoinactivation and incorporation data are consistent with our earlier conclusions from the kinetic data that these inhibitors are specific for the free form of RT in competition with template/primer, and thus represent a novel class of inhibitors.

Adenosine Triphosphate↗
PubMed2004

Antigenicity and immunogenicity of peptide analogues of a low affinity peptide of the human telomerase reverse transcriptase tumor antigen.

Human telomerase reverse transcriptase (hTRT) is a potential target for therapeutic vaccination against cancer. Therefore, it is critically important to identify T cell epitopes useful to induce cytotoxic T cell responses. Here we used a positional scanning combinatorial peptide library to identify peptide analogues for a previously characterized low affinity hTRT peptide (p572). From an initial library containing over 300 billion different peptides and through successive rounds of selection, we retained 72 candidate peptide analogues for further assessment of antigenicity and in vivo immunogenicity in HLA A2.1-transgenic mice. While antigenically cross-reactive with p572, only a fraction of these peptides was immunogenic in mice. Immunogenicity appeared to correlate with the stability of binding to the MHC molecule and the presence of HLA A2.1 anchor residues in position 2 and 9. Two peptides differing by five residues from the reference p572 (p49 and p50) were more effective than p572 in inducing CTL cross-reacting with p572 in HLA A2.1-transgenic mice. Both peptides also expanded specific CTL in peripheral blood lymphocytes of normal human volunteers ex vivo. The present study shows that positional scanning combinatorial peptide libraries can be used to identify hTRT peptide analogues for inclusion in a cancer vaccine.

Animals↗
PubMed1991

How do viral reverse transcriptases recognize their RNA genome?

Reverse transcription is not solely a retroviral mechanism. Hepadnaviruses and caulimoviruses have RNA intermediates that are reverse transcribed into DNA. Moreover non-viral retroelements, retrotransposons, use reverse transcription in their transposition. All these retroelements encode reverse transcriptase but each group developed their own expression modes capable of assuring a specific and efficient replication of their genomes.

Capsid↗
PubMed1997

Elimination of background signals in a modified polymerase chain reaction-based reverse transcriptase assay.

Three highly sensitive reverse transcriptase (RT) assays were recently published that are at least one million times more sensitive than conventional RT assays. These assays derive their high sensitivities through the ability to amplify the complementary DNA (cDNA) product of the RT reaction by the polymerase chain reaction (PCR). We describe a modified PCR-based RT (PBRT) assay that retains the high sensitivities of the original assays while reducing their inherent background signals. The background signal of the PBRT assay was found to be due to an intrinsic RNA-dependent DNA polymerase activity of the Taq DNA polymerase, the enzyme used for the PCR. It could be eliminated by inserting a ribonuclease digestion step prior to amplifying the cDNA product of the RT reaction by PCR and by using a thermostable DNA polymerase identified as having reduced RNA-dependent DNA polymerase activity. Comparable results were obtained using three RNA templates with two purified RT enzymes. This modified assay is capable of detecting reliably between 10 and 100 molecules of RT, which is equivalent to between 1 and 10 retrovirus particles.

DNA-Directed DNA Polymerase↗
PubMed1990

Two independent retrons with highly diverse reverse transcriptases in Myxococcus xanthus.

A reverse transcriptase (RT) was recently found in Myxococcus xanthus, a Gram-negative soil bacterium. This RT has been shown to be associated with a chromosomal region designated a retron responsible for the synthesis of a peculiar extrachromosomal DNA called msDNA (multicopy single-stranded DNA). We demonstrate that M. xanthus contains two independent, unlinked retrons, one for the synthesis of msDNA-Mx162 and the other for msDNA-Mx65. The structural analysis of the retron for msDNA-Mx65 revealed that the coding regions for msdRNA (msr) and msDNA (msd), and an open reading frame (ORF) downstream of msr are arranged in the same manner as found for the Mx162 retron. The ORF encodes a polypeptide of 427 amino acid residues. The amino-terminal domain (residues 1-138) shows no striking similarity to these proteins presently available in the data bases including the msDNA-Mx162 ORF, while the sequence from residues 139-394 can be aligned with various known RT sequences and has 47% identity with the RT domain of the msDNA-Mx162 ORF. On the basis of these findings, possible origins of two highly diverse retrons on the M. xanthus chromosome are discussed.

Amino Acid Sequence↗
PubMed1976

Assay for type C virus in mouse sera based on particulate reverse transcriptase activity.

Assay of particulate reverse transcriptase activity in the sera from feral mice naturally infected with type C virus provides a sensitive and rapid procedure for the determination of in vivo virus infection. The results compare well with assays for infectious virus and with complement fixation or competitive radio-immunoassays for the p30 internal antigen of the virus.

Animals↗
PubMed1992

RNase H activity associated with reverse transcriptase from feline immunodeficiency virus.

Reverse transcription of retroviral genomes requires the action of an RNase H for template switching and primer generation. In this report, we compare enzymatic properties of the RNase H associated with the reverse transcriptase (RT) from feline immunodeficiency virus (FIV) and that from human immunodeficiency virus (HIV). Both enzymes displayed substrate preference for poly[3H](rG) . poly(dC) hybird over poly[3H](rA) . poly(dT) and cation preference for Mg2+ over Mn2+. Activity of the FIV RNase H upon poly(rG) . poly(dC) produced hydrolysis products from 1 to 6 nucleotides in length, similar to that reported for HIV. Dextran sulfates were effective inhibitors of both the FIV and HIV RNase H and RT activities. Nearly identical inhibition constants (0.12 nM) were obtained for all enzyme activities with dextran sulfate 500,000, while different inhibition constants were observed with dextran sulfate 8,000. Our results suggest that FIV and HIV RTs contain a conserved region that is sensitive to the larger dextran sulfate and that dextran sulfate 8,000 may interact at a different site or by a different mechanism.

Animals↗
PubMed2000

In vitro reconstitution of a functional duck hepatitis B virus reverse transcriptase: posttranslational activation by Hsp90.

Reverse transcription in hepatitis B viruses is initiated through a unique protein priming mechanism whereby the viral reverse transcriptase (RT) first assembles into a ribonucleoprotein (RNP) complex with its RNA template and then initiates DNA synthesis de novo using the RT itself as a protein primer. RNP formation and protein priming require the assistance of host cell factors, including the molecular chaperone heat shock protein 90 (Hsp90). To better understand the mechanism of RT activation by Hsp90, we have now mapped the minimal RT sequences of the duck hepatitis B virus that are required for chaperone binding, RNP formation, and protein priming. Furthermore, we have reconstituted in vitro both RNP formation and protein priming using purified RT proteins and host factors. Our results show that (i) Hsp90 recognizes two independent domains of the RT, both of which are necessary for RNP formation and protein priming; (ii) Hsp90 function is required not only to establish, but also to maintain, the RT in a state competent for RNA binding; and (iii) Hsp90 is not required during RT synthesis and can activate the RT posttranslationally. Based on these findings, we propose a model for Hsp90 function whereby the chaperone acts as an active interdomain bridge to bring the two RT domains into a poised but labile conformation competent for RNP formation. It is anticipated that the reconstitution system established here will facilitate the isolation of additional host factors required for RT functions and further elucidation of the mechanisms of RT activation.

HSP90 Heat-Shock Proteins↗
PubMed2004

Requirement of heat shock protein 90 for human hepatitis B virus reverse transcriptase function.

The initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA (pgRNA) by the viral reverse transcriptase (RT). RT-epsilon interaction in the duck hepatitis B virus (DHBV) was recently shown to require the molecular chaperone complex, the heat shock protein 90 (Hsp90). However, the requirement for RT-epsilon interaction in the human HBV has remained unknown due to the inability to obtain a purified RT protein active in specific epsilon binding. We now report that Hsp90 is also required for HBV RT-epsilon interaction. Inhibition of Hsp90 led to diminished HBV pgRNA packaging into nucleocapsids in cells, which depends on RT-epsilon interaction. Furthermore, using truncated HBV RT proteins purified from bacteria and five purified Hsp90 chaperone factors, we have developed an in vitro RT-epsilon binding assay. Our results demonstrate that Hsp90, in a dynamic process that was dependent on ATP hydrolysis, facilitated RT-epsilon interaction in HBV, as in DHBV. Specific epsilon binding required sequences from both the amino-terminal terminal protein and the carboxy-terminal RT domain. Only the cognate HBV epsilon, but not the DHBV epsilon, could bind the HBV RT proteins. Furthermore, the internal bulge, but not the apical loop, of epsilon was required for RT binding. The establishment of a defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT-epsilon interaction and chaperone activation.

DNA Replication↗
PubMed2000

Template definition by Tetrahymena telomerase reverse transcriptase.

The ribonucleoprotein enzyme telomerase extends chromosome ends by copying a specific template sequence within its integral RNA component. An active recombinant telomerase RNP is minimally composed of this RNA and the telomerase reverse transcriptase (TERT) protein, which contains sequence motifs conserved among viral reverse transcriptases (RTs), flanked by N- and C-terminal extensions specific to TERTs. We have used site-directed mutagenesis to explore the roles of Tetrahymena TERT in determining features of telomerase activity in general and in establishing the boundaries and use of an internal RNA template in specific. We identify a new ciliate-specific motif in the TERT N-terminus required for template definition. Moreover, several residues in reverse transcriptase motifs 1, 2, A and D are critical for specific aspects of internal template use. Our results indicate that the unique specificity of telomerase activity is conferred to a reverse transcriptase active site by TERT residues both within and beyond the RT motif region.

Amino Acid Motifs↗
PubMed2003

A novel single-stranded DNA enzyme expression system using HIV-1 reverse transcriptase.

In this study, we exploited a DNA enzyme expression system using the mechanism of HIV-1 reverse transcription in vitro. HIV-1 reverse transcription is initiated when its cognate primer tRNA (Lys-3) binds to the primer binding site (PBS) of the viral RNA template. Therefore, this RNA contains the HIV-1 PBS, the DNA enzyme, and a tRNA (Lys-3) at the 3(')-end of its RNA transcript, such that a single-stranded DNA (ssDNA) is synthesized by the HIV-1 reverse transcriptase. We constructed RNA expression vectors including the HIV-1 PBS, the DNA enzyme, and either a native tRNA (Lys-3) or one of two truncated tRNAs (Lys-3), Delta tRNA (Lys-3) and Delta Delta tRNA (Lys-3). The reactions of the pVAX1-Dz-tRNA (Lys-3), pVAX1-Dz-Delta tRNA (Lys-3), and pVAX1-Dz-Delta Delta tRNA (Lys-3) vectors with T7 RNA polymerase in vitro gave the corresponding RNAs. The liberated RNAs were treated with HIV-1 reverse transcriptase (HIV-1 RT) in vitro, which yielded the corresponding ssDNA. The cleavage assay results demonstrated that the expressed DNA enzyme has cleavage ability against the target sequence. Thus, we have found a new DNA enzyme oligonucleotide expression system using the HIV-1 reverse transcriptase in vitro.

Binding Sites↗
PubMed1995

Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase.

The level of genetic variation of human immunodeficiency virus type 1 (HIV-1), a member of the lentivirus genus of the Retroviridae family, is high relative to that of retroviruses in some other genera. The high error rates of purified HIV-1 reverse transcriptase in cell-free systems suggest an explanation for this high genetic variation. To test whether the in vivo rate of mutation during reverse transcription of HIV-1 is as high as predicted by cell-free studies, and therefore higher than that rates of mutation of retroviruses in other genera, we developed an in vivo assay for detecting forward mutations in HIV-1, using the lacZ alpha peptide gene as a reporter for mutations. This system allows the rates and types of mutations that occur during a single cycle of replication to be studied. We found that the forward mutation rate for HIV-1 was 3.4 x 10(-5) mutations per bp per cycle. Base substitution mutations predominated; G-to-A transition mutations were the most common base substitution. The in vivo mutation rates for HIV-1 are three and seven times higher than those previously reported for two other retroviruses, spleen necrosis virus and bovine leukemia virus, respectively. In contrast, our calculated in vivo mutation rate for HIV-1 is about 20-fold lower than the error rate of purified HIV-1 reverse transcriptase, with the same target sequence. This finding indicates that HIV-1 reverse transcription in vivo is not as error prone as predicted from the fidelity of purified reverse transcriptase in cell-free studies. Our data suggest that the fidelity of purified HIV-1 reverse transcriptase may not accurately reflect the level of genetic variation in a natural infection.

Animals↗
PubMed1988

Functional organization of the murine leukemia virus reverse transcriptase: characterization of a bacterially expressed AKR DNA polymerase deficient in RNase H activity.

The functional organization of the murine leukemia virus reverse transcriptase was investigated by expressing a molecular clone containing AKR MuLV reverse transcriptase-coding sequences in Escherichia coli. A purified preparation of the expressed enzyme (pRT250 reverse transcriptase) consisted primarily of a 69-kilodalton protein that has normal levels of murine leukemia virus polymerase activity but 10-fold-reduced levels of RNase H compared with the viral enzyme. The deficit in RNase H activity was correlated with the absence of 60 to 65 amino acids normally present at the carboxyl end of murine leukemia virus reverse transcriptase. The results provide additional experimental evidence for the localization of polymerase and RNase H domains to the N- and C-terminal regions of reverse transcriptase, respectively.

AKR murine leukemia virus↗
PubMed2001

Three-drug combinations of emivirine and nucleoside reverse transcriptase inhibitors in vitro: long-term culture of HIV-1-infected cells and breakthrough viruses.

Emivirine (EMV) is a non-nucleoside reverse transcriptase inhibitor currently undergoing Phase III clinical trials in HIV-1-infected patients. In this study, the anti-HIV-1 activity of EMV in combination with two nucleoside reverse transcriptase inhibitors was examined in cell cultures. The combinations EMV plus stavudine (d4T) plus lamivudine (3TC) and EMV plus d4T plus didanosine (ddI) synergistically inhibited HIV-1 replication in MT-4 cells. Although not statistically significant, EMV plus d4T plus 3TC appeared to be more synergistic than EMV plus d4T plus ddI. Synergism was also observed with any two-drug combinations, such as EMV plus d4T, EMV plus 3TC, EMV plus ddI, d4T plus 3TC, or d4T plus ddI. The three-drug combinations completely suppressed HIV-1 replication for at least 40 days after virus infection. Except for d4T, virus emerged in the presence of every compound alone or some combinations at lower concentrations. Susceptibility tests of the breakthrough viruses to each compound showed that the viruses obtained in the presence of EMV alone and 3TC alone were significantly less susceptible to EMV and 3TC, respectively. These viruses had specific amino acid mutations in their reverse transcriptase.

Anti-HIV Agents↗
PubMed1996

Reverse transcriptase in situ polymerase chain reaction in atypical mycobacterial adenitis.

OBJECTIVE: To determine whether reverse transcriptase (RT) in situ polymerase chain reaction (PCR) can facilitate the diagnosis of nontuberculous ("atypical") mycobacterial (NTM) cervical adenitis. DESIGN: Retrospective review of 12 patients with neck masses clinically diagnosed as NTM cervical adenitis. SETTING: University medical center caring for both ambulatory and hospitalized children. PATIENTS: Twelve pediatric patients (all younger than 9 years) with cervicofacial masses. INTERVENTION: Surgical excision of the presenting mass. MAIN OUTCOME MEASURES: Reverse transcriptase in situ PCR was used to detect mycobacterial RNA in excised tissue. All specimens were also cultured and stained for acid-fast bacilli. RESULTS: Reverse transcriptase in situ PCR was positive for NTM in 7 of 12 cases. CONCLUSIONS: Infection with NTM may be an extremely indolent process, and the success of RT in situ PCR depends on the presence of mycobacterial nucleic acids. Even in cases in which the findings of RT in situ PCR were positive, infected cells were few in number. Because of the sparsity of infection in the positive cases, NTM may be even more rare in the negative cases, ie, those in which mycobacterial nucleic acids do not exist and cannot be detected by any means, including RT in situ PCR. Although RT in situ PCR, cultures, stains for acid-fast bacilli, and tuberculin tests using purified protein derivative are all helpful in diagnosing NTM cervical adenitis, when nucleic acids are present RT in situ PCR is the simplest, most reliable, and quickest to perform and the results are easiest to interpret.

Child↗