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PubMed2001

Regulation of telomerase activity by alternate splicing of human telomerase reverse transcriptase mRNA in a subset of neuroblastomas.

It has been proposed that the regulation of telomerase takes place at the transcriptional level, the expression of the catalytic subunit human telomerase reverse transcriptase (hTERT) being crucial for telomerase activity (TA). Recently, differential splicing of hTERT mRNA has been demonstrated in various tissues during embryonal development, and it has been suggested that only full-length transcripts translate into functionally active telomerase. With this in view, we analyzed the different hTERT transcripts by reverse transcriptase-polymerase chain reaction in neuroblastic tumors and compared the results with the TA, the tumor growth fraction, and the MYCN status. In a series of 38 neuroblastic tumors, high TA and full-length hTERT transcripts were found in nine samples, whereas nine samples showed absence of both enzymatic activity and hTERT transcripts. Interestingly, in another eight samples, low or absent TA coincided with a lack of full-length hTERT transcripts. Eleven samples contained hTERT transcripts with low or undetectable TA and one sample had low TA but no hTERT transcripts. TA correlated with MYCN amplification and was weakly associated with the proliferative activity. Moreover, a significant correlation with tumor progression was observed. Our findings point at a posttranscriptional regulation of TA in a subset of neuroblastic tumors. Because high TA was detected only in tumors with full-length hTERT transcripts, reverse transcriptase-polymerase chain reaction analysis of archival neuroblastic tumor samples might help to appraise the malignant potential in individual cases.

Alternative Splicing↗
PubMed1998

Characterization of human immunodeficiency virus type-1 (HIV-1) particles that express protease-reverse transcriptase fusion proteins.

We have selectively mutagenized specific residues at the junction between the protease (PR) and reverse transcriptase (RT) genes of human immunodeficiency virus type 1 (HIV-1) to study the effects of PR-RT fusion proteins in the context of a full-length, infectious proviral construct. Mutant viruses derived from COS-7 cells transfected with this construct were analyzed in regard to each of viral replication, maturation, and infectivity. Immunoblot analysis revealed that the mutation prevented cleavage between the PR and RT proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. Interestingly, intracellular PR that existed within the PR-RT fusion protein remained functionally active, whereby HIV-1 precursor proteins were processed efficiently. Furthermore, the RT component of the fusion protein also retained its enzymatic activity as shown in RT assays. Electron microscopy revealed that the mutant viruses containing the PR-RT fusion protein possessed wild-type morphology. These viruses also displayed wild-type sensitivities to inhibitors of each of the HIV-1 PR and RT activities. However, viruses containing the PR-RT fusion protein were 20 times less infectious than wild-type viruses. This defect was further pronounced when mutated Gag-Pol proteins were overexpressed as a consequence of an additional mutation that interfered with frameshifting. Thus, unlike cleavage site mutations at the N terminus of PR, a cleavage site mutation between PR and RT did not affect the enzymatic activities of either PR or RT and viruses containing PR-RT fusion proteins were viable.

Animals↗
PubMed1998

Insertions into the beta3-beta4 hairpin loop of HIV-1 reverse transcriptase reveal a role for fingers subdomain in processive polymerization.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) displays a characteristic poor processivity during DNA polymerization. Structural elements of RT that determine processivity are poorly understood. The three-dimensional structure of HIV-1 RT, which assumes a hand-like structure, shows that the fingers, palm, and thumb subdomains form the template-binding cleft and may be involved in determining the degree of processivity. To assess the influence of fingers subdomain of HIV-1 RT in polymerase processivity, two insertions were engineered in the beta3-beta4 hairpin of HIV-1NL4-3 RT. The recombinant mutant RTs, named FE20 and FE103, displayed wild type or near wild type levels of RNA-dependent DNA polymerase activity on all templates tested and wild type or near wild type-like sensitivities to dideoxy-NTPs. When polymerase activities were measured under conditions that allow a single cycle of DNA polymerization, both of the mutants displayed 25-30% greater processivity than wild type enzyme. Homology modeling the three-dimensional structures of wild type HIV-1NL4-3 RT and its finger insertion mutants revealed that the extended loop between the beta3 and beta4 strands protrudes into the cleft, reducing the distance between the fingers and thumb subdomains to approximately 12 A. Analysis of the models for the mutants suggests an extensive interaction between the protein and template-primer, which may reduce the degree of superstructure in the template-primer. Our data suggest that the beta3-beta4 hairpin of fingers subdomain is an important determinant of processive polymerization by HIV-1 RT.

Amino Acid Sequence↗
PubMed1994

An improved non-radioisotopic reverse transcriptase assay and its evaluation.

We developed an improved, highly sensitive non-radioisotopic (non-RI) reverse transcriptase (RT) assay (RTA). While the original non-RI method previously reported made use of primer immobilization, our improved method was based on a primer-template immobilization procedure. We tested the template specificity, reproducibility and linearity of the new method in assays of human immunodeficiency virus type-1 (HIV-1) RT. The sensitivities of the method previously reported, the improved method and the sensitive radioisotopic (RI-) RTA were compared in assays of recombinant HIV-1 RT, partially purified HIV-1 particles, and the culture supernatant derived from HIV-1-infected cells. For each of these samples except the culture supernatant the improved method was the most sensitive. It appeared that the fetal bovine serum presented in the culture medium interfered with the assay reaction. The curve describing inhibition of the assay reaction by fetal bovine serum showed that the highest degree of sensitivity in the assay was obtained when the culture supernatant sample was diluted four times. With this degree of dilution, the sensitivity of the new method for assay of culture supernatant sample was still half that of the sensitive RI-RTA. Culture supernatants of five peripheral blood mononuclear cell samples obtained from HIV-1-seropositive carriers were assayed by both the improved method and the sensitive RI-RTA; and with each of the methods, however all virus-positive cultures could be detected. The improved non-RI RTA was considered especially useful for assay of culture supernatants for purposes of virus isolation because of its advantages of excellent sensitivity and lack of requirement for radioisotopes.

HIV Reverse Transcriptase↗
PubMed1992

Potential anti-AIDS naphthalenesulfonic acid derivatives. Synthesis and inhibition of HIV-1 induced cytopathogenesis and HIV-1 and HIV-2 reverse transcriptase activities.

Several naphthalenedi- and trisulfonic acids have been synthesized and evaluated for inhibitory potential against cytopathogenesis and purified recombinant human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) reverse transcriptase (RT). The most potent derivative that emerged from the anti-RT study was a small molecule 6 (MW = 840), a dipalmitoylated derivative of 2,7-naphthalenedisulfonic acid. Analog 6 demonstrated 50% inhibitory concentration (IC50) values of 2.42 and 0.86 microM for HIV-1 and HIV-2 RT, respectively. The second most active compound was also a derivative of the same naphthalenedisulfonic acid but contained only one palmitoyl moiety. This compound 9 displayed IC50 values of 4.8 and 3.7 microM for HIV-1 and HIV-2 RT, respectively. Both analogs 6 and 9 are active at noncytotoxic doses, exhibit slightly higher potencies for the RT of HIV-2 over HIV-1, and demonstrate activities superior to the hexasulfonic acid derivative suramin (IC50 values of 9.4 and 15.5 microM for HIV-1 and HIV-2 RT, respectively). In the cytopathogenesis assay, the most active compound is a bis naphthalenedisulfonic acid derivative 17, containing a flexible octamethylene spacer and exhibiting an in vitro therapeutic index of 29.7. Most striking, however, is the influence of the palmitoyl functionality in the naphthalenedisulfonic acid series to confer activity against both HIV-1 and HIV-2 RT.

Enzyme Inhibitors↗
PubMed2006

Effects of trimethoprim on the clearance of apricitabine, a deoxycytidine analog reverse transcriptase inhibitor, and Lamivudine in the isolated perfused rat kidney.

Apricitabine (ATC) is a novel deoxycytidine analog reverse transcriptase inhibitor in development for the treatment of human immunodeficiency virus infection. Studies were performed to characterize the excretion of ATC and its metabolite, BCH-335 (-1-(2-hydroxymethyl-[1,3]oxathiolan-4-yl)-1H-pyrimidine-2,4-dione), in the isolated perfused rat kidney (IPK). A second objective was to investigate the effect of trimethoprim on ATC excretion because trimethoprim inhibits the excretion of lamivudine, structurally similar to ATC, in the IPK. ATC excretion was nonlinear at doses of 80 to 1600 microg. The excretion ratio (ratio of clearance to glomerular filtration rate, assuming negligible protein binding) was greater than 1.0, indicating net tubular secretion. In contrast, the excretion of BCH-335 was independent of the dose of BCH-335. Concomitant administration of ATC and BCH-335 did not affect the excretion of either compound. Trimethoprim significantly inhibited the excretion of both ATC and BCH-335, with IC(50) values of 0.45 and 0.54 microg/ml, respectively. In the presence of trimethoprim, the excretion ratios for both compounds were less than 1.0, indicating tubular reabsorption. Trimethoprim inhibited the excretion of ATC and lamivudine to similar extents. Following concomitant administration of ATC, lamivudine, and trimethoprim, there was no evidence of an interaction between ATC and lamivudine. These results suggest that ATC undergoes active tubular secretion in the kidney. Because the renal excretion of both ATC and lamivudine is inhibited by trimethoprim to similar extents, in clinical practice exposure to ATC, it would be expected to be increased in the presence of therapeutic concentrations of trimethoprim to a similar extent as has been shown previously for lamivudine.

Animals↗
PubMed1996

Effects of zidovudine-selected human immunodeficiency virus type 1 reverse transcriptase amino acid substitutions on processive DNA synthesis and viral replication.

Certain amino acid substitutions in the reverse transcriptase (RT), including D67N, K70R, T215Y, and K219Q, cause high-level resistance of human immunodeficiency virus type 1 (HIV-1) to zidovudine (3'-azidothymidine; AZT) and appear to approximate the template strand of the enzyme-template-primer complex in structural models. We studied whether this set of mutations altered RT-template-primer interaction as well as their effect on virus replication in the absence of inhibitor. When in vitro polymerization was limited to a single association of an RT with an oligodeoxynucleotide-primed heteropolymeric RNA template (a single processive cycle), recombinant-expressed mutant 67/70/215/219 RT synthesized 5- to 10-fold more high-molecular-weight DNA products (>200 nucleotides in length) than wild-type RT. This advantage was maintained as deoxynucleoside triphosphate (dNTP) concentrations were decreased to limiting levels. In contrast, no difference was seen between wild-type and mutant RTs under conditions allowing repeated associations of enzyme with template-primer. Because intracellular dNTP concentrations are low prior to mitogenic stimulation, we compared replication of mutant 67/70/215/219 virus and wild-type virus in peripheral blood mononuclear cells (PBMC) stimulated before and after infection. In the absence of inhibitor, mutant 67/70/215/219 virus had a replication advantage in PBMC stimulated with phytohemagglutinin and interleukin-2 after infection, but virus replication was similar in PBMC stimulated before infection in vitro. The results confirm that RT mutations D67N, K70R, T215Y, and K219Q affect an enzyme-template-primer interaction in vitro and suggest that such substitutions may affect HIV-1 pathogenesis during therapy by increasing viral replication capacity in cells stimulated after infection.

Base Sequence↗
PubMed1994

Mutagenicity and pausing of HIV reverse transcriptase during HIV plus-strand DNA synthesis.

The unusually high frequency of misincorporation by HIV-1 reverse transcriptase (HIV RT) is likely to be the major factor in the rapid accumulation of viral mutations in AIDS, especially in the env gene. To investigate the ability of HIV RT to copy the env gene, we subcloned an HIV env gene fragment into a single-stranded DNA vector and measured the progression of synthesis by HIV RT. We observed that HIV RT, but not RT from avian myeloblastosis virus, DNA polymerase-alpha or T7 DNA polymerase, pauses specifically at poly-deoxyadenosine stretches within the env gene. The frequency of bypassing the polyadenosine stretches by HIV RT is enhanced by increasing the ratio of enzyme to template. We measured the fidelity of DNA synthesis within a segment of the hypervariable region 1 of the env gene (V-1) containing a poly-deoxyadenosine sequence by repetitively copying the DNA by HIV RT, and then cloning and sequencing the copied fragments. We found that 27% of the errors identified in V-1 sequence were frameshift mutations opposite the poly-adenosine tract, a site where strong pausing was observed. Pausing of HIV RT at the polyadenosine tract could be enhanced by either distamycin A or netropsin, (A-T)-rich minor groove binding peptides. Moreover, netropsin increases the frequency of frameshift mutations in experiments in which HIV RT catalyzes gap filling synthesis within the lacZ gene in double-stranded circular M13mp2 DNA. These combined results suggest that the enhanced mutation frequency may be due to increased pausing at netropsin-modified polyadenosine tracts. Therefore, netropsin and related A-T binding chemicals may selectively enhance frameshift mutagenesis induced by HIV RT and yield predominantly non-viable virus.

Base Composition↗
PubMed2000

Intranasal immunization with HIV reverse transcriptase: effect of dose in the induction of helper T cell type 1 and 2 immunity.

HIV reverse transcriptase (RT) is immunologically recognized by the host after natural infection with HIV. To study the immune response to this nonenvelope protein and the effect of the antigen dose and the immunization route on the differential induction of cellular or humoral responses, CB6F1 mice were immunized intranasally with various doses of recombinant RT (0.025-25 microg/dose) in the presence of the mucosal adjuvant cholera toxin (CT). An antigen-specific, dose-dependent, in vitro proliferative response was observed in splenic cells from all mouse groups immunized with RT and CT. Proliferative responses in salivary gland-associated lymph node (SGALN) cells from the same mice were detected only with higher antigen dose immunizations (2.5, 25 microg/dose). IFN-gamma, a Th1-type cytokine, was detected in RT-stimulated culture supernatants from splenic and SGALN cells from all groups of mice immunized with RT and CT in a dose-dependent fashion. IL-4, a Th2-type cytokine, was detected in RT-stimulated culture supernatants from splenic and SGALN cells from mice immunized with higher doses of RT and CT. RT-specific IgG2a, a Th1-type-related antibody, was detected consistently in sera from all animals immunized with RT and CT and was predominant in mice immunized with lower antigen doses (0.025, 0.25 microg/dose). RT-specific IgG1, a Th2-type-related antibody, was detected consistently in mice immunized with higher antigen doses and was predominant in these groups. These studies demonstrate the immunogenicity of recombinant RT and the effect of the antigen dose in the induction of Th1-type and Th2-type immune responses after mucosal immunization.

AIDS Vaccines↗
PubMed1997

Metabolism of the HIV-1 reverse transcriptase inhibitor delavirdine in mice.

Delavirdine mesylate (U-90152T) is a highly specific nonnucleoside HIV-1 reverse transcriptase inhibitor currently under development for the treatment of AIDS. The excretion, disposition, brain penetration, and metabolism of delavirdine were investigated in CD-1 mice after oral administration of [14C]delavirdine mesylate at single doses of 10 and/or 250 mg/kg and multiple doses of 200 mg/kg/day. Studies were conducted with 14C-carboxamide and 2-14C-pyridine labels, as well as 13C3-labeled drug to facilitate metabolite identification. Excretion was dose dependent with 57-70% of the radioactivity eliminated in feces and 25-36% in urine. Pharmacokinetic analyses of delavirdine and its N-desisopropyl metabolite (desalkyl delavirdine) in plasma showed that delavirdine was absorbed and metabolized rapidly, that it constituted a minor component in circulation, that its pharmacokinetics were nonlinear, and that its metabolism to desalkyl delavirdine was capacity limited or inhibitable. Delavirdine did not significantly cross the blood-brain barrier; however, its N-isopropylpyridinepiperazine metabolite arising from amide bond cleavage-was present in brain at levels 2- to 3-fold higher than in plasma. The metabolism of delavirdine in the mouse was extensive and involved amide bond cleavage, N-desalkylation, hydroxylation at the C-6' position of the pyridine ring, and pyridine ring-cleavage as determined by MS and/or 1H and 13C NMR spectroscopies. N-desalkylation and amide bond cleavage were the primary metabolic pathways at low drug doses and, as the biotransformation of delavirdine to desalkyl delavirdine reached saturation or inhibition, amide bond cleavage became the predominant pathway at higher doses and after multiple doses.

Animals↗
PubMed1997

Initial appearance of the 184Ile variant in lamivudine-treated patients is caused by the mutational bias of human immunodeficiency virus type 1 reverse transcriptase.

Treatment of human immunodeficiency virus type 1-infected patients with lamivudine (3TC) results in the appearance of drug-resistant virus variants with a mutation at the 184Met codon (ATG) of the reverse transcriptase (RT) gene. The 184Ile (ATA) variant appears first, but subsequently the 184Val (GTG) variant outcompetes the 184Ile variant. We demonstrated previously that the 184Val enzyme and the corresponding virus are more fit than 184Ile, thereby explaining eventual outgrowth of 184Val. In this study, we set out to determine why 184Ile is usually observed first after initiation of 3TC therapy. With a limiting dilution approach during in vitro selection with 3TC, we measured a significantly higher frequency of the G-->A substitution toward the ATA codon (184Ile; 56%) than the A-->G substitution toward GTG (184Val; 12.5%). This result indicates that the initial appearance of the 184Ile variant in patients is a consequence of the mutation bias of the RT enzyme. Interestingly, a novel 3TC-resistant variant which was generated by T-->C substitution (184Thr; 28%) was also observed. The RT enzyme of the 184Thr variant was less than 10% active compared with the wild-type enzyme, and the replication capacity of this variant was severely reduced. Selection of the 184Thr variant illustrates that the limiting dilution approach allows the selection of drug-resistant variants with suboptimal fitness.

Anti-HIV Agents↗
PubMed1996

Highly sensitive qualitative and quantitative detection of reverse transcriptase activity: optimization, validation, and comparative analysis with other detection systems.

An ultra-sensitive assay for reverse transcriptase (RT) activity called Amp-RT has been developed. An in vitro transcribed heteropolymeric RNA sequence was used as a template, and polymerase chain reaction (PCR) amplification with Southern-blot hybridization served as a detection system for the cDNA product of the reaction. Titration of Mg2+ and Mn2+ concentrations using the human immunodeficiency virus type 1 (HIV-1) and the human T lymphotropic virus type 1 (HTLV-I), respectively, showed optimal assay reactivity for both viruses at 2-20 mM of Mg2+. Analysis of density banded HIV-1 showed that the peak RT activity of the assay was associated with the fractions consistent with retrovirus particles. The sensitivity of Amp-RT was also compared with that of three conventional RT assays by using seven different retroviruses including HIV-1, simian immunodeficiency virus (SIV), caprine arthritis-encephalitis virus (CAEV), HTLV-I and HTLV-II, simian retrovirus type 2 (SRV-2), and gibbon ape leukemia virus (GALV). HTLV-I, HTLV-II, and GALV could not be detected by the three conventional RT assays. Amp-RT was able to detect all these viruses in 10(1)-10(3)-fold dilutions. Similarly, Amp-RT was found to be 10(3)-10(6)-fold more sensitive than the other RT assays in detecting HIV-1, SIV< or CAEV. Culture supernatants from uninfected cell lines were all Amp-RT negative. A quantitative Amp-RT assay was also developed by using recombinant HIV-1 RT and signal quantitation. The assay was found to have a 5 log linear range, and therefore, provides a useful tool for quantitating RT and retroviruses. Amp-RT offers a sensitive generic tool for the qualitative and quantitative detection of known and unknown retroviruses.

Animals↗
PubMed2000

Non-nucleoside HIV-1 reverse transcriptase inhibitors: synthesis and biological evaluation of novel quinoxalinylethylpyridylthioureas as potent antiviral agents.

New heterocyclic derivatives of ethylpyridylthiourea, quinoxalinylethylpyridylthiourea (QXPT) and analogues, inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and prevented HIV-1 cytopathogenicity in T4 lymphocytes. Several of these novel non-nucleoside RT inhibitors, with a substituted pyrroloquinoxalinone heteroaromatic skeleton, showed inhibitory activity against wild-type RT as well as against mutant RTs containing the single amino acid substitutions L1001, K103N, V106A, Y1811 and Y188L that was much greater than other non-nucleoside inhibitors such as nevirapine. Maximum potency in enzymatic assays was achieved with a fluoropyrroloquinoxaline skeleton linked to the ethylpyridylthiourea moiety (FQXPT). In cell-based assays on different cell lines and on human monocyte-macrophages, 6-FQXPT exhibited EC50 values in the nanomolar range, with a promising selectivity index. Moreover, 6-FQXPT showed synergistic antiviral activity with zidovudine.

Amino Acid Substitution↗
PubMed2006

Intravaginal ring delivery of the reverse transcriptase inhibitor TMC 120 as an HIV microbicide.

TMC 120 (Dapivirine) is a potent non-nucleoside reverse transcriptase inhibitor that is presently being developed as a vaginal HIV microbicide. To date, most vaginal microbicides under clinical investigation have been formulated as single-dose semi-solid gels, designed for application to the vagina before each act of intercourse. However, a clear rationale exists for providing long-term, controlled release of vaginal microbicides in order to afford continuous protection against heterosexually transmitted HIV infection and to improve user compliance. In this study we report on the incorporation of various pharmaceutical excipients into TMC 120 silicone, reservoir-type intravaginal rings (IVRs) in order to modify the controlled release characteristics of the microbicide. The results demonstrate that TMC 120 is released in zero-order fashion from the rings over a 28-day period and that release parameters could be modified by the inclusion of release-modifying excipients in the IVR. The hydrophobic liquid excipient isopropyl myristate had little effect on steady-state daily release rates, but did increase the magnitude and duration of burst release in proportion to excipient loading in the IVR. By comparison, the hydrophobic liquid poly(dimethylsiloxane) had little effect on TMC 120 release parameters. A hydrophilic excipient, lactose, had the surprising effect of decreasing TMC 120 burst release while increasing the apparent steady-state daily release in a concentration-dependent manner. Based on previous cell culture data and vaginal physiology, TMC120 is released from the various ring formulations in amounts potentially capable of maintaining a protective vaginal concentration. It is further predicted that the observed release rates may be maintained for at least a period of 1 year from a single ring device. TMC 120 release profiles and the mechanical properties of rings could be modified by the physicochemical nature of hydrophobic and hydrophilic excipients incorporated into the IVRs.

Administration, Intravaginal↗
PubMed2000

Comparison of human telomerase reverse transcriptase messenger RNA and telomerase activity as urine markers for diagnosis of bladder carcinoma.

BACKGROUND: Human telomerase reverse transcriptase (hTERT) has been identified as the catalytic subunit of telomerase ribonucleoprotein complex known to be required for cellular immortality and oncogenesis. Although human telomerase activity (hTA) is considered as a general marker for malignancy based on its presence in most malignant tumors including bladder cancer, its detection in urine is affected by many factors. The objective of this study was to compare the clinical utility of detecting urine hTERT messenger RNA (mRNA) by multiplex hTERT/GAPDH RT-PCR and urine hTA by telomerase repeat amplification protocol (TRAP) in the diagnosis of bladder cancer. METHODS AND RESULTS: Cystoscopy urine samples or bladder washes prospectively collected from 35 patients with confirmed (35) or clinically suspected (5) transitional cell carcinoma (TCC) of the bladder were examined by TRAP, hTERT/GAPDH RT-PCR, and urine cytology. The control group comprised 21 healthy volunteers and 3 patients without TCC. The hTERT/GAPDH RT-PCR test showed significantly higher diagnostic sensitivity than TRAP assay (94.3% vs 48.6%, P <.001) and urine cytology (95.2% vs 61.9%, P =.008) for confirmed TCCs. In particular, for superficial TCCs low grade (I-II), the hTERT/GAPDH RT-PCR test outperformed TRAP (90% vs 25%, P <.001) and urine cytology (91.7% vs 58.3%, P =.46). The overall specificity of the hTERT/GAPDH RT-PCR, TRAP and urine cytology was 92% (22/24), 100% (24/24), and 100% (3/3), respectively. A positive hTERT mRNA expression was also detected in urologic specimens from 3 patients with previous history of TCC, 3 to 6 months before cystoscopic evidence of cancer. CONCLUSION: In this pilot study, the hTERT mRNA expression in urine sediments is a more sensitive marker for diagnosis of TCC of the bladder than hTA and cytology. However, there is a higher false-positive rate.

Adult↗
PubMed1998

Analysis of a coded panel of licensed vaccines by polymerase chain reaction-based reverse transcriptase assays: a collaborative study [seecomments].

BACKGROUND: A recent publication reporting the presence of low levels of reverse transcriptase (RT) activity in certain vaccines for human use necessitated that regulatory agencies address the issue of whether this RT activity presented a risk to humans. Detection of low levels of RT activity corresponding to fewer than ten virions became possible with the development of highly-sensitive polymerase chain reaction (PCR)-based RT (PBRT) assays. Variations of the PBRT assay were developed in three laboratories. These assays were reported as being at least one million-fold more sensitive than conventional RT assays. OBJECTIVE: To ascertain the sensitivity and reliability of PBRT assays in different laboratories and to determine which vaccine samples possessed RT activity. STUDY DESIGN: Coded panels of licensed vaccines together with positive and negative controls was assembled at the Center for Biologics Evaluation and Research (CBER) of the Food and Drug Administration (FDA) and distributed to five cooperating laboratories as well as to our laboratory at CBER. Each laboratory carried out their version of the PBRT assay and submitted the results to the coordinator at CBER. RESULTS: Results of the PBRT analyses carried out in the six laboratories are presented. Five of the six laboratories reported results that were highly consistent. RT activity was detected in live attenuated vaccines that were prepared in chick embryo cells (mumps, measles and yellow fever), but very low or undetectable RT activity was found in vaccines produced in mammalian cells (rabies and rubella). Influenza vaccines from several manufacturers included in the panel displayed the most variability, with different products of this inactivated vaccine having differing amounts of RT activity. CONCLUSIONS: Only vaccines produced in chick embryo cells had significant RT activity. Because RT activity was present in the allantoic fluid of uninfected chick embryos and culture medium from chick embryo fibroblasts, the RT activity arises from the cell substrate used for vaccine production. The PBRT assays were reliably able to detect the low levels of RT activity in chicken-derived vaccines.

Animals↗
PubMed2006

Site-directed mutagenesis in the fingers subdomain of HIV-1 reverse transcriptase reveals a specific role for the beta3-beta4 hairpin loop in dNTP selection.

HIV-1 reverse transcriptase shares the key features of high fidelity polymerases, such as a closed architecture of the active site, but displays a level of fidelity that is intermediate to that of high fidelity, replicative polymerases and low fidelity translesion synthesis (TLS) polymerases. The beta3-beta4 loop of the HIV-1 RT fingers subdomain makes transient contacts with the dNTP and template base. To investigate the role of active site architecture in HIV-1 RT fidelity, we truncated the beta3-beta4 loop, eliminating contact between Lys65 and the gamma-phosphate of dNTP. The mutant, in a manner reminiscent of TLS polymerases, was only able to incorporate a nucleotide that was capable of base-pairing with the template nucleotide, but not a nucleotide shape-analog incapable of Watson-Crick hydrogen bonding. Unexpectedly, however, the deletion mutant differed from the TLS polymerases in that it displayed an increased fidelity. The increased fidelity was associated with reduced dNTP binding affinity as measured using the dead end complex formation. In an effort to delineate the specific amino acid residue in the deleted segment responsible for this phenotype, we examined the K65 residue. Two substitution mutants, K65R and K65A were studied. The K65A mutant behaved similarly to the deletion mutant displaying dependence on Watson-Crick hydrogen bonding, increased fidelity and reduced dNTP-binding, while the K65R was more akin to wild-type enzyme. These results underscore the key role of the K65 residue in the phenotype observed in the deletion mutant. Based on the well-known electrostatic interaction between K65 and the gamma-phosphate moiety of incoming dNTP substrate in the ternary complex structure of HIV-1 RT, we conclude that non-discriminatory interactions between beta3-beta4 loop and the dNTP in wild-type HIV-1 RT help lower dNTP selectivity. Our results show that the fidelity of dNTP insertion is influenced by protein interactions with the triphosphate moiety.

Alanine↗
PubMed1998

Dominance of the E89G substitution in HIV-1 reverse transcriptase in regard to increased polymerase processivity and patterns of pausing.

The substitution of a glycine for glutamic acid at position 89 in human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) (E89G) confers resistance to several nucleoside and non-nucleoside inhibitors of RT. As residue 89 contacts the template strand, it has been suggested that this mutation may modulate the conformation of the RT.template/primer complex. In addition, certain mutations in RT that confer resistance to nucleoside analogs, such as M184V, are located near the polymerase active site. To characterize further these substitutions, we performed processivity assays alongside an analysis of pausing profiles with wild-type (wt) RT and recombinant RTs containing substitutions at E89G, M184V, or both. We now show that E89G RT has higher processivity than wt enzyme as well as a different pattern of pausing sites. Similar findings were obtained with the doubly mutated RT, although enzyme containing only the M184V mutation had lower processivity than wt. Consistent with these observations, and from a mechanistic standpoint, both E89G-containing as well as doubly mutated RT had decreased dissociation constants from a complex consisting of RT and template-primer, in comparison with either wt RT or M184V-containing RT. No significant differences were observed among the various enzymes in regard to Km values for the heteropolymeric RNA template used in these studies. Viruses containing the E89G mutation synthesized longer strand DNA products than either wt viruses or viruses containing only the M184V mutation in endogenous RT assays. Thus, the E89G substitution is a dominant determinant in regard to each of the koff values from an RT.template/primer complex, RT processivity, and specific patterns of pausing during DNA polymerization.

Amino Acid Substitution↗