An evaluation by midwives and gynecologists of treatability of cervical lesions by cryotherapy among human papillomavirus-positive women.

OBJECTIVES: To estimate efficacy of a visual triage of human papillomavirus (HPV)-positive women to either immediate cryotherapy or referral if not treatable (eg, invasive cancer, large precancers). METHODS: We evaluated visual triage in the HPV-positive women aged 25 to 55 years from the 10,000-woman Guanacaste Cohort Study (n = 552). Twelve Peruvian midwives and 5 international gynecologists assessed treatability by cryotherapy using digitized high-resolution cervical images taken at enrollment. The reference standard of treatability was determined by 2 lead gynecologists from the entire 7-year follow-up of the women. Women diagnosed with histologic cervical intraepithelial neoplasia grade 2 or worse or 5-year persistence of carcinogenic HPV infection were defined as needing treatment. RESULTS: Midwives and gynecologists judged 30.8% and 41.2% of women not treatable by cryotherapy, respectively (P < 0.01). Among 149 women needing treatment, midwives and gynecologists correctly identified 57.5% and 63.8% (P = 0.07 for difference) of 71 women judged not treatable by the lead gynecologists and 77.6% and 59.7% (P < 0.01 for difference) of 78 women judged treatable by cryotherapy. The proportion of women judged not treatable by a reviewer varied widely and ranged from 18.6% to 61.1%. Interrater agreement was poor with mean pairwise overall agreement of 71.4% and 66.3% and kappa's of 0.33 and 0.30 for midwives and gynecologists, respectively. CONCLUSIONS: In future "screen-and-treat" cervical cancer prevention programs using HPV testing and cryotherapy, practitioners will visually triage HPV-positive women. The suboptimal performance of visual triage suggests that screen-and-treat programs using cryotherapy might be insufficient for treating precancerous lesions. Improved, low-technology triage methods and/or improved safe and low-technology treatment options are needed.

Treatability by cryotherapy in a screen-and-treat strategy.

OBJECTIVES: We estimated the percentage of women infected with human papillomavirus (HPV+) who cannot be immediately treated with cryotherapy. MATERIALS AND METHODS: In a 10,000-woman Costa Rican cohort, we analyzed the 559 HPV+ women aged 25 to 55 years and estimated the proportion for whom immediate cryotherapy was not indicated (i.e., invasive cancer, large precancerous lesions, or benign abnormalities that risk failure such as large ectopy, squamocolumnar junction not visualized, polyps, ulcers, or distorted or atrophied cervix). To determine whether cryotherapy at time of baseline HPV screening would effectively treat HPV+ women, 2 expert gynecologists independently judged entire clinical histories (5-7 years of cytology, histology, and HPV tests) and a full longitudinal series of digitized cervical images. RESULTS: Reviewers judged 144 (25.8%) of 559 HPV+ women as not treatable by immediate cryotherapy. Among 72 women with cervical intraepithelial neoplasia grade 3 who would benefit most from a screening program, 35 (48.6%) were not treatable. In particular, 29 women (40.3%) were determined not treatable for reasons most likely associated with cryotherapy's inadequacy (lesion was large, suspected cancerous or in the endocervical canal or fornix). CONCLUSIONS: "Screen-and-treat" programs in low-resource settings will soon use a rapid HPV test to screen older women once or twice in their lifetime, identifying women at higher risk for precancer. Our findings suggest that cryotherapy might not effectively treat many precancers, and other safe, low-technology treatment options could be required, in a scenario where all HPV+ women in this targeted group would receive cryotherapy at the same visit.

Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones.

High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives beta-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). beta-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC50 of 0.2 microM, while the IC50 for Escherichia coli and human RNases H was 50 microM and 5.7 microM, respectively. In contrast, the related tropolone analog beta-thujaplicin (2-hydroxy-4-(methylethyl)-2,4,6-cycloheptatrien-1-one), which lacks the 7-OH group of the heptatriene ring, was inactive, while manicol, which possesses a 7-OH group, inhibited HIV-1 and E.coli RNases H with IC50 = 1.5 microM and 40 microM, respectively. Such a result highlights the importance of the 2,7-dihydroxy function of these tropolone analogs, possibly through a role in metal chelation at the RNase H active site. Inhibition of HIV-2 RT-associated RNase H indirectly indicates that these compounds do not occupy the nonnucleoside inhibitor-binding pocket in the vicinity of the DNA polymerase domain. Both beta-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 microM, suggesting that they are specific for the C-terminal RNase H domain, while surface plasmon resonance studies indicated that the inhibition was not due to intercalation of the analog into the nucleic acid substrate. Finally, we have demonstrated synergy between beta-thujaplicinol and calanolide A, a nonnucleoside inhibitor of HIV-1 RT, raising the possibility that both enzymatic activities of HIV-1 RT can be simultaneously targeted.

A capillary electrophoretic assay for ribonuclease H activity.

A capillary electrophoretic assay was developed to measure the ribonuclease (RNase) H activity of human immunodeficiency virus (HIV) type 1 reverse transcriptase. Cleavage of a fluorescein-labeled RNA-DNA heteroduplex was monitored by capillary electrophoresis. This new assay was used as a secondary assay to confirm hits from a high-throughput screening program. Since autofluorescent compounds in samples migrated differently from both substrate and product in most cases, the assay was extremely robust for assaying enzymatic inhibition of such samples, in contrast to a simple well-based approach. The assay was broadly applicable to other RNases H, specifically those from human, Escherichia coli, and HIV-2, although product profiles varied for each enzyme.

Identification of an inhibitor-binding site to HIV-1 integrase with affinity acetylation and mass spectrometry.

We report a methodology that combines affinity acetylation with MS analysis for accurate mapping of an inhibitor-binding site to a target protein. For this purpose, we used a known HIV-1 integrase inhibitor containing aryl di-O-acetyl groups (Acetylated-Inhibitor). In addition, we designed a control compound (Acetylated-Control) that also contained an aryl di-O-acetyl group but did not inhibit HIV-1 integrase. Examination of the reactivity of these compounds with a model peptide library, which collectively contained all 20 natural amino acids, revealed that aryl di-O-acetyl compounds effectively acetylate Cys, Lys, and Tyr residues. Acetylated-Inhibitor and Acetylated-Control exhibited comparable chemical reactivity with respect to these small peptides. However, these two compounds differed markedly in their interactions with HIV-1 integrase. In particular, Acetylated-Inhibitor specifically acetylated K173 at its inhibitory concentration (3 microM) whereas this site remained unrecognized by Acetylated-Control. Our data enabled creation of a detailed model for the integrase:Acetylated-Inhibitor complex, which indicated that the inhibitor selectively binds at an architecturally critical region of the protein. The methodology reported herein has a generic application for systems involving a variety of ligand-protein interactions.

A fluorescence-based high-throughput screening assay for inhibitors of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H activity.

A fluorescence resonance energy transfer assay readily applicable to 96-well and 384-well microplate formats with robotic operation was developed to enable high-throughput screening for inhibitors of human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT)-associated RNase H activity, an underexplored target for antiretroviral development. The assay substrate is an 18-nucleotide 3'-fluorescein-labeled RNA annealed to a complementary 18-nucleotide 5'-Dabcyl-modified DNA. The intact duplex has an extremely low background fluorescent signal and provides up to 50-fold fluorescent signal enhancement following hydrolysis. The size and sequence of the duplex are such that HIV-1 RT-RNase H cuts the RNA strand close to the 3' end. The fluorescein-labeled ribonucleotide fragment readily dissociates from the complementary DNA at room temperature with immediate generation of a fluorescent signal. This assay is rapid, inexpensive, and robust, providing Z' factors of 0.8 and coefficients of variation of about 5%. The assay can be carried out both in real-time (continuous) and in "quench" modes; the latter requires only two addition steps with no washing and is thus suitable for robotic operation. Several chemical libraries totaling more than 106,000 compounds were screened with this assay in approximately 1 month.

Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagent.

We have devised a high-resolution protein footprinting methodology to dissect HIV-1 reverse transcriptase (RT) contacts to the viral RNA:tRNA complex. The experimental strategy included modification of surface-exposed lysines in RT and RT-viral RNA:tRNA complexes by the primary amine selective reagent NHS-biotin, SDSPAGE separation of p66 and p51 polypeptides, in gel proteolysis, and comparative mass spectrometric analysis of peptide fragments. The lysines modified in free RT but protected from biotinylation in the nucleoprotein complex were readily revealed by this approach. Results of a control experiment examining the RT-DNA:DNA complex were in excellent agreement with the crystal structure data on the identical complex. Probing the RT-viral RNA:tRNA complex revealed that a majority of protein contacts are located in the primer-template binding cleft in common with the RT-DNA:DNA and RT-RNA:DNA species. However, our footprinting data indicate that the p66 fingers subdomain makes additional contacts to the viral RNA:tRNA specific for this complex and not detected with DNA:DNA. The protein footprinting method described herein has a generic application for high-resolution solution structural studies of multiprotein-nucleic acid contacts.

Mutating conserved residues in the ribonuclease H domain of Ty3 reverse transcriptase affects specialized cleavage events.

The reverse transcriptase-associated ribonuclease H (RT/RNase H) domains from the gypsy group of retrotransposons, of which Ty3 is a member, share considerable sequence homology with their retroviral counterparts. However, the gypsy elements have a conserved tyrosine (position 459 in Ty3 RT) instead of the conserved histidine in the catalytic center of retroviral RTs such as at position 539 of HIV-1. In addition, the gypsy group shows conservation of histidine adjacent to the third of the metal-chelating carboxylate residues, which is Asp-426 of Ty3 RT. The role of these and additional catalytic residues was assessed with purified recombinant enzymes and through the ability of Ty3 mutants to support transposition in Saccaromyces cerevisiae. Although all mutations had minimal impact on DNA polymerase function, amidation of Asp-358, Glu-401, and Asp-426 eliminated Mg(2+)- and Mn(2+)-dependent RNase H function. Replacing His-427 and Tyr-459 with Ala and Asp-469 with Asn resulted in reduced RNase H activity in the presence of Mg(2+), whereas in the presence of Mn(2+) these mutants displayed a lack of turnover. Despite this, mutations at all positions were lethal for transposition. To reconcile these apparently contradictory findings, the efficiency of specialized RNase H-mediated events was examined for each enzyme. Mutants retaining RNase H activity on a heteropolymeric RNA.DNA hybrid failed to support DNA strand transfer and release of the (+) strand polypurine tract primer from (+) RNA, suggesting that interrupting one or both of these events might account for the transposition defect.

Pre-existing distortions in nucleic acid structure aid polypurine tract selection by HIV-1 reverse transcriptase.

Precise cleavage at the polypurine tract (PPT)/U3 junction by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase RNase H is critical for generating a correct viral DNA end for subsequent integration. Using potassium permanganate (KMnO(4)) modification, we have identified a significant distortion in the nucleic acid structure at the HIV-1 PPT/U3 junction in the absence of trans-acting factors. Unusually high reactivity of template thymine +1 is detected when the PPT primer is extended by DNA or RNA at its 3' terminus. Chemical footprinting suggests that the extent of base unstacking in the wild-type species is comparable when the +1 A:T base pair is replaced by a C:T mismatch. However, reactivity of this template base is diminished after alterations to upstream (rA)(4):(dT)(4) or (rG)(6):(dC)(6) tracts. Importantly, there is a correlation between the structural deformation at base pair +1 and precise cleavage at the PPT/U3 junction by HIV-1 reverse transcriptase/RNase H. KMnO(4) modification also revealed unusually high reactivity for one of two (dT)(4):(rA)(4) duplexes upstream of the PPT/U3 junction, suggesting a significant structural distortion within the PPT itself in the absence of the retroviral polymerase. Structural abnormalities in this region are not only essential for resistance of the PPT to hydrolysis but also significantly impact the conformation of the PPT/U3 junction. Our data collectively suggest that the entire PPT sequence contributes to the structural distortion at the PPT/U3 junction, potentially providing a mechanism for its selective processing.