Expression of high-affinity laminin receptor mRNA correlates with cell proliferation rather than invasion in human papillomavirus-associated cervical neoplasms.

Induction of the expression of the Mr 67,000 high-affinity laminin receptor gene has been postulated as playing a role in the progression of human tumors to invasive cancers. We tested this hypothesis by examining histopathological sections of a large number of epithelial lesions of the genital tract associated with human papillomaviruses. In situ hybridization was performed with a riboprobe generated from a laminin receptor complementary DNA. Laminin receptor mRNA was expressed primarily in the less differentiated cells in normal squamous tissues and in a spectrum of squamous neoplasms. There was no net induction of mRNA per cell in intraepithelial or invasive squamous neoplasms relative to normal tissue. In contrast, laminin receptor mRNA was not expressed at a detectable level in normal glands of the uterine cervix but was dramatically induced in morphologically abnormal, human papillomavirus-positive glands, irrespective of the genotype of human papillomaviruses present. The induction occurred before any evidence of invasion, and there was no further increase during the transition from adenocarcinoma in situ to invasive carcinoma. We conclude that induction of high-affinity laminin receptor gene expression is associated with the development of malignancies of cervical glandular epithelia, but the increased expression appears to correlate with the proliferative rather than the invasive properties of these cells.

Induction of proliferating cell nuclear antigen in differentiated keratinocytes of human papillomavirus-infected lesions.

The expression of proliferating cell nuclear antigen (PCNA) was studied in human papillomavirus (HPV)-infected, benign and malignant lesions of the genital tract and larynx using immunocytochemical staining of formalin-fixed clinical specimens. We observed the induction of PCNA in squamous carcinomas and adenocarcinomas, as has been demonstrated with other malignancies. In addition, the differentiated keratinocytes of the upper spinous cells and granulocytes in condylomata acuminata and low-grade intraepithelial neoplasias showed a consistent induction of PCNA compared with the normal squamous epithelium, in which only some of the parabasal and basal cells were positive. This reactivation of PCNA synthesis correlated with the presence of high copy numbers of HPV DNA and was independent of the oncogenic risk potential of the infecting HPV genotype. We postulate that HPV gene products induce the expression of PCNA and other components of the host DNA replication machinery in differentiated cells of squamous lesions to facilitate vegetative viral replication.

Concentration-targeted phase I trials of atevirdine mesylate in patients with HIV infection: dosage requirements and pharmacokinetic studies. The ACTG 187 and 199 study teams.

RATIONALE: To determine the dosage requirements and pharmacokinetics of atevirdine, a non-nucleoside reverse transcriptase inhibitor and its N-dealkylated metabolite (N-ATV) during phase I studies in patients receiving atevirdine alone or in combination with zidovudine. DESIGN: Two open label, phase I studies conducted by the adult AIDS Clinical Trials Group (ACTG) in which atevirdine was administered every 8 h with weekly dosage adjustments to attain targeted trough plasma atevirdine concentrations. SETTING: Five Adult AIDS Clinical Trials Units. PATIENTS: Fifty patients (ACTG 199; n = 20 and ACTG 187; n = 30) with HIV-1 infection and < or =500 CD4+ lymphocytes/mm3. INTERVENTION: ACTG 199; 12 weeks of therapy with atevirdine (dose-adjusted to achieve plasma trough atevirdine concentrations of 5-10 microM) and zidovudine (200 mg every 8 h). ACTG 187: 12 weeks of atevirdine monotherapy with atevirdine doses adjusted to achieve escalating, targeted trough plasma concentration ranges (5-13, 14-22, and 23-31 microM). MEASUREMENTS: ACTG 199: atevirdine, N-ATV and zidovudine trough determinations weekly (all patients) and intensive pharmacokinetics (selected patients) prior to and at 6 and 12 weeks during combination therapy. ACTG 187: atevirdine and N-ATV trough concentrations over a 12 week period. Intensive pharmacokinetic studies were conducted prior to and at 4 and/or 8 weeks during atevirdine monotherapy in female patients. RESULTS: Atevirdine plasma concentrations demonstrated considerable interpatient variability which was minimized by the adjustment of maintenance doses (range: 600-3900 mg/day) to achieve the desired trough concentrations. In ACTG 187, the mean number of weeks to attain the target value, and the percentage of patients who attained the target, was group I (5-11 microM): 2.7+/-2.4 weeks (92%); group II (12-21 microM): 2.6+/-1.8 (64%); and group III (22-31 microM): 7.0+/-5.6 weeks (27%). In ACTG 199 it was 3.2+/-5.2 weeks (95%) to achieve a 5-10 microM trough. Atevirdine demonstrated a mono- or bi-exponential decline among most of the patients studied after the first dose. During multiple-dosing a number of patterns of atevirdine disposition were observed including; rapid absorption with Cmax at 0.5-1 h, delayed absorption with Cmax at 3-4 h; minimal Cmax to Cmin fluctuation and Cmax to Cmin ratios of > 4. N-ATV (an inactive metabolite) patterns were characterized on day one by rapid appearance of the metabolite which peaked at 2-3 h after the dose and declined in a mono- or bi-exponential manner. At steady-state N-ATV patterns demonstrated minimal Cmax to Cmin fluctuations with some of the patients having more stable plasma N-ATV concentrations, while others had greater fluctuations week to week. CONCLUSIONS: Considerable interpatient variability was noted in the pharmacokinetics of atevirdine. The variation in drug disposition was reflected in the range of daily doses required to attain the targeted trough concentrations. Atevirdine metabolism did not appear to reach saturation during chronic dosing in many of our patients, as reflected by the pattern of N-ATV/ATV ratios in plasma and saturation was not an explanation for the variation in dosing requirements. No apparent differences were noted between males and females, and atevirdine did not appear to influence zidovudine disposition.

Interlaboratory concordance of DNA sequence analysis to detect reverse transcriptase mutations in HIV-1 proviral DNA. ACTG Sequencing Working Group. AIDS Clinical Trials Group.

Thirteen laboratories evaluated the reproducibility of sequencing methods to detect drug resistance mutations in HIV-1 reverse transcriptase (RT). Blinded, cultured peripheral blood mononuclear cell pellets were distributed to each laboratory. Each laboratory used its preferred method for sequencing proviral DNA. Differences in protocols included: DNA purification; number of PCR amplifications; PCR product purification; sequence/location of PCR/sequencing primers; sequencing template; sequencing reaction label; sequencing polymerase; and use of manual versus automated methods to resolve sequencing reaction products. Five unknowns were evaluated. Thirteen laboratories submitted 39043 nucleotide assignments spanning codons 10-256 of HIV-1 RT. A consensus nucleotide assignment (defined as agreement among > or = 75% of laboratories) could be made in over 99% of nucleotide positions, and was more frequent in the three laboratory isolates. The overall rate of discrepant nucleotide assignments was 0.29%. A consensus nucleotide assignment could not be made at RT codon 41 in the clinical isolate tested. Clonal analysis revealed that this was due to the presence of a mixture of wild-type and mutant genotypes. These observations suggest that sequencing methodologies currently in use in ACTG laboratories to sequence HIV-1 RT yield highly concordant results for laboratory strains; however, more discrepancies among laboratories may occur when clinical isolates are tested.

The non-nucleoside reverse transcriptase inhibitor efavirenz stimulates replication of human immunodeficiency virus type 1 harboring certain non-nucleoside resistance mutations.

We measured the effects of non-nucleoside reverse transcriptase (RT) inhibitor-resistant mutations K101E+G190S, on replication fitness and EFV-resistance of HIV(NL4-3). K101E+G190S reduced fitness in the absence of EFV and increased EFV resistance, compared to either single mutant. Unexpectedly, K101E+G190S also replicated more efficiently in the presence of EFV than in its absence. Addition of the nucleoside resistance mutations L74V or M41L+T215Y to K101E+G190S improved fitness and abolished EFV-dependent stimulation of replication. D10, a clinical RT backbone containing M41L+T215Y and K101E+G190S, also demonstrated EFV-dependent stimulation that was dependent on the presence of K101E. These studies demonstrate that non-nucleoside reverse transcriptase inhibitors can stimulate replication of NNRTI-resistant HIV-1 and that nucleoside-resistant mutants can abolish this stimulation. The ability of EFV to stimulate NNRTI-resistant mutants may contribute to the selection of HIV-1 mutants in vivo. These studies have important implications regarding the treatment of HIV-1 with combination nucleoside and non-nucleoside therapies.

The HIV-1 reverse transcriptase mutants G190S and G190A, which confer resistance to non-nucleoside reverse transcriptase inhibitors, demonstrate reductions in RNase H activity and DNA synthesis from tRNA(Lys, 3) that correlate with reductions in replication efficiency.

We evaluated the replication efficiency of the HIV reverse transcriptase (RT) mutants K103N, G190A, and G190S, which confer resistance to the non-nucleoside RT inhibitor efavirenz, using growth competition assays in cell culture. In the absence of efavirenz, the fitness hierarchy was G190S < G190A < K103N < wild-type. The fitness reduction of G190S relative to K103N was less evident at high efavirenz concentrations, although K103N still replicated more efficiently. Efficiency of RNase H cleavage and RNA-dependent DNA synthesis from tRNA(Lys, 3) correlated with relative fitness, in biochemical studies of mutant RTs. Presteady state and steady state polymerization assays using DNA primers detected no abnormalities. This work is consistent with previous studies demonstrating that initiation of viral DNA synthesis is reduced in mutants with slowed RNase H cleavage, and suggests that both abnormalities contribute to the replication defect of these mutants. It also suggests that high concentrations of efavirenz are unlikely to favor the selection of G190S clinically.

Association of human papillomavirus infection and disease with magnitude of human immunodeficiency virus type 1 (HIV-1) RNA plasma level among women with HIV-1 infection.

Ninety-three women with human immunodeficiency virus type 1 (HIV-1) infection were enrolled in a cross-sectional study to evaluate the relationship between plasma HIV-1 RNA levels and coincident cervical infection and disease caused by human papillomaviruses (HPVs). HIV-1 RNA plasma levels of >10,000 copies/mL were highly associated with the presence in cervical specimens of HPV DNA of oncogenic (high risk) virus genotypes (P=.006; relative risk, 2.57). In addition, similar HIV-1 RNA plasma levels were associated with abnormal Pap smears (P=.01; relative risk, 2.11). In this study, 81% of women with high-risk HPV cervical infection had abnormal Pap smears. Measurement of HIV-1 RNA plasma levels may help to identify a subgroup of HIV-1-infected women at increased risk for cervical HPV infection and disease.

Impact of clinical reverse transcriptase sequences on the replication capacity of HIV-1 drug-resistant mutants.

We have shown that the HIV-1 laboratory strain NL4-3 that contains P236L [a reverse transcriptase mutation conferring resistance to the nonnucleoside reverse transcriptase inhibitor (NRTI) delavirdine] replicates more slowly than wild-type NL4-3. Other NNRTI-resistance mutations, such as K103N and Y181C, do not reduce the replication capacity of NL4-3 as much as P236L and develop more frequently in HIV-1 isolates from patients failing delavirdine. However, a minority of patients on delavirdine therapy still have isolates with P236L. We postulated that reverse transcriptase (RT) sequences from these patient isolates contain other mutations that compensate for the adverse effect of P236L. To test this hypothesis, we created 15 chimeric NL4-3 isolates that contained delavirdine-resistant RT sequences derived from eight patient isolates and characterized their replication kinetics. Nine of 10 patient-derived clones containing P236L replicated as slowly as NL4-3 with P236L. In contrast, three of five clones that did not have P236L (but had either K103N or Y181C) replicated significantly better than NL4-3 with P236L. Thus, the majority of patients who acquire P236L during delavirdine therapy do not have RT mutations that compensate for the replication defect conferred by P236L. We hypothesize that HIV-1 isolates with P236L may have a compensatory mutation outside RT. Alternatively, variants of HIV-1 with reduced replication fitness may be selected during antiretroviral therapy, suggesting that stochastic events rather than viral replication fitness may determine which drug-resistant mutants emerge early during antiretroviral failure. In some isolates, it appears that the background RT sequence can contribute significantly to the replication fitness of drug-resistant HIV-1 variants.

The Y181C mutant of HIV-1 reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors alters the size distribution of RNase H cleavages.

We investigated the effects of the nonnucleoside reverse transcriptase inhibitor-resistant mutant Y181C on RNA 5'-end-directed RNase H cleavage by HIV-1 reverse transcriptase, using an RNA.DNA hybrid in which a radiolabeled RNA 5' end was recessed. Y181C produced a higher ratio of secondary (9 nucleotide long) to primary (18 nucleotide long) products than wild type. When the RNA was 3'-end-labeled, Y181C generated a long product, which results when secondary cleavage precedes the primary. When using an RNA.DNA hybrid in which the labeled RNA 5' end and DNA 3' end were flush, formation of secondary product by both enzymes was inhibited. Under these conditions, Y181C cleaved closer to the RNA 5' end than wild type. Studies with this substrate labeled at the RNA 3' end showed that Y181C is no more likely than wild type to cleave toward the RNA 3' end. Thus, Y181C RT has a strong preference to cleave in the direction of the RNA 5' end even when secondary cleavage is prevented, resulting in a disruption of the normal sequence of primary followed by secondary cleavages.

Development of zidovudine resistance mutations in patients receiving prolonged didanosine monotherapy.

Human immunodeficiency virus type 1 (HIV-1) isolates from 2 patients who received didanosine (ddI) monotherapy for > 2 years were analyzed for reverse transcriptase (RT) mutations by sequencing of proviral DNA from peripheral blood mononuclear cell cultures. One patient was otherwise antiretroviral-naive; the other had received zidovudine for 5 months before beginning ddI therapy. Isolates obtained from both patients before initiation of ddI monotherapy were free of HIV-1 RT mutations associated with zidovudine or ddI resistance. However, after prolonged ddI monotherapy, mutations associated with zidovudine resistance (M41L, D67N, K70R, and/or T215Y) were detected in HIV-1 isolates from both patients. There was no evidence that surreptitious use of zidovudine or technical artifact caused these findings. This observation suggests that prolonged ddI monotherapy may decrease the efficacy of subsequent zidovudine therapy in some patients.

Analysis of env sequence evolution in human immunodeficiency virus-infected patients receiving therapy with nonnucleoside reverse-transcriptase inhibitors.

Nonnucleoside reverse-transcriptase inhibitors (NNRTIs) can rapidly select for drug-resistant human immunodeficiency virus type 1 (HIV-1) variants, although their effect on HIV-1 quasi-species diversity is unknown. To determine if changes in env gene diversification occur with NNRTI therapy, we used the heteroduplex tracking assay (HTA) to study HIV-1 env sequence diversity in 2 groups of patients: those who were on no therapy or were on chronic antiretroviral therapy and those who had just initiated NNRTIs. Forty-nine paired samples from 46 patients were analyzed. Fourteen of 32 paired samples from the NNRTI group and 9 of 17 paired samples from the control group had HTA changes (P>.10). There was no correlation between HTA change and sampling time interval, baseline virus load, change in virus load, or development of NNRTI resistance. Thus, we found no significant correlation of NNRTI therapy with changes in env HTA patterns, suggesting that these treatments had little short-term impact on HIV-1 quasi-species diversity.

Penile intraepithelial neoplasia: clinical presentation and an analysis of the physical state of human papillomavirus DNA.

Forty-four men with penile intraepithelial neoplasia (PIN) and a matched control group of 88 men with condyloma acuminatum were evaluated in three centers studying anogenital human papillomavirus (HPV) infections. PIN and condyloma groups could not be distinguished on the basis of historical features or clinical presentation. Although PINs were more likely than condylomata to be pigmented (31/46 [67%] vs. 33/97 [34%], P < .001), 43% of PIN III were not pigmented, suggesting that pigmentation is not a sensitive indicator of high-grade PIN. HPV-16 infection, as determined by in situ hybridization, was closely associated with PIN III (0/24 PIN I contained HPV-16 vs. 12/13 PIN III, P < .001). Southern blot analysis demonstrated only episomal viral genomes, suggesting that integration is not an early event in penile neoplasia.

Mutation of the methylated tRNA(Lys)(3) residue A58 disrupts reverse transcription and inhibits replication of human immunodeficiency virus type 1.

Cellular tRNA(Lys)(3) serves as the primer for reverse transcription of human immunodeficiency virus type 1 (HIV-1). tRNA(Lys)(3) interacts directly with HIV-1 reverse transcriptase (RT), is packaged into viral particles, and anneals to the primer-binding site (PBS) of the HIV-1 genome in order to initiate reverse transcription. Residue A58 of tRNA(Lys)(3), which lies outside the PBS-complementary region, is posttranscriptionally methylated to form 1-methyladenosine 58 (M(1)A58). This methylation is thought to serve as a pause signal for plus-strand strong-stop DNA synthesis during reverse transcription. However, formal proof that the methylation is necessary for the pausing of RT has not been obtained in vivo. In the present study, we investigated the role of tRNA(Lys)(3) residue A58 in the replication cycle of HIV-1 in living cells. We have developed a mutant tRNA(Lys)(3) derivative, tRNA(Lys)(3)A58U, in which A58 was replaced by U. This mutant tRNA was expressed in CEM cells. We demonstrate that the presence of M(1)A58 is necessary for the appropriate termination of plus-strand strong-stop DNA synthesis and that the absence of M(1)A58 allows RT to read the tRNA sequences beyond residue 58. In addition, we show that replacement of M(1)A58 with U inhibits the replication of HIV-1 in vivo. These results highlight the importance of tRNA primer residue A58 in the reverse transcription process. Inhibition of reverse transcription with mutant tRNA primers constitutes a novel approach for therapeutic intervention against HIV-1.

Phase I study of atevirdine, a nonnucleoside reverse transcriptase inhibitor, in combination with zidovudine for human immunodeficiency virus type 1 infection. ACTG 199 Study Team.

Twenty patients were enrolled in a phase I clinical trial of atevirdine, a nonnucleoside reverse transcriptase inhibitor (NNRTI), given in combination with zidovudine for treatment of human immunodeficiency virus type 1 (HIV-1) infection. Fifteen patients had received no previous antiretroviral therapy. HIV-1 isolates obtained at 6-week intervals were tested for sensitivity to atevirdine and zidovudine. Two patients developed a rash within 2 weeks of enrollment, and 1 of these developed concomitant fever and hepatitis. No hematopoietic, neurologic, or pancreatic toxicities were observed. Atevirdine had considerable initial interpatient pharmacokinetic variability. Forty-seven percent of patients treated with atevirdine plus zidovudine had increased CD4 lymphocyte counts, and HIV isolates from 62% of patients remained sensitive to atevirdine after 24 weeks of therapy. Atevirdine plus zidovudine was well-tolerated. Additional studies should be done to determine the role of atevirdine in the therapy for HIV infection.

HIV-1 drug susceptibilities and reverse transcriptase mutations in patients receiving combination therapy with didanosine and delavirdine.

Previous studies have shown that the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase mutation Y181C, which confers high-level resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), develops rarely during therapy with NNRTIs plus zidovudine. To determine whether didanosine (ddI) is also effective in preventing the emergence of Y181C, we analyzed delavirdine (DLV) susceptibilties and reverse transcriptase sequences of isolates obtained from patients enrolled in a pharmacokinetic study of DLV and ddI. Nine NNRTI-naive patients were evaluated. Seven received DLV/ddI and two received DLV/ddI/zidovudine. Median durations of prior zidovudine and ddI were 26 and 15 months, respectively. Isolates from eight of nine patients had a mutation(s) associated with nucleoside resistance at entry. After treatment with DLV and ddI alone, isolates from five of seven patients developed Y181C, four in combination with K103N. Thus, in this group of nucleoside-experienced patients, combination therapy with DLV/ddI did not prevent the emergence of Y181C.

Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.

Human papillomaviruses trophic for anogenital epithelia cause benign warts, and certain genotypes are closely associated with cervical neoplasia. By using our modifications of the epithelial raft culture system, we were able to recapitulate and modulate the infectious program of a papillomavirus in vitro for the first time. Small pieces of a condyloma containing human papillomavirus type 11 were explanted onto a dermal equivalent consisting of a collagen matrix with fibroblasts and were cultured at the medium-air interface. The infected stem cells proliferated rapidly across the matrix, stratified, and differentiated, as judged by histology. The results correlated with the state of epithelial differentiation, which, in turn, was dependent on the type of fibroblast in the matrix. Under conditions where the epithelial outgrowth underwent terminal differentiation, the entire productive program took place, leading to virion assembly. In contrast, using an alternative condition where the outgrowth failed to achieve terminal differentiation, only the E-region RNAs from the E1 promoter accumulated to any appreciable extent. The proliferating cell nuclear antigen was induced in the differentiated suprabasal cells in the productive cyst growth, which also exhibited high copy viral DNA and abundant E6-E7 RNAs. Comparable cells in the nonproductive cyst outgrowth were negative for all three. These results suggest that the E6 and E7 proteins may play a role in establishing a cellular environment conducive to vegetative viral replication. The culture conditions described should be useful for genetic analysis of this family of important human pathogens and for testing potential pharmacological agents.

The P236L delavirdine-resistant human immunodeficiency virus type 1 mutant is replication defective and demonstrates alterations in both RNA 5'-end- and DNA 3'-end-directed RNase H activities.

The nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) delavirdine (DLV) selects in vitro for the human immunodeficiency virus type 1 (HIV-1) RT mutation P236L, which confers high-level resistance to DLV but not other NNRTIs. Unexpectedly, P236L has developed infrequently in HIV-1 isolates obtained from patients receiving DLV; K103N is the predominant resistance mutation observed in that setting. We characterized the replication fitness of viruses derived from pNL4-3 containing P236L or K103N in both H9 and primary human peripheral blood mononuclear cell cultures infected in parallel with the two mutants. In the absence of DLV, p24 production by wild-type virus occurred more rapidly and to higher levels than with either mutant; P236L consistently demonstrated a two- to threefold decrease in p24 relative to K103N. At low levels of DLV, growth of wild-type virus was severely inhibited, and K103N replicated two- to threefold more efficiently than P236L. At high concentrations of DLV, P236L replication and K103N replication were both inhibited. Recombinant RTs containing K103N or P236L were analyzed for DNA polymerization on heteropolymeric RNA templates and RNase H degradation of RNA-DNA hybrids. Neither mutant demonstrated defects in polymerization. K103N demonstrated normal RNA 5'-end-directed RNase H cleavage and slowed DNA 3'-end-directed RNase H cleavage compared to wild-type RT. P236L demonstrated slowing of both DNA 3'-end- and RNA 5'-end-directed RNase H cleavage, consistent with its reduced replication efficiency relative to K103N. These data suggest that NNRTI resistance mutations can lead to reductions in the efficiency of RNase H cleavage, which may contribute to a reduction in the replication fitness of HIV-1.

Effects of zidovudine use during pregnancy on resistance and vertical transmission of human immunodeficiency virus type 1.

The resistance of human immunodeficiency virus type 1 (HIV-1) to zidovudine and the vertical transmission of the virus were assessed among all 62 HIV-1-infected pregnant women identified prior to delivery at one institution. HIV-1 was transmitted to infants from 11 (26%) of 42 women who did not receive oral zidovudine but from only 1 of 20 women given such treatment (P = .04). Isolates of HIV-1 from 16 of the 20 zidovudine-treated women were available. Twelve of 16 isolates were wild-type for pol codons 41, 67, 70, 215, and 219; two (one susceptible and one moderately resistant to zidovudine) had mutations at codon 70; and two (both highly resistant to zidovudine) had mutations at codons 41 and 215. The virus was vertically transmitted from a woman infected with one of the highly resistant strains, and the infant's isolate was highly resistant to zidovudine. These limited data suggest that maternal treatment with oral zidovudine reduces the rate of vertical transmission of HIV-1 but that vertical transmission of virus resistant to zidovudine can occur.

Therapeutic efficacy and complications of excisional biopsy of condyloma acuminatum.

BACKGROUND AND OBJECTIVES: Among the various treatment modalities for condyloma acuminatum, excisional cold-blade surgery appears excellent but it has been little studied and little used, particularly for lesions not located in the perianal area. GOALS: To examine the efficacy and complications of scissors excision of single anogenital warts. STUDY DESIGN: Retrospective analysis of single warts completely excised with scissors for the purpose of biopsy before patient entry in a randomized, placebo-controlled study of the efficacy and safety of various parenteral interferons in combination with cryotherapy. RESULTS: Of 152 patients entered in the main study, 85 patients were available for analysis. At 4 and 16 weeks after excision, 16 of 85 (19%) and 14 of 68 (21%) of the excised lesions recurred. After at 6 least months of follow-up, 2 of 11 (18%) of the excision sites demonstrated some evidence of pigmentation changes. CONCLUSIONS: Scissors excision of single anogenital warts has a high rate of success and acceptable long-term side-effects.

Mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors demonstrate altered rates of RNase H cleavage that correlate with HIV-1 replication fitness in cell culture.

Three mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (V106A, V179D, and Y181C), which occur in clinical isolates and confer resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), were analyzed for RNA- and DNA-dependent DNA polymerization and RNase H cleavage. All mutants demonstrated processivities of polymerization that were indistinguishable from wild-type enzyme under conditions in which deoxynucleoside triphosphates were not limiting. The V106A reverse transcriptase demonstrated a three- to fourfold slowing of both DNA 3'-end-directed and RNA 5'-end-directed RNase H cleavage relative to both wild-type and V179D enzymes, similar to what was observed for P236L in a previously published study (P. Gerondelis et al., J. Virol. 73:5803-5813, 1999). In contrast, the Y181C reverse transcriptase demonstrated a selective acceleration of the secondary RNase H cleavage step during both modes of RNase H cleavage. The relative replication fitness of these mutants in H9 cells was assessed in parallel infections as well as in growth competition experiments. Of the NNRTI-resistant mutants, V179D was more fit than Y181C, and both of these mutants were more fit than V106A, which demonstrated the greatest reduction in RNase H cleavage. These findings, in combination with results from previous work, suggest that abnormalities in RNase H cleavage are a common characteristic of HIV-1 mutants resistant to NNRTIs and that combined reductions in the rates of DNA 3'-end- and RNA 5'-end-directed cleavages are associated with significant reductions in the replication fitness of HIV-1.