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Members of SEPALLATA ( SEP ) and APETALA1 ( AP1 )/ SQUAMOSA ( SQUA ) MADS-box transcription factor subfamilies play key roles in floral organ identity determination and floral meristem determinacy in the rosid species Arabidopsis ( Arabidopsis thaliana ). Here, we present a functional characterization of the seven SEP / AGL6 and four AP1 / SQUA genes in the distant asterid species petunia ( Petunia × hybrida ). Based on the analysis of single and higher order mutants, we report that the petunia SEP1 / SEP2 / SEP3 orthologs together with AGL6 encode classical SEP floral organ identity and floral termination functions, with a master role for the petunia SEP3 ortholog FLORAL BINDING PROTEIN2 ( FBP2 ). By contrast, the FBP9 subclade members FBP9 and FBP23 , for which no clear ortholog is present in Arabidopsis, play a major role in determining floral meristem identity together with FBP4 , while contributing only moderately to floral organ identity. In turn, the four members of the petunia AP1 / SQUA subfamily redundantly are required for inflorescence meristem identity and act as B-function repressors in the first floral whorl, together with BEN / ROB genes. Overall, these data together with studies in other species suggest major differences in the functional diversification of the SEP / AGL6 and AP1 / SQUA MADS-box subfamilies during angiosperm evolution.plantcell;31/12/3033/FX1F1fx1., (© 2019 American Society of Plant Biologists. All rights reserved.)

Translationally Controlled Tumor Protein (TCTP) controls growth by regulating the G1/S transition during cell cycle progression. Our genetic interaction studies show that TCTP fulfills this role by interacting with CSN4, a subunit of the COP9 Signalosome complex, known to influence CULLIN-RING ubiquitin ligases activity by controlling CULLIN (CUL) neddylation status. In agreement with these data, downregulation of CSN4 in Arabidopsis and in tobacco cells leads to delayed G1/S transition comparable to that observed when TCTP is downregulated. Loss-of-function of AtTCTP leads to increased fraction of deneddylated CUL1, suggesting that AtTCTP interferes negatively with COP9 function. Similar defects in cell proliferation and CUL1 neddylation status were observed in Drosophila knockdown for dCSN4 or dTCTP, respectively, demonstrating a conserved mechanism between plants and animals. Together, our data show that CSN4 is the missing factor linking TCTP to the control of cell cycle progression and cell proliferation during organ development and open perspectives towards understanding TCTP's role in organ development and disorders associated with TCTP miss-expression., Competing Interests: The authors have declared that no competing interests exist.

Roses have high cultural and economic importance as ornamental plants and in the perfume industry. We report the rose whole-genome sequencing and assembly and resequencing of major genotypes that contributed to rose domestication. We generated a homozygous genotype from a heterozygous diploid modern rose progenitor, Rosa chinensis 'Old Blush'. Using single-molecule real-time sequencing and a meta-assembly approach, we obtained one of the most comprehensive plant genomes to date. Diversity analyses highlighted the mosaic origin of 'La France', one of the first hybrids combining the growth vigor of European species and the recurrent blooming of Chinese species. Genomic segments of Chinese ancestry identified new candidate genes for recurrent blooming. Reconstructing regulatory and secondary metabolism pathways allowed us to propose a model of interconnected regulation of scent and flower color. This genome provides a foundation for understanding the mechanisms governing rose traits and should accelerate improvement in roses, Rosaceae and ornamentals.

The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT (HWS) affects organ growth and the timing of floral organ abscission. The loss-of-function hws-1 mutant exhibits fused sepals and increased organ size. To understand the molecular mechanisms of HWS during plant development, we mutagenized hws-1 seeds with ethylmethylsulphonate (EMS) and screened for mutations suppressing hws-1 associated phenotypes. We isolated the shs1/hws-1 (suppressor of hws-1) mutant in which hws-1 sepal fusion phenotype was suppressed. The shs1/hws-1 mutant carries a G→A nucleotide substitution in the MIR164 binding site of CUP-SHAPED COTYLEDON 1 (CUC1) mRNA. CUC1 and CUP-SHAPED COTYLEDON 2 (CUC2) transcript levels were altered in shs1, renamed cuc1-1D, and in hws-1 mutant. Genetic interaction analyses using single, double and triple mutants of cuc1-1D, cuc2-1D (a CUC2 mutant similar to cuc1-1D), and hws-1, demonstrate that HWS, CUC1 and CUC2 act together to control floral organ number. Loss of function of HWS is associated with larger petal size due to alterations in cell proliferation and mitotic growth, a role shared with the CUC1 gene.

Background and Aims: SUPERMAN is a cadastral gene controlling the sexual boundary in the flower. The gene's functions and role in flower development and evolution have remained elusive. The analysis of a contrasting SUP allelic series (for which the names superman, superwoman and supersex have been coined) makes it possible to distinguish early vs. late regulatory processes at the flower meristem centre to which SUP is an important contributor. Their understanding is essential in further addressing evolutionary questions linking bisexuality and flower meristem homeostasis., Methods: Inter-allelic comparisons were carried out and SUP interactions with other boundary factors and flower meristem patterning and homeostasis regulators (such as CLV, WUS, PAN, CUC, KNU, AG, AP3/PI, CRC and SPT) have been evaluated at genetic, molecular, morphological and histological levels., Key Results: Early SUP functions include mechanisms of male-female (sexual) boundary specification, flower mersitem termination and control of stamen number. A SUP-dependent flower meristem termination pathway is identified and analysed. Late SUP functions play a role in organ morphogenesis by controlling intra-whorl organ separation and carpel medial region formation. By integrating early and late SUP functions, and by analyzing in one single experiment a series of SUP genetic interactions, the concept of meristematic 'transference' (cascade) - a regulatory bridging process redundantly and sequentially co-ordinating the triggering and completion of flower meristem termination, and carpel margin meristem and placenta patterning - is proposed., Conclusions: Taken together, the results strongly support the view that SUP(-type) function(s) have been instrumental in resolving male/female gradients into sharp male and female identities (whorls, organs) and in enforcing flower homeostasis during evolution. This has probably been achieved by incorporating the meristem patterning system of the floral axis into the female/carpel programme., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Transcription factors of the plant-specific homeodomain leucine zipper IV (HD-ZIP IV) family have been found from moss to higher plants, and several family members have been associated with epidermis-related expression and/or function. In maize (Zea mays), four of the five characterized HD-ZIP IV family members are expressed specifically in the epidermis, one contributes to trichome development, and target genes of another one are involved in cuticle biosynthesis. Assessing the phylogeny, synteny, gene structure, expression, and regulation of the entire family in maize, 12 novel ZmHDZIV genes were identified in the recently sequenced maize genome. Among the 17 genes, eight form homeologous pairs duplicated after the split of maize and sorghum (Sorghum bicolor), whereas a fifth duplication is shared with sorghum. All 17 ZmHDZIV genes appear to be derived from a basic module containing seven introns in the coding region. With one possible exception, all 17 ZmHDZIV genes are expressed and show preferential expression in immature reproductive organs. Fourteen of 15 ZmHDZIV genes with detectable expression in laser-dissected tissues exhibit a moderate to very strong expression preference for the epidermis, suggesting that at least in maize, the majority of HD-ZIP IV family members may have epidermis-related functions. Thirteen ZmHDZIV genes carry conserved motifs of 19 and 21 nucleotides in their 3' untranslated region. The strong evolutionary conservation and the size of the conserved motifs in the 3' untranslated region suggest that the expression of HD-ZIP IV genes may be regulated by small RNAs.

Here, we describe an HIV-infected patient with pretreatment resistance to raltegravir, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors, and the ultimate ability to achieve viral suppression. Pretreatment integrase resistance testing is not routinely performed because transmitted integrase mutations conferring resistance to raltegravir are currently thought to be negligible. We suggest obtaining a pretreatment integrase genotype in patients with transmitted multiclass drug resistance in order to create an optimal first regimen and increase the chance for virological suppression.

Cultivated for centuries, the varieties of rose have been selected based on a number of flower traits. Understanding the genetic and molecular basis that contributes to these traits will impact on future improvements for this economically important ornamental plant. In this study, we used scanning electron microscopy and sections of meristems and flowers to establish a precise morphological calendar from early rose flower development stages to senescing flowers. Global gene expression was investigated from floral meristem initiation up to flower senescence in three rose genotypes exhibiting contrasted floral traits including continuous versus once flowering and simple versus double flower architecture, using a newly developed Affymetrix microarray (Rosa1_Affyarray) tool containing sequences representing 4765 unigenes expressed during flower development. Data analyses permitted the identification of genes associated with floral transition, floral organs initiation up to flower senescence. Quantitative real time PCR analyses validated the mRNA accumulation changes observed in microarray hybridizations for a selection of 24 genes expressed at either high or low levels. Our data describe the early flower development stages in Rosa sp, the production of a rose microarray and demonstrate its usefulness and reliability to study gene expression during extensive development phases, from the vegetative meristem to the senescent flower.

Background: Roses have been cultivated for centuries and a number of varieties have been selected based on flower traits such as petal form, color, and number. Wild-type roses have five petals (simple flowers), whereas high numbers of petals (double flowers) are typical attributes of most of the cultivated roses. Here, we investigated the molecular mechanisms that could have been selected to control petal number in roses., Methodology/principal Findings: We have analyzed the expression of several candidate genes known to be involved in floral organ identity determination in roses from similar genetic backgrounds but exhibiting contrasting petal numbers per flower. We show that the rose ortholog of AGAMOUS (RhAG) is differentially expressed in double flowers as compared to simple flowers. In situ hybridization experiments confirm the differential expression of RhAG and demonstrate that in the double-flower roses, the expression domain of RhAG is restricted toward the center of the flower. Conversely, in simple-flower roses, RhAG expression domain is wider. We further show that the border of RhAG expression domain is labile, which allows the selection of rose flowers with increased petal number. Double-flower roses were selected independently in the two major regions for domestication, China and the peri-Mediterranean areas. Comparison of RhAG expression in the wild-type ancestors of cultivated roses and their descendants both in the European and Chinese lineages corroborates the correlation between the degree of restriction of RhAG expression domain and the number of petals. Our data suggests that a restriction of RhAG expression domain is the basis for selection of double flowers in both the Chinese and peri-Mediterranean centers of domestication., Conclusions/significance: We demonstrate that a shift in RhAG expression domain boundary occurred in rose hybrids, causing double-flower phenotype. This molecular event was selected independently during rose domestication in Europe/Middle East and in China.

Background: Patients on long-term highly active antiretroviral therapy (HAART) were studied to determine persistence, drug resistance development, and evolution of HIV-1 proviral DNA., Methods: Peripheral blood mononuclear cells were obtained by large volume blood drawn (500 mL) from 8 clinically successfully treated patients who had received uninterrupted HAART for up to 8.9 years. HIV-1 load was determined by Taqman real-time polymerase chain reaction. Drug resistance mutations were determined by sequencing and ultrasensitive, allele-specific, reverse transcriptase (RT)-polymerase chain reaction., Results: HIV-1 DNA load was significantly higher in aged memory (CD45RO CD57) when compared with memory (CD45RO CD57) and naive (CD27 CD45RO) CD4 T cells after HAART. Sequencing revealed no major drug resistance mutations in protease in all patients and appearance of resistance mutations in RT in just 1 patient. In 1 of 5 patients with undetectable viremia during treatment, RT M184 substitutions were detected. Phylogenetic analysis showed short genetic distances between patient sequences., Conclusions: During long-term HAART, HIV-1 is able to persist in terminally differentiated CD4 T cells as proviral DNA. Viral evolution was restricted, and in 80% of the patients with undetectable viremia, no sign of viral replication could be detected.

Using standard and ultrasensitive techniques, we detected nonnucleoside reverse-transcriptase inhibitor-associated resistance mutations in 11 (20%) of 54 subjects who discontinued virologically suppressive nonnucleoside reverse-transcriptase inhibitor-containing antiretroviral therapy. Resistance was detected in 45% and 14% of subjects with a baseline human immunodeficiency virus type 1 RNA level of 51-400 copies/mL and

Background: Understanding the selection and decay of drug-resistant HIV-1 variants is important for designing optimal antiretroviral therapy., Objective: To develop a high-throughput, real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay to quantify non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant variants K103N (AAT or AAC alleles) at frequencies as low as 0.1%, and to apply this to monitor these variants before, during, and after NNRTI therapy., Methods: HIV-1 RNA in longitudinal plasma samples obtained from patients starting and stopping NNRTI therapy was converted to cDNA and the target sequence region amplified and quantified by real-time PCR. Approximately 10 copies/reaction provided a template for a second round of PCR using primers that discriminated between the mutant and wild-type alleles. Amplification specificity was confirmed by thermal denaturation analysis., Results: Frequencies of 103N similar to assay background (0.029%) were observed in longitudinal samples from 9 of 12 treatment-naive patients; three patients had transient increases in 103N frequency to a range of 0.21-0.48%, which was 7-16.5 times assay background. Analysis of longitudinal plasma samples from six NNRTI-experienced patients showed three patterns: persistence of 103N variants after stopping NNRTI therapy, codon switching of 103N between AAC and AAT during NNRTI therapy, and decay of 103N variants to below assay background after cessation of NNRTI therapy., Conclusions: Allele-specific RT-PCR quantified the emergence and decay of drug-resistant variants in patients over a broad range of frequencies (0.1-100%). The rate of decay of K103N variants after stopping NNRTI therapy was highly variable.

Antiviral resistance is a significant obstacle in the treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals. Because nonnucleoside reverse transcriptase inhibitors (NNRTIs) specifically target HIV-1 reverse transcriptase (RT) and do not effectively inhibit simian immunodeficiency virus (SIV) RT, the development of animal models to study the evolution of antiviral resistance has been problematic. To facilitate in vivo studies of NNRTI resistance, we examined whether a SIV that causes immunopathogenesis in pigtail macaques could be made sensitive to NNRTIs. Two simian-human immunodeficiency viruses (SHIVs) were derived from the genetic background of SIV(mne): SIV-RT-YY contains RT substitutions intended to confer NNRTI susceptibility (V181Y and L188Y), and RT-SHIV(mne) contains the entire HIV-1 RT coding region. Both mutant viruses grew to high titers in vitro but had reduced fitness relative to wild-type SIV(mne). Although the HIV-1 RT was properly processed into p66 and p51 subunits in RT-SHIV(mne) particles, the RT-SHIV(mne) virions had lower levels of RT per viral genomic RNA than HIV-1. Correspondingly, there was decreased RT activity in RT-SHIV(mne) and SIV-RT-YY particles. HIV-1 and RT-SHIV(mne) were similarly susceptible to the NNRTIs efavirenz, nevirapine, and UC781. However, SIV-RT-YY was less sensitive to NNRTIs than HIV-1 or RT-SHIV(mne). Classical NNRTI resistance mutations were selected in RT-SHIV(mne) after in vitro drug treatment and were monitored in a sensitive allele-specific real-time RT-PCR assay. Collectively, these results indicate that RT-SHIV(mne) may be a useful model in macaques for the preclinical evaluation of NNRTIs and for studies of the development of drug resistance in vivo.

We have identified and characterized a potent new nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) of human immunodeficiency virus type 1 (HIV-1) that also is active against HIV-2 and which interferes with virus replication by two distinct mechanisms. 1-(3-Cyclopenten-1-yl)methyl-6-(3,5-dimethylbenzoyl)-5-ethyl-2,4-pyrimidinedione (SJ-3366) inhibits HIV-1 replication at concentrations of approximately 1 nM, with a therapeutic index of greater than 4 x 10(6). The efficacy and toxicity of SJ-3366 are consistent when evaluated with established or fresh human cells, and the compound is equipotent against all strains of HIV-1 evaluated, including syncytium-inducing, non-syncytium-inducing, monocyte/macrophage-tropic, and subtype virus strains. Distinct from other members of the pharmacologic class of NNRTIs, SJ-3366 inhibited laboratory and clinical strains of HIV-2 at a concentration of approximately 150 nM, yielding a therapeutic index of approximately 20,000. Like most NNRTIs, the compound was less active when challenged with HIV-1 strains possessing the Y181C, K103N, and Y188C amino acid changes in the RT and selected for a virus with a Y181C amino acid change in the RT after five tissue culture passages in the presence of the compound. In combination anti-HIV assays with nucleoside and nonnucleoside RT and protease inhibitors, additive interactions occurred with all compounds tested with the exception of dideoxyinosine, with which a synergistic interaction was found. Biochemically, SJ-3366 exhibited a K(i) value of 3.2 nM, with a mixed mechanism of inhibition against HIV-1 RT, but it did not inhibit HIV-2 RT. SJ-3366 also inhibited the entry of both HIV-1 and HIV-2 into target cells. On the basis of its therapeutic index and multiple mechanisms of anti-HIV action, SJ-3366 represents an exciting new compound for use in HIV-infected individuals.

(+)-Calanolide A (NSC 650886) has previously been reported to be a unique and specific nonnucleoside inhibitor of the reverse transcriptase (RT) of human immunodeficiency virus (HIV) type 1 (HIV-1) (M. J. Currens et al., J. Pharmacol. Exp. Ther., 279:645-651, 1996). Two isomers of calanolide A, (-)-calanolide B (NSC 661122; costatolide) and (-)-dihydrocalanolide B (NSC 661123; dihydrocostatolide), possess antiviral properties similar to those of calanolide A. Each of these three compounds possesses the phenotypic properties ascribed to the pharmacologic class of nonnucleoside RT inhibitors (NNRTIs). The calanolide analogs, however, exhibit 10-fold enhanced antiviral activity against drug-resistant viruses that bear the most prevalent NNRTI resistance that is engendered by amino acid change Y181C in the RT. Further enhancement of activity is observed with RTs that possess the Y181C change together with mutations that yield resistance to AZT. In addition, enzymatic inhibition assays have demonstrated that the compounds inhibit RT through a mechanism that affects both the K(m) for dTTP and the V(max), i.e., mixed-type inhibition. In fresh human cells, costatolide and dihydrocostatolide are highly effective inhibitors of low-passage clinical virus strains, including those representative of the various HIV-1 clade strains, syncytium-inducing and non-syncytium-inducing isolates, and T-tropic and monocyte-tropic isolates. Similar to calanolide A, decreased activities of the two isomers were observed against viruses and RTs with amino acid changes at residues L100, K103, T139, and Y188 in the RT, although costatolide exhibited a smaller loss of activity against many of these NNRTI-resistant isolates. Comparison of cross-resistance data obtained with a panel of NNRTI-resistant virus strains suggests that each of the three stereoisomers may interact differently with the RT, despite their high degree of structural similarity. Selection of viruses resistant to each of the three compounds in a variety of cell lines yielded viruses with T139I, L100I, Y188H, or L187F amino acid changes in the RT. Similarly, a variety of resistant virus strains with different amino acid changes were selected in cell culture when the calanolide analogs were used in combination with other active anti-HIV agents, including nucleoside and nonnucleoside RT and protease inhibitors. In assays with combinations of anti-HIV agents, costatolide exhibited synergy with these anti-HIV agents. The calanolide isomers represent a novel and distinct subgroup of the NNRTI family, and these data suggest that a compound of the calanolide A series, such as costatolide, should be evaluated further for therapeutic use in combination with other anti-HIV agents.

A series of compounds related to oxathiin carboxanilide has been identified as nonnucleoside reverse transcriptase inhibitors (NNRTIs) of HIV-1, and structure-activity relationships have been described (Buckheit RW, et al.: Antimicrob Agents Chemother 1995;39:2718-2727). Three new analogs (UC040, UC82, and UC781) inhibited laboratory and clinical isolates of HIV-1, including isolates representative of the various clades of HIV-1 found worldwide, in both established and fresh human cells. Virus isolates with the amino acid changes L100I, K103N, V106I, and Y181C in the reverse transcriptase were partially resistant to these compounds. However, UC781 inhibited these virus isolates at low nontoxic concentrations, presenting a broad in vitro therapeutic index. As with other NNRTIs, each of the compounds synergistically interacted with AZT to inhibit HIV-1 replication. UC781 possesses a favorable pharmacokinetic profile in mice with a high level of oral bioavailability. Plasma concentrations reached maximum levels within 2 to 4 hr of oral administration and remained in excess of those required for in vitro anti-HIV activity for at least 24 hr after a single oral dose. When evaluated in a murine hollow fiber implant model of HIV infection, UC781 dosed orally or parenterally was able to suppress HIV replication completely in this model system, providing evidence of the in vivo efficacy of the compound.

Transforming growth factor beta s (TGF beta) are a family of multifunctional growth factors that are important embryonic morphogens. Because TGF beta s may regulate the development of epitheliochorial placentas, we investigated the location, expression, secretion, and effects of TGF beta s in bovine placentomes and cell cultures derived from chorionic and endometrial epithelia. Placentomes from early second-trimester pregnancies were examined by immunohistochemistry for TGF beta 1, TGF beta 2, and TGF beta 3, and for TGF beta expression in Northern slot-blots. Effects of TGF beta s were assessed in trophoblastic and endometrial epithelial cell lines by DNA synthesis assays. Secretion of TGF beta s by trophoblastic and endometrial epithelial cells was determined using bioassays. All forms of TGF beta were immunolocalized in bovine placentomes. TGF beta mRNA was expressed in chorioallantois, caruncles, and in cultured trophoblastic and endometrial epithelial cells. Endometrial and trophoblastic cells secreted active and latent TGF beta s, and these cells had a transient proliferative response to all forms of TGF beta. These results indicate that TGF beta s are present at the fetal-maternal interface of the bovine placentome and may promote endometrial and chorionic growth.

Current thrust in controlling the Acquired Immune Deficiency Syndrome (AIDS) focuses on antiviral drug development targeting the infection and replication of the human immunodeficiency virus (HIV), the causative agent of AIDS. To date, treatment of AIDS has relied on nucleoside reverse transcriptase inhibitors such as AZT, ddI, and ddC, which eventually become ineffective upon the emergence of resistant mutants bearing specific nucleotide substitutions. The Anti-AIDS Drug Screening Program of the NCI conducts and coordinates a high-capacity semi-robotic in vitro screening of synthetic or natural compounds submitted by academic, research and pharmaceutical institutions world-wide. About 10,000 synthetic compounds are screened annually for anti-HIV activity. Confirmed active agents are subjected to in-depth studies on range and mechanism of action. Emerging from this intense screening activity were a number of potentially promising categories of nonnucleoside reverse transcriptase inhibitors (NNRTI) with structural diversity but strong and reproducible anti-HIV activity. Over 2500 active compounds were evaluated for their inhibitory activity against a panel of both laboratory and clinical virus isolates in the appropriate established cell line or fresh human peripheral blood leukocyte and macrophage preparations. Out of these, 40 agents could be placed structurally in nine categories with an additional 16 unique compounds that share the characteristics of NNRTI. These NNRTIs were shown to inhibit reverse transcriptase enzymatically using homopolymeric or ribosomal RNA as templates. NNRTIs demonstrated similarity in their inhibitory pattern against the HIV-1 laboratory strains IIIB and RF, and an AZT-resistant strain; all were inactive against HIV-2. These compounds were further tested against NNRTI-resistant HIV-1 isolates. NNRTI-resistant HIV-1 isolates were selected and characterized with respect to the change(s) in the viral reverse transcriptase nucleotide sequence. Also, differential cross-resistance or sensitivity patterns to NNRTIs were studied in detail among NNRTI-resistant mutants. When tested in combination with AZT, all of the NNRTI's uniformly exhibited synergistic inhibition of HIV-1, suggesting that combination antiviral therapy of NNRTIs with AZT may be therapeutically promising for AIDS treatment.

Virus isolates resistant to 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) and a highly potent HEPT derivative, [1-benzyloxymethyl-5-ethyl-6-(alpha-pyridylthio)uracil] (NSC 648400, E-BPTU), were selected in cell culture. Cross-resistance evaluation indicated that the two drug-resistant virus isolates were phenotypically distinct from one another although each of the virus isolates was resistant to both of the HEPT derivatives. The virus isolate resistant to NSC 648400 had a single amino acid change in the reverse transcriptase (Y181C) which resulted in cross-resistance to all of the nonnucleoside reverse transcriptase inhibitors evaluated, with the exception of calanolide A. The NSC 648400-resistant virus isolate exhibited 15-fold enhanced sensitivity to calanolide A. The virus isolate selected in the presence of HEPT exhibited a single amino acid change (P236L) which was not cross-resistant to other nonnucleoside RT inhibitors tested with the exception of the two HEPT derivatives. This HEPT-resistant virus isolate exhibited enhanced sensitivity (5- to 10-fold) to thiazolobenzimidazole. We have used both virus isolates with defined single amino acid changes in the RT and bacterially expressed RTs with site-directed amino acid substitutions to test the effects of a wide variety of mutations on the activity of NSC 648400. Single mutations at amino acids 101, 103, 106, 181, or 236 yielded virus with high resistance (> 20-fold) to NSC 648400, while lower levels of resistance were seen with mutations at amino acids 98, 100, or 108. These results suggest that several changes in the conformation of the nonnucleoside inhibitor binding site of the HIV-1 reverse transcriptase can affect the inhibitory activity of the HEPT class of compounds.