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Detailed clonal phenotypic/genotypic analyses explored viral-escape mechanisms during maraviroc-based therapy in highly treatment-experienced participants from the MOTIVATE trials. To allow real-time assessment of samples while maintaining a blind trial, the first 267 enrolled participants were selected for evaluation. At failure, plasma samples from 20/50 participants (16/20 maraviroc-treated) with CXCR4-using virus and all 38 (13 maraviroc-treated) with CCR5-tropic virus were evaluated. Of those maraviroc-treated participants with CXCR4-using virus at failure, genotypic and phenotypic clonal tropism determinations showed >90% correspondence in 14/16 at Day 1 and 14/16 at failure. Phylogenetic analysis of clonal sequences detected pre-treatment progenitor CXCR4-using virus, or on-treatment virus highly divergent from the Day 1 R5 virus, excluding possible co-receptor switch through maraviroc-mediated evolution. Re-analysis of pre-treatment samples using the enhanced-sensitivity Trofile® assay detected CXCR4-using virus pre-treatment in 16/20 participants failing with CXCR4-using virus. Post-maraviroc reversion of CXCR4-use to CCR5-tropic occurred in 7/8 participants with follow-up, suggesting selective maraviroc inhibition of CCR5-tropic variants in a mixed-tropic viral population, not emergence of de novo mutations in CCR5-tropic virus, as the main virologic escape mechanism. Maraviroc-resistant CCR5-tropic virus was observed in plasma from 5 treated participants with virus displaying reduced maximal percent inhibition (MPI) but no evidence of IC50 change. Env clones with reduced MPI showed 1-5 amino acid changes specific to each V3-loop region of env relative to Day 1. However, transferring on-treatment resistance-associated changes using site-directed mutagenesis did not always establish resistance in Day 1 virus, and key 'signature' mutation patterns associated with reduced susceptibility to maraviroc were not identified. Evolutionary divergence of the CXCR4-using viruses is confirmed, emphasizing natural selection not influenced directly by maraviroc; maraviroc simply unmasks pre-existing lineages by inhibiting the R5 virus. For R5-viral failure, resistance development through drug selection pressure was uncommon and manifested through reduced MPI and with virus strain-specific mutational patterns., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: RM is an employee of Pfizer and owns stock in the company. CC, ML, and EV are not employed but are affiliated to The Research Network, Ltd, which administers the contract with Pfizer for work on the maraviroc program, including the finalization of the analysis and preparation of the submitted work. CC, ML, and EV were previously employed by Pfizer; EV, JM, ML, MM, and MW were employed during the conduct of the trial. CC owns stock in GlaxoSmithKline. JT, WH, and JMW are employees of Monogram Biosciences. DLR reports a grant from the Medical Research Council (MRC; G1001806/1) and consultancy fees from Pfizer during the conduct of the study. JT reports a grant from Small Business Innovation Research (Grant number: 1 R21 AI114399) outside the scope of the submitted work, and a fee for service from Pfizer during the conduct of the study; in addition, JT has a patent US 9,581,595 B2 issued. Pfizer is one of the beneficiaries of the ViiV Healthcare Joint Venture along with GlaxoSmithKline and Shinogi. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The emergence and transmission of antiretroviral drug resistance have been and remain a concern among people living with human immunodeficiency virus (HIV)-1 infection. The protease inhibitor (PI) darunavir has been approved for use in the United States for more than 10 years and has demonstrated a high barrier to resistance. Previous analyses identified significant reductions in the prevalence of samples with darunavir resistance-associated mutations (RAMs) and with phenotypic resistance to darunavir and other PIs between 2006 and 2012. This analysis extends those findings by evaluating darunavir and PI resistance among clinical samples submitted for routine drug resistance testing (combined genotyping and phenotyping) in the United States from 2010 to 2017. Frequencies of 11 darunavir and 23 primary PI RAMs, and phenotypic susceptibility, were assessed yearly among all samples and in a subset of samples with distinct phenotypic resistance to one or more PIs. Among all samples (N = 60,760), the proportion with 0 darunavir RAMs was 91.7% in 2010 and 95.8% in 2017. The proportions of all samples with phenotypic susceptibility to darunavir, atazanavir, and lopinavir were, respectively, 97.4%, 94.2%, and 94.7% in 2010 and 98.6%, 97.7%, and 97.5% in 2017. Among the 4,799 samples with phenotypic resistance to one or more PIs, the proportions with phenotypic susceptibility to darunavir, atazanavir, and lopinavir were, respectively, 73.3%, 41.5%, and 46.0% in 2010 and 70.7%, 53.7%, and 48.8% in 2017. The prevalence of darunavir RAMs among commercially tested HIV-1 samples remained low and generally stable from 2010 to 2017, and high proportions showed phenotypic darunavir susceptibility.

In January 2015, an outbreak of undiagnosed human immunodeficiency virus (HIV) infections among persons who inject drugs (PWID) was recognized in rural Indiana. By September 2016, 205 persons in this community of approximately 4400 had received a diagnosis of HIV infection. We report results of new approaches to analyzing epidemiologic and laboratory data to understand transmission during this outbreak. HIV genetic distances were calculated using the polymerase region. Networks were generated using data about reported high-risk contacts, viral genetic similarity, and their most parsimonious combinations. Sample collection dates and recency assay results were used to infer dates of infection. Epidemiologic and laboratory data each generated large and dense networks. Integration of these data revealed subgroups with epidemiologic and genetic commonalities, one of which appeared to contain the earliest infections. Predicted infection dates suggest that transmission began in 2011, underwent explosive growth in mid-2014, and slowed after the declaration of a public health emergency. Results from this phylodynamic analysis suggest that the majority of infections had likely already occurred when the investigation began and that early transmission may have been associated with sexual activity and injection drug use. Early and sustained efforts are needed to detect infections and prevent or interrupt rapid transmission within networks of uninfected PWID., (Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Background: Drug resistance testing and co-receptor tropism determination are key components of the management of antiretroviral therapy for HIV-1-infected individuals. The purpose of this study was to examine trends of HIV-1 resistance and viral evolution in the past decade by surveying a large commercial patient testing database., Methods: Temporal trends of drug resistance, viral fitness and co-receptor usage among samples submitted for routine phenotypic and genotypic resistance testing to protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), as well as for tropism determination were investigated., Results: Within 62,397 resistant viruses reported from 2003 to 2012, we observed a decreasing trend in the prevalence of three-class resistance (from 25% to 9%) driven by decreased resistance to PIs (43% to 21%) and NRTIs (79% to 57%), while observing a slight increase in NNRTI resistance (68% to 75%). The prevalence of CXCR4-mediated entry among tropism testing samples (n=52,945) declined over time from 47% in 2007 to 40% in 2012. A higher proportion of CXCR4-tropic viruses was observed within samples with three-class resistance (50%) compared with the group with no resistance (36%)., Conclusions: Decreased prevalence of three-class resistance and increased prevalence of one-class resistance was observed within samples reported between 2003 and 2012. The fraction of CXCR4-tropic viruses has decreased over time; however, CXCR4 usage was more prevalent among multi-class-resistant samples, which may be due to the more advanced disease stage of treatment-experienced patients. These trends have important implications for clinical practice and future drug discovery and development.

Although fitness landscapes are central to evolutionary theory, so far no biologically realistic examples for large-scale fitness landscapes have been described. Most currently available biological examples are restricted to very few loci or alleles and therefore do not capture the high dimensionality characteristic of real fitness landscapes. Here we analyze large-scale fitness landscapes that are based on predictive models for in vitro replicative fitness of HIV-1. We find that these landscapes are characterized by large correlation lengths, considerable neutrality, and high ruggedness and that these properties depend only weakly on whether fitness is measured in the absence or presence of different antiretrovirals. Accordingly, adaptive processes on these landscapes depend sensitively on the initial conditions. While the relative extent to which mutations affect fitness on their own (main effects) or in combination with other mutations (epistasis) is a strong determinant of these properties, the fitness landscape of HIV-1 is considerably less rugged, less neutral, and more correlated than expected from the distribution of main effects and epistatic interactions alone. Overall this study confirms theoretical conjectures about the complexity of biological fitness landscapes and the importance of the high dimensionality of the genetic space in which adaptation takes place., Competing Interests: The authors have declared that no competing interests exist.

Human immunodeficiency virus (HIV-1) is, like most pathogens, under selective pressure to escape the immune system of its host. In particular, HIV-1 can avoid recognition by cytotoxic T lymphocytes (CTLs) by altering the binding affinity of viral peptides to human leukocyte antigen (HLA) molecules, the role of which is to present those peptides to the immune system. It is generally assumed that HLA escape mutations carry a replicative fitness cost, but these costs have not been quantified. In this study, we assess the replicative cost of mutations which are likely to escape presentation by HLA molecules in the region of HIV-1 protease and reverse transcriptase. Specifically, we combine computational approaches for prediction of in vitro replicative fitness and peptide binding affinity to HLA molecules. We find that mutations which impair binding to HLA-A molecules tend to have lower in vitro replicative fitness than mutations which do not impair binding to HLA-A molecules, suggesting that HLA-A escape mutations carry higher fitness costs than non-escape mutations. We argue that the association between fitness and HLA-A binding impairment is probably due to an intrinsic cost of escape from HLA-A molecules, and these costs are particularly strong for HLA-A alleles associated with efficient virus control. Counter-intuitively, we do not observe a significant effect in the case of HLA-B, but, as discussed, this does not argue against the relevance of HLA-B in virus control. Overall, this article points to the intriguing possibility that HLA-A molecules preferentially target more conserved regions of HIV-1, emphasizing the importance of HLA-A genes in the evolution of HIV-1 and RNA viruses in general.

HIV-1 replicative capacity (RC) provides a measure of within-host fitness and is determined in the context of phenotypic drug resistance testing. However it is unclear how these in-vitro measurements relate to in-vivo processes. Here we assess RCs in a clinical setting by combining a previously published machine-learning tool, which predicts RC values from partial pol sequences with genotypic and clinical data from the Swiss HIV Cohort Study. The machine-learning tool is based on a training set consisting of 65000 RC measurements paired with their corresponding partial pol sequences. We find that predicted RC values (pRCs) correlate significantly with the virus load measured in 2073 infected but drug naïve individuals. Furthermore, we find that, for 53 pairs of sequences, each pair sampled in the same infected individual, the pRC was significantly higher for the sequence sampled later in the infection and that the increase in pRC was also significantly correlated with the increase in plasma viral load and with the length of the time-interval between the sampling points. These findings indicate that selection within a patient favors the evolution of higher replicative capacities and that these in-vitro fitness measures are indicative of in-vivo HIV virus load.

As many as 15 million children and adolescents in the United States are in need of behavioral health services, and it is often the pediatric primary care system that is their first contact with formal assessment and intervention services. However, pediatric primary care providers (PPCPs) face challenges to assessing and managing children with behavioral health concerns, including lack of time, lack of training, and lack of behavioral health specialists to whom they can refer. The Connecticut Behavioral Health Partnership has forged relationships between primary care and behavioral health providers through its enhanced care clinic (ECC) initiative, which focuses on improved access to behavioral health services and coordination of care for children insured by Medicaid. We report on interviews with 24 PPCPs and 8 staff/administrators from 12 pediatric practices throughout the state about their experiences with the ECCs. The majority of participants expressed satisfaction with the behavioral health partnerships and, based on their experience, would join the partnership again., (©2011 APA)

The development of a quantitative understanding of viral evolution and the fitness landscape in HIV-1 drug resistance is a formidable challenge given the large number of available drugs and drug resistance mutations. We analyzed a dataset measuring the in vitro fitness of 70,081 virus samples isolated from HIV-1 subtype B infected individuals undergoing routine drug resistance testing. We assayed virus samples for in vitro replicative capacity in the absence of drugs as well as in the presence of 15 individual drugs. We employed a generalized kernel ridge regression to estimate main fitness effects and epistatic interactions of 1,859 single amino acid variants found within the HIV-1 protease and reverse transcriptase sequences. Models including epistatic interactions predict an average of 54.8% of the variance in replicative capacity across the 16 different environments and substantially outperform models based on main fitness effects only. We find that the fitness landscape of HIV-1 protease and reverse transcriptase is characterized by strong epistasis.

Ibalizumab (formerly TNX-355) is a first-in-class, monoclonal antibody inhibitor of CD4-mediated human immunodeficiency type 1 (HIV-1) entry. Multiple clinical trials with HIV-infected patients have demonstrated the antiviral activity, safety, and tolerability of ibalizumab treatment. A 9-week phase Ib study adding ibalizumab monotherapy to failing drug regimens led to transient reductions in HIV viral loads and the evolution of HIV-1 variants with reduced susceptibility to ibalizumab. This report characterizes these variants by comparing the phenotypic susceptibilities and envelope (env) sequences of (i) paired baseline and on-treatment virus populations, (ii) individual env clones from selected paired samples, and (iii) env clones containing site-directed mutations. Viruses with reduced susceptibility to ibalizumab were found to exhibit reduced susceptibility to the anti-CD4 antibody RPA-T4. Conversely, susceptibility to soluble CD4, which targets the HIV-1 gp120 envelope protein, was enhanced. No changes in susceptibility to the fusion inhibitor enfuvirtide or the CCR5 antagonist maraviroc were observed. Functionally, viruses with reduced ibalizumab susceptibility also displayed high levels of infectivity relative to those of paired baseline viruses. Individual env clones exhibiting reduced ibalizumab susceptibility contained multiple amino acid changes in different regions relative to the paired baseline clones. In particular, clones with reduced susceptibility to ibalizumab contained fewer potential asparagine-linked glycosylation sites (PNGSs) in variable region 5 (V5) than did paired ibalizumab-susceptible clones. The reduction in ibalizumab susceptibility due to the loss of V5 PNGSs was confirmed by site-directed mutagenesis. Taken together, these findings provide important insights into resistance to this new class of antiretroviral drug.

To examine mutational pathways that lead to CXCR4 use of HIV-1, we analyzed the genotypic and phenotypic characteristics of envelope sequences from a large panel of patient virus populations and individual clones containing different V3 mutations. Basic amino acid substitutions at position 11 were strong determinants of CXCR4-mediated entry but required multiple compensatory mutations to overcome associated reductions in infectivity. In contrast, basic amino acid substitutions at position 25, or substitutions at positions 6-8 resulting in the loss of a potential N-linked glycosylation site, contributed to CXCR4-mediated entry but required additional substitutions acting cooperatively to confer efficient CXCR4 use. Our assumptions, based upon examination of patient viruses, were largely confirmed by characterizing the coreceptor utilization of five distinct panels of isogenic envelope sequences containing V3 amino acid substitutions introduced by site-directed mutagenesis. These results further define the mutational pathways leading to CXCR4 use and their associated genetic barriers., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objective(s): Dual HIV-1 utilizes cellular CCR5 and CXCR4 coreceptors to enter host cells. Recent studies indicate that the ability of these viruses to use both coreceptors varies significantly in cell lines expressing CXCR4 or CCR5; however, it is not clear whether differences in coreceptor mediated infection in vitro reflect infection of primary cells in vivo., Methods: We evaluated coreceptor usage of dual envelope clones from patient viruses using a single-cycle pseudovirus assay conducted in cell lines and a replication-competent assay performed using peripheral blood mononuclear cells. Dual envelope clones were selected and classified into three groups, R5>X4, R5 approximately X4, and X4>R5, based on their ability to mediate entry by using CXCR4 and CCR5 in a pseudovirus assay., Results: We observed a high degree of concordance between measurements of coreceptor-mediated entry in pseudovirus and peripheral blood mononuclear cell assays. R5>X4 viruses were efficiently inhibited by a CCR5 antagonist, but not a CXCR4 antagonist, whereas X4>R5 viruses were efficiently inhibited by a CXCR4 antagonist, but not a CCR5 antagonist. R5 approximately X4 viruses were not inhibited, or only partially inhibited, by either a CCR5 or a CXCR4 antagonist alone., Conclusions: These observations indicate that measurements of coreceptor use determined using pseudoviruses and coreceptor-expressing cell lines are generally concordant with the results obtained using replication-competent assays and peripheral blood mononuclear cell. This suggests that a considerable fraction of dual viruses preferentially infect either CCR5 or CXCR4 target cells in vivo. The clinical implications of preferential coreceptor utilization by dual viruses, that is, HIV-1 pathogenesis and response to coreceptor antagonists, require additional studies.

We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7-10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH).

To detect general patterns and temporal trends of HIV-1 resistance, we apply principal component analysis (PCA) to in vitro fitness data. Twenty-eight thousand virus samples, obtained between 2002 and 2008, were assayed for fitness in 16 to 21 selective environments. Fitness measurements are based on replication capacity (RC), which quantifies in vitro viral replication in a single cycle of infection. RC is determined both in the absence of drugs and in the presence of 6-7 nucleoside analog reverse transcriptase inhibitors (NRTIs), 3-4 non-nucleoside reverse transcriptase inhibitors (NNRTIs), and 6-9 protease inhibitors (PIs). PCA shows remarkable structure in RC across the different environments, which reveals differences in the patterns of resistance and cross-resistance between drugs or between drug classes. To probe the causes of the observed patterns, we develop a model to generate simulated data and subject these simulated data to an equivalent analysis. By comparing the outcomes of PCA on the original and the simulated data, we quantify which part of the total variance of the original data is due to non-specific effects, class-specific effects, and drug-specific effects of resistance mutations. We find that relative fitness is mainly drug-independent and that drug-specific effects are substantially bigger than class-specific effects for NRTIs, but not for NNRTIs or PIs. The observed patterns remain remarkably stable over the period of observation. Comparison with known potent combination therapies suggests that PCA helps to identify combinations that act synergistically in preventing the emergence of resistance., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: We previously reported the existence of CXCR4-using HIV-1 in 6-14 week-old Ugandan infants. Whether these viruses were transmitted from the mother perinatally or evolved after transmission is not known. In the current study, we investigated the origin of the CXCR4-using viruses in these infants by comparing HIV-1 envelope clones from the infants to those from their mothers at or near the time of delivery., Methods: Envelope clones were isolated from five Ugandan infant plasma samples that harbored CXCR4-using viruses, collected at the time of HIV diagnosis (four at birth, one at week 6), and from their mothers at delivery. Coreceptor usage and phylogenetic relatedness of HIV-1 populations in mother-infant pairs were analyzed in detail using the Trofile assay and sequence analysis of envelope clones, respectively., Results: X4-tropic clones were identified in two mother-infant pairs and dual-tropic clones were found in three pairs, either alone or in combination with R5-tropic viruses. Dual-tropic clones varied in their ability to infect CXCR4-expressing cells. In each mother-infant pair, X4-tropic or dual-tropic clones shared similar phenotypic profiles and V3 sequence patterns; gp160 sequences of X4-tropic and dual-tropic clones from infants were phylogenetically indistinguishable from those of their mothers. The virus populations were phylogenetically homogenous in three infants and segregated according to coreceptor tropism in the remaining two infants., Conclusions: This study demonstrates that X4-tropic and dual-tropic HIV-1 can be transmitted from mother to infant, before, during or shortly after delivery, and establishes vertical transmission as an important source of CXCR4-using viruses in infants., (2009 Wolters Kluwer Health | Lippincott Williams & Wilkins)

The present study reports on the preliminary psychometric characteristics of a new personality and neuropsychological, 250-item, self-report measure, the Coolidge Correctional Inventory (CCI), in an archival de-identified sample of 3,962 prison inmates. The median internal reliability for the 33 CCI scales and subscales was alpha = .79 (range: alpha = .49 to .93). A prevalence estimate, based on the polythetic criteria in DSM-IV-TR, of at least one personality disorder was 61% of the entire sample, and the prevalence of ADHD was estimated to be 16%. Drug and alcohol problems were also highly prevalent (60%). These results appear to support the preliminary reliability and validity of the CCI and also reveal a high rate of psychopathology and neuropsychological dysfunction among prison inmates., ((c) 2009 John Wiley & Sons, Ltd.)

We screened 150 individuals from two recent seroconverter cohorts and found that six (4%) had CXCR4-using viruses. Clonal analysis of these six individuals, along with a seventh individual identified during clinical care as a recent seroconverter, revealed the presence of both X4- and dual-tropic variants in these recently infected adults. The ability of individual CXCR4-using variants to infect cells expressing CD4/CXCR4 or CD4/CCR5 varied dramatically. These data demonstrate that virus populations in some newly infected individuals can consist of either heterogeneous populations containing both CXCR4-using and CCR5-tropic viruses, or homogeneous populations containing only CXCR4-using viruses. The presence of CXCR4-using viruses at early stages of infection suggests that testing for viral tropism before using CCR5 antagonists may be important even in persons with known recent infection. The presence of CXCR4-using viruses in a subset of newly infected individuals could impact the efficacies of vaccine and microbicide strategies that target CCR5-tropic viruses.

In a phase I/II evaluation of the CXCR4 antagonist AMD3100, human immunodeficiency virus RNA levels were significantly reduced in a single study subject who harbored CXCR4 (X4)-tropic virus, but not in subjects who harbored either dual/mixed (DM)-tropic or CCR5 (R5)-tropic virus (C. W. Hendrix et al., J. Acquir. Immune Defic. Syndr. 37:1253-1262, 2004). In this study, we analyzed the envelope clones of DM-tropic virus in baseline and treated virus populations from 14 subjects. Ten subjects exhibited significant reductions in CXCR4-mediated infectivity after 10 days of AMD3100 therapy relative to baseline (X4 suppressor group), while four subjects had no reduction of CXCR4-mediated infectivity (X4 nonsuppressor group). The baseline viruses of the X4 suppressor group infected CXCR4-expressing cells less efficiently than those of the X4 nonsuppressor group. Clonal analysis indicated that the baseline viruses from the X4 suppressor group contained a higher proportion of R5-tropic variants mixed with CXCR4-using variants, while the X4 nonsuppressor group was enriched for CXCR4-using variants. AMD3100 suppressed X4-tropic variants in all subjects studied, but not all dualtropic variants. Furthermore, dualtropic variants that used CXCR4 efficiently were suppressed by AMD3100, while dualtropic variants that used CXCR4 poorly were not. This study demonstrated that AMD3100 has the ability to suppress both X4-tropic and certain dualtropic variants in vivo. The suppression of CXCR4-using variants by AMD3100 is dependent on both the tropism composition of the virus population and the efficiency of CXCR4 usage of individual variants.

Many studies have demonstrated that the third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is a major determinant of coreceptor tropism. Other regions in the surface gp120 subunit of Env can modulate coreceptor tropism in a manner that is not fully understood. In this study, we evaluated the effect of env determinants outside of V3 on coreceptor usage through the analysis of (i) patient-derived env clones that differ in coreceptor tropism, (ii) chimeric env sequences, and (iii) site-directed mutants. The introduction of distinct V3 sequences from CXCR4-using clones into an R5-tropic env backbone conferred the inefficient use of CXCR4 in some but not all cases. Conversely, in many cases, X4- and dual-tropic env backbones containing the V3 sequences of R5-tropic clones retained the ability to use CXCR4, suggesting that sequences outside of the V3 regions of these CXCR4-using clones were responsible for CXCR4 use. The determinants of CXCR4 use in a set of dual-tropic env sequences with V3 sequences identical to those of R5-tropic clones mapped to the gp41 transmembrane (TM) subunit. In one case, a single-amino-acid substitution in the fusion peptide of TM was able to confer CXCR4 use; however, TM substitutions associated with CXCR4 use varied among different env sequences. These results demonstrate that sequences in TM can modulate coreceptor specificity and that env sequences other than that of V3 may facilitate efficient CXCR4-mediated entry. We hypothesize that the latter plays an important role in the transition from CCR5 to CXCR4 coreceptor use.

In human immunodeficiency virus type 1 (HIV-1) subtype B, CXCR4 coreceptor use ranges from approximately 20% in early infection to approximately 50% in advanced disease. Coreceptor use by non-subtype B HIV is less well characterized. We studied coreceptor tropism of subtype A and D HIV-1 collected from 68 pregnant, antiretroviral drug-naive Ugandan women (HIVNET 012 trial). None of 33 subtype A or 10 A/D-recombinant viruses used the CXCR4 coreceptor. In contrast, nine (36%) of 25 subtype D viruses used both CXCR4 and CCR5 coreceptors. Clonal analyses of the nine subtype D samples with dual or mixed tropism revealed heterogeneous viral populations comprised of X4-, R5-, and dual-tropic HIV-1 variants. In five of the six samples with dual-tropic strains, V3 loop sequences of dual-tropic clones were identical to those of cocirculating R5-tropic clones, indicating the presence of CXCR4 tropism determinants outside of the V3 loop. These dual-tropic variants with R5-tropic-like V3 loops, which we designated "dual-R," use CCR5 much more efficiently than CXCR4, in contrast to dual-tropic clones with X4-tropic-like V3 loops ("dual-X"). These observations have implications for pathogenesis and treatment of subtype D-infected individuals, for the association between V3 sequence and coreceptor tropism phenotype, and for understanding potential mechanisms of evolution from exclusive CCR5 use to efficient CXCR4 use by subtype D HIV-1.

Most human immunodeficiency virus type 1 (HIV-1) strains require either the CXCR4 or CCR5 chemokine receptor to efficiently enter cells. Blocking viral binding to these coreceptors is an attractive therapeutic target. Currently, several coreceptor antagonists are being evaluated in clinical trials that require characterization of coreceptor tropism for enrollment. In this report, we describe the development of an automated and accurate procedure for determining HIV-1 coreceptor tropism (Trofile) and its validation for routine laboratory testing. HIV-1 pseudoviruses are generated using full-length env genes derived from patient virus populations. Coreceptor tropism is determined by measuring the abilities of these pseudovirus populations to efficiently infect CD4+/U87 cells expressing either the CXCR4 or CCR5 coreceptor. Viruses exclusively and efficiently infecting CXCR4+/CD4+/U87 cells are designated X4-tropic. Conversely, viruses exclusively and efficiently infecting CCR5+/CD4+/U87 cells are designated R5-tropic. Viruses capable of infecting both CXCR4+/CD4+/U87 and CCR5+/CD4+/U87 cells are designated dual/mixed-tropic. Assay accuracy and reproducibility were established by evaluating the tropisms of well-characterized viruses and the variability among replicate results from samples tested repeatedly. The viral subtype, hepatitis B virus or hepatitis C virus coinfection, and the plasma viral load did not affect assay performance. Minority subpopulations with alternate tropisms were reliably detected when present at 5 to 10%. The plasma viral load above which samples can be amplified efficiently in the Trofile assay is 1,000 copies per ml of plasma. Trofile has been automated for high-throughput use; it can be used to identify patients most likely to benefit from treatment regimens that include a coreceptor inhibitor and to monitor patients on treatment for the emergence of resistant virus populations that switch coreceptor tropism.

Background: Chemokine receptors serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry, influence cell tropism, and may critically determine central nervous system infection pathogenesis. Using an in vitro functional entry assay, we examined utilization of 2 principal coreceptors in cerebrospinal fluid (CSF) and plasma in 46 subjects., Methods: Paired CSF and plasma samples were selected from subjects with a range of CD4 T cell counts. Amplified populations of env sequences were characterized as using CCR5 (R5), CXCR4 (X4), or both receptors (R5+X4). Individual clones derived from 3 subjects were analyzed for viral tropism and phylogeny., Results: CSF and plasma pairs were mainly concordant for R5 (36/46) or R5+X4 (5/46) viruses. However, 5 pairs were discordant, 2 of which had the R5+X4 phenotype in CSF despite having the R5 phenotype in plasma. Although R5+X4 tropism was associated with advanced immunodeficiency, all 4 subjects with acquired immunodeficiency syndrome dementia complex had R5 tropism in CSF. Clones derived from R5+X4-tropic populations revealed mixtures of R5 and X4 viruses and viruses able to utilize either coreceptor, suggesting both virus exchange between compartments and autonomous CSF virus evolution., Conclusions: Although R5 viruses predominate in the CSF, HIV-1 populations able to utilize CXCR4 are also present. Discordant tropism in CSF and plasma may have implications for R5 inhibitor therapy.

Purpose of Review: HIV entry into cells is mediated through sequential interactions between HIV envelope proteins (Env) and two cellular molecules: CD4 and a co-receptor, typically either CCR5 or CXCR4. Co-receptor preference has been associated with other viral traits; specifically, CXCR4-tropic viruses have been associated with increased host cell pathogenicity and more rapid progression of disease. Recently, much attention has been focused on the development of CCR5 and CXCR4 antagonists as antiviral agents and several are set to enter phase III trials in 2004 and 2005. The development of assays to assess the co-receptor tropism of HIV populations is critical for the optimal design and performance of clinical trials to evaluate these agents. In addition, the use of these agents in a clinical setting is likely to benefit from a reliable methodology for tropism determination prior to the selection of an optimal antiviral therapy or to evaluate continued efficacy of a regimen., Recent Findings: Tropism assays that use recombinant viruses and pseudotyped HIV are becoming more commonly employed and comparative data with standard assays have begun to be accumulated. These assays are being used to expand on earlier studies of the epidemiology and natural history of HIV tropism. In addition, tropism assays have facilitated the study of co-receptor inhibitors in vitro and in phase I and II trials., Summary: The development of rapid, reliable tropism assays has been useful in advancing the development of novel antiviral agents. Defining the role of these assays in routine clinical practice will be the next important step.

Reproductive strategies such as sexual reproduction and recombination that involve the shuffling of parental genomes for the production of offspring are ubiquitous in nature. However, their evolutionary benefit remains unclear. Many theories have identified potential benefits, but progress is hampered by the scarcity of relevant data. One class of theories is based on the assumption that mutations affecting fitness exhibit negative epistasis. Retroviruses recombine frequently and thus provide a unique opportunity to test these theories. Using amino acid sequence data and fitness values from 9466 human immunodeficiency virus 1 (HIV-1) isolates, we find in contrast to these theories strong statistical evidence for a predominance of positive epistasis in HIV-1.

Background: Cross resistance is common among the non-nucleoside reverse transcriptase inhibitors (NNRTIs). G190A appears in 5-15% of the patients treated with nevirapine or efavirenz who develop clinical resistance., Objectives: In this study we investigated the effect of G190A and other NNRTI substitutions on the phenotypic susceptibility to this class of drugs., Study Design: We identified 15 individuals, who after treatment with NNRTIs (nevirapine or efavirenz; median exposure of 20 months), developed isolated G190A, G190A in combination with K103N, or K103N alone. Phenotypic and genotypic analyses of stored plasma specimens were performed before and after the mutations occurred to assess NNRTI susceptibility., Results: All isolates that developed only G190A substitution became less susceptible to nevirapine (median: 125-fold) and efavirenz (median: 10-fold) but were 2.5-fold more sensitive to delavirdine (Wilcoxon P = 0.06). In the group with only K103N substitution, acquisition of resistance to all NNRTIs was observed. In the group with the double substitutions, G190A and K103N, delavirdine susceptibility decreased 13-fold, while resistance to nevirapine and efavirenz decreased by 239- and 154-folds, respectively (Kruskal-Wallis H P = 0.009)., Conclusions: The data suggest that the presence of a G190A substitution attenuates the phenotypic resistance associated with a K103N substitution, although resistance is still present. The in vivo significance of the increased phenotypic susceptibility to delavirdine is not known but could be evaluated in a clinical trial.

Wild-type viruses from the ViroLogic phenotype-genotype database were evaluated to determine the upper confidence limit of the drug susceptibility distributions, or "biological cutoffs," for the PhenoSense HIV phenotypic drug susceptibility assay. Definition of the natural variation in drug susceptibility in wild-type human immunodeficiency virus (HIV) type 1 isolates is necessary to determine the prevalence of innate drug resistance and to assess the capability of the PhenoSense assay to reliably measure subtle reductions in drug susceptibility. The biological cutoffs for each drug, defined by the 99th percentile of the fold change in the 50% inhibitory concentration distributions or the mean fold change plus 2 standard deviations, were lower than those previously reported for other phenotypic assays and lower than the clinically relevant cutoffs previously defined for the PhenoSense assay. The 99th percentile fold change values ranged from 1.2 (tenofovir) to 1.8 (zidovudine) for nucleoside reverse transcriptase RT inhibitors (RTIs), from 3.0 (efavirenz) to 6.2 (delavirdine) for nonnucleoside RTIs, and from 1.6 (lopinavir) to 3.6 (nelfinavir) for protease inhibitors. To evaluate the potential role of intrinsic assay variability in the observed variations in the drug susceptibilities of wild-type isolates, 10 reference viruses with different drug susceptibility patterns were tested 8 to 30 times each. The median coefficients of variation in fold change for the reference viruses ranged from 12 to 18% for all drugs except zidovudine (32%), strongly suggesting that the observed differences in wild-type virus susceptibility to the different drugs is related to intrinsic virus variability rather than assay variability. The low biological cutoffs and assay variability suggest that the PhenoSense HIV assay may assist in defining clinically relevant susceptibility cutoffs for resistance to antiretroviral drugs.

Nucleoside reverse-transcriptase inhibitors (NRTIs) are important components of most antiretroviral combination treatment regimens. Using a large collection of clinical isolates, we characterized patterns of cross-resistance among all NRTIs. Drugs were grouped by the effect of the M184V mutation: susceptibility to group 1 drugs (zidovudine, stavudine, tenofovir, and adefovir) increased when M184V was present, whereas susceptibility to group 2 drugs (didanosine, zalcitabine, abacavir, and lamivudine) decreased. Significant cross-resistance was observed among all NRTIs and was most notable when samples with or without M184V were analyzed separately. An increasing number of thymidine-analogue mutations (TAMs) was associated with a progressive reduction in drug susceptibility for all NRTIs. The modulating effect of M184I/V on drug susceptibility was present regardless of the number of TAMs. The broad range of susceptibility observed for viruses containing the same number of TAMs indicates that the genetic correlates of NRTI resistance remain to be fully elucidated.

Suboptimal treatment of human immunodeficiency virus type 1 (HIV-1) infection with nonnucleoside reverse transcriptase inhibitors (NNRTI) often results in the rapid selection of drug-resistant virus. Several amino acid substitutions at position 190 of reverse transcriptase (RT) have been associated with reduced susceptibility to the NNRTI, especially nevirapine (NVP) and efavirenz (EFV). In the present study, the effects of various 190 substitutions observed in viruses obtained from NNRTI-experienced patients were characterized with patient-derived HIV isolates and confirmed with a panel of isogenic viruses. Compared to wild-type HIV, which has a glycine at position 190 (G190), viruses with 190 substitutions (A, C, Q, S, V, E, or T, collectively referred to as G190X substitutions) were markedly less susceptible to NVP and EFV. In contrast, delavirdine (DLV) susceptibility of these G190X viruses increased from 3 to 300-fold (hypersusceptible) or was only slightly decreased. The replication capacity of viruses with certain 190 substitutions (C, Q, V, T, and E) was severely impaired and was correlated with reduced virion-associated RT activity and incomplete protease (PR) processing of the viral p55(gag) polyprotein. These defects were the result of inadequate p160(gagpol) incorporation into virions. Compensatory mutations within RT and PR improved replication capacity, p55(gag) processing, and RT activity, presumably through increased incorporation of p160(gagpol) into virions. We observe an inverse relationship between the degree of NVP and EFV resistance and the impairment of viral replication in viruses with substitutions at 190 in RT. These observations may have important implications for the future design and development of antiretroviral drugs that restrict the outgrowth of resistant variants with high replication capacity.

Objective: To evaluate the clinical significance of hypersusceptibility to non-nucleoside reverse transcriptase inhibitors (NNRTI)., Design: Analysis of a prospective clinical trial cohort., Patients: NNRTI-naive patients failing a stable antiretroviral regimen., Measurements: HIV phenotype, HIV RNA, and CD4 cell counts were prospectively collected after patients changed to a new regimen. Hypersusceptibility to NNRTI was defined as a fold-change (FC) in IC50 (inhibitory concentration of 50%) of < 0.4., Results: The 177 patients had a mean HIV RNA of 4.1 log10 copies/ml, CD4 cell count of 322 x 10(6) cells/l and 41 months of prior antiretroviral treatment. Hypersusceptibility to one or more NNRTI was present in 29%. Both longer duration and reduced susceptibility to nucleoside reverse transcriptase inhibitors were associated with efavirenz hypersusceptibility (P < 0.05). NNRTI-containing regimens were initiated in 106 patients at baseline. The mean change in log HIV RNA after 6 months was greater for patients with hypersusceptibility (-1.2 log10 copies/ml; n = 21) than in patients without (-0.8 log10 copies/ml; n = 77) (P = 0.016). Differences persisted to month 12 (P = 0.023). Multiple linear regression models confirmed that hypersusceptibility to NNRTI was a significant independent predictor of the magnitude of early (months 1-4) HIV RNA reduction, after accounting for the baseline HIV RNA and the number of drugs to which the patient's virus was susceptible (P < 0.02). CD4 cell increases (months 4-10) were 28- 60 x 10(6) cells/l greater in patients with hypersusceptible virus (P < or = 0.1)., Conclusion: NNRTI hypersusceptibility occurred in more than 20% of nucleoside-experienced patients and was associated with greater reduction of HIV RNA and increase in CD4 cells., (Copyright 2002 Lippincott Williams & Wilkins)

Objective: The routine use of phenotypic drug resistance testing in patient management has revealed that many HIV-1 strains possess significantly increased drug sensitivity, or 'hypersusceptibility' compared with wild-type viruses. This study describes hypersusceptibility to non-nucleoside reverse transcriptase inhibitors (NNRTI) and was designed to determine the prevalence of and viral characteristics associated with NNRTI hypersusceptibility in patient-derived viruses., Methods: Retrospective analyses were performed on a large clinical laboratory dataset containing phenotypic drug susceptibility and genotypic sequence results from HIV-1 patient isolates. Genetically engineered viruses were used to confirm the role of certain nucleoside reverse transcriptase inhibitor (NRTI)-resistance mutations in NNRTI hypersusceptibility., Results: Hypersusceptibility to delavirdine, efavirenz and nevirapine was detected in 10.7, 10.8 and 8.0% of more than 17,000 consecutive plasma samples submitted for phenotypic susceptibility testing. In analyses limited to a subset of viruses derived from patients with known treatment histories, NNRTI hypersusceptibility was observed significantly more frequently among viruses from NRTI experienced/NNRTI-naive patients compared with viruses from NRTI/NNRTI-naive patients. Significant inverse correlations between NRTI and NNRTI susceptibility exist among the viruses from NRTI-experienced patients. Analyses of viruses classified according to their NNRTI susceptibility identified 18 positions in reverse transcriptase where substitutions were significantly associated with NNRTI hypersusceptibility., Conclusions: NNRTI hypersusceptibility is common among patient HIV-1 isolates, especially in NRTI-resistant viruses. Genotypic correlates of hypersusceptibility are complex and not easily defined by a simple analysis of NRTI-associated resistance mutations. NNRTI hypersusceptibility may provide an explanation for the superior virologic response to NNRTI-containing salvage regimens observed in NRTI-experienced patients in several clinical trials., (Copyright 2002 Lippincott Williams & Wilkins)

To identify the level of phenotypic susceptibility for stavudine (d4T) that is associated with a diminished virologic response to d4T therapy, phenotyping was performed on archived baseline HIV isolates from 26 subjects who received d4T monotherapy in AIDS Clinical Trials Group (ACTG) 302 who had received >3 years of prior zidovudine (ZDV) monotherapy. Seven of 26 subjects achieved a virologic response of >0.3-log10 copies/mL reduction in plasma HIV RNA after 8 weeks of d4T. Responders had lower fold changes in susceptibility to d4T (1.0 vs. 1.6, p=.003), lower baseline viral loads (4.26 vs. 4.74 log10 copies/mL, p=.004), and fewer thymidine analog mutations (TAMS) (1 vs. 2, p=.059). Lower baseline d4T fold change in susceptibility predicted greater reductions in HIV RNA from baseline to week 8 after adjusting for baseline HIV RNA, ZDV fold change in susceptibility, and number of TAMS. Using the same phenotypic assay, drug susceptibility among 240 antiretroviral-naive patients found all HIV isolates to have d4T susceptibility

Cross-resistance between zidovudine, stavudine, and lamivudine was studied, using purified recombinant reverse transcriptase from a zidovudine-susceptible and -resistant pair of clinical isolates of human immunodeficiency virus type 1. The zidovudine-resistant isolate exhibited low-level cross-resistance to both stavudine and lamivudine in drug susceptibility assays. Enzyme from the resistant isolate demonstrated reduced inhibition by zidovudine triphosphate and stavudine triphosphate and, to a lesser extent, lamivudine triphosphate. These findings provide additional evidence at the viral and enzyme level for cross-resistance between zidovudine and stavudine, and they suggest a possible effect of zidovudine resistance on susceptibility to lamivudine.

We have determined the 3.0 A resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The 'RNase H primer grip', consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual 'unzipping' of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

Recently, significant numbers of individuals with primary human immunodeficiency virus (HIV) infection have been found to harbor viral strains with reduced susceptibility to antiretroviral drugs. In one study, HIV from 16% of such antiretroviral-naive individuals was shown to have a susceptibility to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) between 2.5- and 10-fold lower than that of a wild-type control. Mutations in the RT domain that had previously been associated with antiretroviral resistance were not shared by these strains. We have analyzed by logistic regression 46 variable amino acid sites in RT for their effect on susceptibility and have identified two novel sites influencing susceptibility to NNRTIs: amino acids 135 and 283 in RT. Eight different combinations of amino acids at these sites were observed among these patients. These combinations showed a 14-fold range in mean susceptibility to both nevirapine and delavirdine. In vitro mutagenesis of the control strain combined with a phenotypic assay confirmed the significance of amino acid variation at these sites for susceptibility to NNRTIs.

Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.

Context: Loss of viral suppression in patients infected with human immunodeficiency virus (HIV), who are receiving potent antiretroviral therapy, has been attributed to outgrowth of drug-resistant virus; however, resistance patterns are not well characterized in patients whose protease inhibitor combination therapy fails afterachieving viral suppression., Objective: To characterize drug susceptibility of virus from HIV-infected patients who are failing to sustain suppression while taking an indinavir-containing antiretroviral regimen., Design and Setting: Substudy of the AIDS Clinical Trials Group 343, a multicenter clinical research trial conducted between February 1997 and October 1998., Patients: Twenty-six subjects who experienced rebound (HIV RNA level > or =200 copies/mL) during indinavir monotherapy (n = 9) or triple-drug therapy (indinavir, lamivudine, and zidovudine; n = 17) after initially achieving suppression while receiving all 3 drugs, and 10 control subjects who had viral suppression while receiving triple-drug therapy., Main Outcome Measure: Drug susceptibility, determined by a phenotypic assay and genotypic evidence of resistance assessed by nucleotide sequencing of protease and reverse transcriptase, compared among the 3 patient groups., Results: Indinavir resistance was not detected in the 9 subjects with viral rebound during indinavir monotherapy or in the 17 subjects with rebound during triple-drug therapy, despite plasma HIV RNA levels ranging from 10(2) to 10(5) copies/mL. In contrast, lamivudine resistance was detected by phenotypic assay in rebound isolates from 14 of 17 subjects receiving triple-drug therapy, and genotypic analyses showed changes at codon 184 of reverse transcriptase in these 14 isolates. Mean random plasma indinavir concentrations in the 2 groups with rebound were similar to those of a control group with sustained viral suppression, although levels below 50 ng/mL were more frequent in the triple-drug group than in the control group (P = .03)., Conclusions: Loss of viral suppression may be due to suboptimal antiviral potency, and selection of a predominantly indinavir-resistant virus population may be delayed for months even in the presence of ongoing indinavir therapy. The results suggest possible value in assessing strategies using drug components of failing regimens evaluated with resistance testing.

Context: The transmission of drug-resistant human immunodeficiency virus (HIV) has been documented, but the prevalence of such transmission is unknown., Objective: To assess the spectrum and frequency of antiretroviral susceptibility among subjects with primary HIV infection., Design, Setting, and Patients: Retrospective analysis of 141 subjects identified from clinical research centers in 5 major metropolitan areas, enrolled from 1989 to 1998, with HIV seroconversion within the preceding 12 months and no more than 7 days' prior antiretroviral (ARV) therapy., Main Outcome Measures: Phenotypic and genotypic ARV susceptibility of HIV from plasma samples., Results: The transmission of drug-resistant HIV as assessed by a greater than 10-fold reduction in ARV susceptibility to 1 or more drugs was observed in 3 (2%) of 141 subjects, including to a nonnucleoside reverse transcriptase inhibitor in 1 patient and to a nucleoside reverse transcriptase inhibitor and a protease inhibitor in 2 patients. Population-based sequence analysis of these 3 samples identified multidrug-resistance mutations in reverse transcriptase (M184V, T215Y, K219K/R) and protease (L101/V, K20R, M361, M46I, G48V, L63P, A71T, V771, V82T, 184V, L90M) in the 2 latter patient samples, along with numerous polymorphisms. A reduction in susceptibility of greater than 2.5- to 10-fold to 1 or more drugs was observed in viral isolates from 36 patients (26%). Sequence analysis of these 36 samples identified well-characterized drug resistance mutation in reverse transcriptase and protease in only 1 of these patients., Conclusions: Reductions in drug susceptibility of more than 10-fold were rare among this cohort of recently HIV-infected subjects and were distributed among each of the 3 major classes of ARV drugs tested. Reductions in susceptibility of more than 2.5- to 10-fold to certain ARV drugs of unknown clinical significance were highly prevalent among newly infected patients. Resistance testing may be warranted to monitor the frequency of drug resistance over time and to assess the potential for geographic variability.

The lack of a well-behaved permanent, adherent, nontransformed chicken cell line has made some experiments with avian leukosis-sarcoma viruses (ASLV) and vectors considerably more difficult. The EV-O-derived line, DF-1, supports the efficient replication of subgroups (A), (B), and (C) ASLV, as well as amphotrophic murine leukemia virus and an ASLV-derived vector that has its env gene derived from the env gene from an amphotrophic murine leukemia virus. The cell line responds appropriately to the expression of a transforming oncogene (v-myc) to a growth suppressor gene [p21(waf1)] and can be sorted (using FACS) if infected by an ASLV vector that expresses GFP., (Copyright 1998 Academic Press.)

In human immunodeficiency virus type 1-infected cells, the efficient expression of viral proteins from unspliced and singly spliced RNAs is dependent on two factors: the presence in the cell of the viral protein Rev and the presence in the viral RNA of the Rev-responsive element (RRE). We show here that the HIV-1 Rev/RRE system can increase the expression of avian leukosis virus (ALV) structural proteins in mammalian cells (D-17 canine osteosarcoma) and promote the release of mature ALV virions from these cells. In this system, the Rev/RRE interaction appears to facilitate the export of full-length unspliced ALV RNA from the nucleus to the cytoplasm, allowing increased production of the ALV structural proteins. Gag protein is produced in the cytoplasm of the ALV-transfected cells even in the absence of a Rev/RRE interaction. However, a functional Rev/RRE interaction increases the amount of Gag present intracellularly and, more strikingly, results in the release of mature ALV particles into the supernatant. RCAS virus containing an RRE is replication-competent in chicken embryo fibroblasts; however, we have been unable to determine whether the particles produced in D-17 cells are as infectious as the particles produced in chicken embryo fibroblasts.

We have tested whether avian leukosis viruses (ALVs) can use tRNAs other than tRNATrp to initiate reverse transcription. The primer binding site (PBS) of a wild-type ALV provirus, which is complementary to the 3' end of tRNA(Trp), was replaced with sequences homologous to the 3' ends of six different chicken tRNAs (tRN(APro), tRNA(Lys), tRNA(Met), tRNA(Ile), tRNA(Phe), and tRNA(Ser)). Transfection of these proviruses into chicken embryo fibroblasts resulted in the production of infectious viruses, all of which apparently used the tRNA specified by the mutated PBS to replicate. However, growth of these viruses resulted in reversion to the wild-type (tRNA(Trp)) PBS. Some of the viruses revert quite quickly, while others are more stable. The relative stability of a given PBS correlated with the concentration of the corresponding tRNA in the virion. We determined the percentage of viral RNA that had a tRNA bound to the PBS and found that the occupancy rate is lower in the mutants than in the wild-type virus. We conclude that many different tRNAs can be used as primers to initiate reverse transcription in ALV. However, ALVs that use tRNA(Trp) have a growth advantage over ALVs that use other tRNAs.

We have developed a simple, PCR-based protocol, random primed/anchored-PCR (RPA-PCR), that allows the selective amplification and efficient cloning of segments that are adjacent to any known sequence. We demonstrate that RPA-PCR can be used to prepare a nested set of evenly spaced deletions suitable for DNA sequencing. However, it should also be possible to use this technique for a number of other purposes: generating deletions for the analysis of eukaryotic promoters, extending cDNA clones in the 5' direction, cloning the insertion sites of retroviral proviruses and transposons, and analyzing intron/exon boundaries.

The sequence of the human immunodeficiency virus type 2 circle junction was determined. The most common sequence found between the conserved CA and TG dinucleotides at the ends of the integrated provirus was five bases long (GGTAC). This suggests that the integration of human immunodeficiency virus type 2 DNA is accompanied by the asymmetric loss of two and three bases, respectively, from the U3 and U5 ends of the linear double-stranded DNA prior to integration.

The sequence of the LTR-LTR circle junction of human immunodeficiency virus type 1 (HIV-1) was determined. The circle junction sequences were amplified by the polymerase chain reaction and cloned into M13 sequencing vectors. The circle junction contains 4 base pairs that are not present in the integrated provirus. We show that reverse transcription in HIV-1 initiates with the addition of a dC to the tRNA primer, suggesting that the tRNA used to initiate reverse transcription ends with the consensus CCA triplet. This indicates that the source of one of the four bases in the circle junction is probably the terminal A of the tRNA primer used to initiate reverse transcription. We propose that, in HIV-1, removal of the tRNA primer by RNase H cleavage shows an unusual specificity such that cleavage occurs between the terminal rA and the adjacent rC of the tRNA primer. These data also imply that the HIV-1 integration protein removes two bases from each end of the linear viral DNA during integration as has been described for other well-studied retroviruses.

The naturally occurring adrenal steroid, dehydroepiandrosterone (DHEA), is a potent non-competitive inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH). Oral administration of DHEA to mice inhibits spontaneous breast cancer and chemically induced tumors of the lung and colon. Topical application of DHEA to mouse skin inhibits 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and tetradecanoylphorbol-13-acetate (TPA)-promoted papillomas and DMBA-induced carcinomas at both the initiation and promotion phase. Evidence is presented that critical steps in the initiation process (mixed-function oxidase activation of a carcinogen) and promotion process (enhanced rates of cell proliferation and superoxide formation) all require NADPH and may be inhibited by DHEA and structural analogs as a result of a lowering of the NADPH cellular pool. Results obtained by others with fibroblasts and lymphocytes from individuals with the Mediterranean variant of G6PDH deficiency also indicate that a reduction in the NADPH cellular pool confers resistance to benzo(a)pyrene. Preliminary data suggest that food restriction may depress G6PDH levels and this may contribute to the tumor preventive effect of underfeeding.

Both dehydroepiandrosterone (DHEA) and the synthetic steroid 16 alpha-Br-epiandrosterone (Br-Epi), a more potent inhibitor of glucose-6-phosphate dehydrogenase (G6PDH) than DHEA, inhibit the 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of superoxide anion (O2-) formation by human neutrophils. DHEA has previously been shown to inhibit the development of spontaneous breast cancer and chemically induced tumors of the lung and colon as well as TPA promoted skin tumors in the mouse. The inhibition of TPA stimulated O2- formation by DHEA may contribute to the cancer preventive activity of this steroid.

Neurons in layer V of the murine posteromedial barrel subfield (PMBSF) project to structures at or caudal to the spinal-medullary junction. During postnatal development a reduction occurs in the density of the neurons which form this projection. In principle, three processes might be expected to contribute to this reduction: cell death, tissue growth, and axon pruning. Three different paradigms in which cells of origin of the projection are labeled retrogradely with True Blue, injected into the spinal-medullary junction, taken together with an estimate of the relative growth of layer V, provide separate estimates of the magnitude and rate of reduction consequent to these different processes during the first 3 postnatal weeks. The density of neurons in an index sector of layer V of the PMBSF which contribute to the projection at varied ages is estimated by injections made at a range of ages from postnatal day 1 (P1) to P16, with a survival of 4 days in each instance. Overall reduction in density is 80%. The component due primarily to axon pruning is estimated to be 50% by injections delivered at graded ages from P1 to P16 with survival to P20 in each instance. The component of the reduction attributable to increase in volume is estimated at 30% by a series of injections delivered at P1 with graded survival times from P5 through P20. A reduction due to cell death is not identified. The reduction in density due to tissue growth is essentially linear through the interval P5-P11. At all ages, neuronal somata of origin of the spinal-medullary projection are located within layer V. Subsequent to P15 they are confined to sublayer Vb; at earlier ages somata in Va and Vc also contribute axons to the projection. Although volume increase due to growth of the neuropil reduces the density of the population contributing to the projection equally in all three sublayers, final elimination of all contributions from Va and Vc depends upon axon pruning.