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Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC50 in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.[Mol Cancer Ther 2009;8(8):2286–95]

Supplementary Data from Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells

Bistable motoneurons of the spinal cord exhibit warmth-activated plateau potential driven by Na+ and triggered by a brief excitation. The thermoregulating molecular mechanisms of bistability and their role in motor functions remain unknown. Here, we identify thermosensitive Na+-permeable Trpm5 channels as the main molecular players for bistability in mouse motoneurons. Pharmacological, genetic or computational inhibition of Trpm5 occlude bistable-related properties (slow afterdepolarization, windup, plateau potentials) and reduce spinal locomotor outputs while central pattern generators for locomotion operate normally. At cellular level, Trpm5 is activated by a ryanodine-mediated Ca2+ release and turned off by Ca2+ reuptake through the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. Mice in which Trpm5 is genetically silenced in most lumbar motoneurons develop hindlimb paresis and show difficulties in executing high-demanding locomotor tasks. Overall, by encoding bistability in motoneurons, Trpm5 appears indispensable for producing a postural tone in hindlimbs and amplifying the locomotor output., The authors show that Trpm5, but not Trpm4, is the main Na+ -permeant channel mediating the warmth-activated ICaN in lumbar motoneurons. Trpm5 is also critical in generating plateau potentials in bistable motoneurons that are essential for producing a postural tone in hindlimbs and amplifying the locomotor output.

The central pattern generator (CPG) for locomotion is a set of pacemaker neurons endowed with inherent bursting driven by the persistent sodium current (INaP). How they proceed to regulate the locomotor rhythm remained unknown. Here, in neonatal rodents, we identified a persistent potassium current critical in regulating pacemakers and locomotion speed. This current recapitulates features of the M-current (IM): a subthreshold noninactivating outward current blocked by 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) and enhanced by N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide (ICA73). Immunostaining and mutant mice highlight an important role of Kv7.2-containing channels in mediating IM. Pharmacological modulation of IM regulates the emergence and the frequency regime of both pacemaker and CPG activities and controls the speed of locomotion. Computational models captured these results and showed how an interplay between IM and INaP endows the locomotor CPG with rhythmogenic properties. Overall, this study provides fundamental insights into how IM and INaP work in tandem to set the speed of locomotion.

International audience; Planarians, which are non-parasitic flatworms, are highly resistant to bacterial infections. To better understand the mechanisms underlying this resistance, we investigated the role of the circadian machinery in the anti-bacterial response of the freshwater planarian Schmidtea mediterranea. We identified Smed-Tim from S. mediterranea as a homolog of the mammalian clock gene Tim. We showed via RNA interference that Smed-Tim is required for the antimicrobial activities of Schmidtea mediterranea against Staphylococcus aureus infection during the light/dark cycle. Indeed, S. aureus infection leads to the expression of Smed-Tim, which in turn promotes Smed-Traf6 and Smed-morn2, but not Smed-p38 MAPK expression, 2 master regulators of planarian anti-microbial responses.

SummaryVariations in lipopolysaccharide (LPS), a bacterial outer membrane component, determine virulence of the obligate intracellular bacterium Coxiella burnetii, but the underlying mechanisms are unknown. We find that while avirulent C. burnetii LPS (avLPS) stimulates host p38α-MAPK signaling required for proper trafficking of bacteria containing compartments to lysosomes for destruction, pathogenic C. burnetii LPS (vLPS) does not. The defect in vLPS and pathogenic C. burnetii targeting to degradative compartments involves an antagonistic engagement of TLR4 by vLPS, lack of p38α-MAPK-driven phosphorylation, and block in recruitment of the homotypic fusion and protein-sorting complex component Vps41 to vLPS-containing vesicles. An upstream activator of p38α-MAPK or phosphomimetic mutant Vps41-S796E expression overrides the inhibition, allowing vLPS and pathogenic C. burnetii targeting to phagolysosomes. Thus, p38α-MAPK and its crosstalk with Vps41 play a central role in trafficking bacteria to phagolysosomes. Pathogenic C. burnetii has evolved LPS variations to evade this host response and thrive intracellularly.

Chronic myelomonocytic leukemia (CMML) is a haematological disease currently classified in the category of myelodysplastic syndromes/myeloproliferative neoplasm (MDS/MPN) because of its dual clinical and biological presentation. The molecular biology of CMML is poorly characterized. We studied a series of 53 CMML samples including 31 cases of myeloproliferative form (MP-CMML) and 22 cases of myelodysplastic forms (MD-CMML) using array-comparative genomic hybridization (aCGH) and sequencing of 13 candidate genes including ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, PTPN11, RUNX1, TET2 and WT1. Mutations in ASXL1 and in the genes associated with proliferation (CBL, FLT3, PTPN11, RAS) were mainly found in MP-CMML cases. Mutations of ASXL1 correlated with an evolution toward an acutely transformed state: all CMMLs that progressed to acute phase were mutated and none of the unmutated patients had evolved to acute leukaemia. The overall survival of ASXL1 mutated patients was lower than that of unmutated patients.

Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC50 in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.[Mol Cancer Ther 2009;8(8):2286–95]

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor β (TGFβ) superfamily. We used two-hybrid screening to map Smad signaling protein–protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

Human immunodeficiency virus type 1 plasma viruses from 29 entry inhibitor-naive patients were characterized for their susceptibilities to T-20, AMD3100, and RANTES. A strikingly wide range of susceptibilities to T-20 was observed that was influenced by coreceptor usage but not by the susceptibilities of the viruses to inhibitors that target the chemokine receptors or by polymorphisms in the gp41 N helix.

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-γ production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity.

To survive in macrophages, Coxiella burnetii hijacks the activation pathway of macrophages. Recently, we have demonstrated that C. burnetii, via its lipopolysaccharide (LPS), avoids the activation of p38α-MAPK through an antagonistic engagement of Toll-like receptor (TLR)-4. We investigated the fine-tuned mechanism leading to the absence of activation of the p38α-MAPK despite TLR-4 engagement. In macrophages challenged with Escherichia coli LPS or with the LPS from the avirulent variants of C. burnetii, TLR-4 and TLR-2 co-immunoprecipitated. This association was absent in cells challenged by the LPS of pathogenic C. burnetii. The disruption makes TLRs unable to signal during the recognition of the LPS of pathogenic C. burnetii. The disruption of TLR-2 and TLR-4 was induced by the re-organization of the macrophage cytoskeleton by C. burnetii LPS. Interestingly, blocking the actin cytoskeleton re-organization relieved the disruption of the association TLR-2/TLR-4 by pathogenic C. burnetii and rescued the p38α-MAPK activation by C. burnetii. We elucidated an unexpected mechanism allowing pathogenic C. burnetii to avoid activating macrophages by the disruption of the TLR-2 and TLR-4 association.

Objective To investigate the role of thymic output and viral fitness in immune reconstitution in HIV-1-infected children on antiretroviral therapy. Methods Thymic output was studied by measuring levels of T-cell receptor rearrangement excision circles (TREC) in peripheral blood lymphocytes, using a real-time quantitative PCR assay. Recombinant viruses containing pre-therapy or post-therapy HIV-1 protease domains were evaluated for viral infectivity in a quantitative single-cycle assay. Results Eighteen HIV-1-infected children who showed a significant increase in CD4 T-cell count after therapy were studied; HIV-1 plasma viraemia was substantially suppressed in 12 children (virological responders), but not in the other six (virological non-responders). TREC were quantified at baseline, and sequentially during the first 12 months of therapy. Both virological responders and non-responders showed an increase in TREC levels that was inversely correlated with baseline TREC and CD4 T cell counts. Changes in TREC positively correlated with CD4 T-cell count increases in virological responders, but not in non-responders; moreover, the ratios between TREC and CD4 T-cell count increases were higher in non-responders than in responders, suggesting a persistence of peripheral CD4 T-cell loss in the former. Drug-resistant viruses with reduced replicative capacity were documented in three out of six non-responders. Conclusions These findings indicate that recovery of thymic function is a pivotal event in immune reconstitution, and suggest that CD4 T-cell increase despite persistent viraemia is sustained by a continuous thymic output that compensates peripheral CD4 T-cell depletion which might be slowed down by emerging viruses with reduced fitness.

International audience; Dugesia japonica planarian flatworms are naturally exposed to various microbes but typically survive this challenge. We show that planarians eliminate bacteria pathogenic to Homo sapiens, Caenorhabditis elegans, and/or Drosophila melanogaster and thus represent a model to identify innate resistance mechanisms. Whole-transcriptome analysis coupled with RNAi screening of worms infected with Staphylococcus aureus or Legionella pneumophila identified 18 resistance genes with nine human orthologs, of which we examined the function of MORN2. Human MORN2 facilitates phagocytosis-mediated restriction of Mycobacterium tuberculosis, L. pneumophila, and S. aureus in macrophages. MORN2 promotes the recruitment of LC3, an autophagy protein also involved in phagocytosis, to M. tuberculosis-containing phagosomes and subsequent maturation to degradative phagolysosomes. MORN2-driven trafficking of M. tuberculosis to single-membrane, LC3-positive compartments requires autophagy-related proteins Atg5 and Beclin-1, but not Ulk-1 and Atg13, highlighting the importance of MORN2 in LC3-associated phagocytosis. These findings underscore the value of studying planarian defenses to identify immune factors.

Human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors (PI) is a major obstacle to the full success of combined antiretroviral therapy. High-level resistance to these compounds is the consequence of stepwise accumulation of amino acid substitutions in the HIV-1 protease (PR), following pathways that usually differ from one inhibitor to another. The selective advantage conferred by resistance mutations may depend upon several parameters: the impact of the mutation on virus infectivity in the presence or absence of drug, the nature of the drug, and its local concentration. Because drug concentrations in vivo are subject to extensive variation over time and display a markedly uneven tissue distribution, the parameters of selection for HIV-1 resistance to PI in treated patients are complex and poorly understood. In this study, we have reconstructed a large series of HIV-1 mutants that carry single or combined mutations in the PR, retracing the accumulation pathways observed in ritonavir-, indinavir-, and saquinavir-treated patients. We have then measured the phenotypic resistance and the drug-free infectivity of these mutant viruses. A deeper insight into the evolutionary value of HIV-1 PR mutants came from a novel assay system designed to measure the replicative advantage of mutant viruses as a function of drug concentration. By tracing the resultant fitness profiles, we determined the range of drug concentrations for which mutant viruses displayed a replicative advantage over the wild type and the extent of this advantage. Fitness profiles were fully consistent with the order of accumulation of resistance mutations observed in treated patients and further emphasise the key importance of local drug concentration in the patterns of selection of drug-resistant HIV-1 mutants.

Tropheryma whipplei, the agent of Whipple's disease, inhibits phago-lysosome biogenesis to create a suitable niche for its survival and replication in macrophages. To understand the mechanism by which it subverts phagosome maturation, we used biochemical and cell biological approaches to purify and characterise the intracellular compartment where Tropheryma whipplei resides using mouse bone-marrow-derived macrophages. We showed that in addition to Lamp-1, the Tropheryma whipplei phagosome is positive for Rab5 and Rab7, two GTPases required for the early to late phagosome transition. Unlike other pathogens, inhibition of PI(3)P production was not the mechanism for Rab5 stabilisation at the phagosome. Overexpression of the inactive, GDP-bound form of Rab5 bypassed the pathogen-induced blockade of phago-lysosome biogenesis. This suggests that Tropheryma whipplei blocks the switch from Rab5 to Rab7 by acting on the Rab5 GTPase cycle. A bio-informatic analysis of the Tropheryma whipplei genome revealed a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologous with the GAPDH of Listeria monocytogenes, and this may be the bacterial protein responsible for blocking Rab5 activity. To our knowledge, Tropheryma whipplei is the first pathogen described to induce a "chimeric" phagosome stably expressing both Rab5 and Rab7, suggesting a novel and specific mechanism for subverting phagosome maturation.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-γ production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity.

Chronic myelomonocytic leukaemia (CMML) is a haematological disease currently classified in the category of myelodysplastic syndromes/myeloproliferative neoplasm (MDS/MPN) because of its dual clinical and biological presentation. The molecular biology of CMML is poorly characterized. We studied a series of 53 CMML samples including 31 cases of myeloproliferative form (MP-CMML) and 22 cases of myelodysplastic forms (MD-CMML) using array-comparative genomic hybridisation (aCGH) and sequencing of 13 candidate genes including ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, PTPN11, RUNX1, TET2 and WT1. Mutations in ASXL1 and in the genes associated with proliferation (CBL, FLT3, PTPN11, NRAS) were mainly found in MP-CMML cases. Mutations of ASXL1 correlated with an evolution toward an acutely transformed state: all CMMLs that progressed to acute phase were mutated and none of the unmutated patients had evolved to acute leukaemia. The overall survival of ASXL1 mutated patients was lower than that of unmutated patients.

International audience; No abstract available

International audience; No abstract available

International audience; The myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal haematological diseases characterized by ineffective haematopoiesis and predisposition to acute myeloid leukaemia (AML). The pathophysiology of MDSs remains unclear. A definition of the molecular biology of MDSs may lead to a better classification, new prognosis indicators and new treatments. We studied a series of 40 MDS/AML samples by high-density array-comparative genome hybridization (aCGH). The genome of MDSs displayed a few alterations that can point to candidate genes, which potentially regulate histone modifications and WNT pathways (e.g. ASXL1, ASXL2, UTX, CXXC4, CXXC5, TET2, TET3). To validate some of these candidates we studied the sequence of ASXL1. We found mutations in the ASXL1 gene in four out of 35 MDS patients (11%). To extend these results we searched for mutations of ASXL1 in a series of chronic myelomonocytic leukaemias, a disease classified as MDS/Myeloproliferative disorder, and found mutations in 17 out of 39 patients (43%). These results show that ASXL1 might play the role of a tumour suppressor in myeloid malignancies.

Background Chronic myelomonocytic leukemia (CMML) is a hematological disease close to, but separate from both myeloproliferative disorders (MPD) and myelodysplastic syndromes and may show either myeloproliferative (MP-CMML) or myelodysplastic (MD-CMML) features. Not much is known about the molecular biology of this disease. Methods We studied a series of 30 CMML samples (13 MP- and 11 MD-CMMLs, and 6 acutely transformed cases) from 29 patients by using Agilent high density array-comparative genomic hybridization (aCGH) and sequencing of 12 candidate genes. Results Two-thirds of samples did not show any obvious alteration of aCGH profiles. In one-third we observed chromosome abnormalities (e.g. trisomy 8, del20q) and gain or loss of genes (e.g. NF1, RB1 and CDK6). RAS mutations were detected in 4 cases (including an uncommon codon 146 mutation in KRAS) and PTPN11 mutations in 3 cases. We detected 11 RUNX1 alterations (9 mutations and 2 rearrangements). The rearrangements were a new, cryptic inversion of chromosomal region 21q21-22 leading to break and fusion of RUNX1 to USP16. RAS and RUNX1 alterations were not mutually exclusive. RAS pathway mutations occurred in MP-CMMLs (~46%) but not in MD-CMMLs. RUNX1 alterations (mutations and cryptic rearrangement) occurred in both MP and MD classes (~38%). Conclusion We detected RAS pathway mutations and RUNX1 alterations. The latter included a new cryptic USP16-RUNX1 fusion. In some samples, two alterations coexisted already at this early chronic stage.

International audience; No abstract available

Gene expression profiling separates chronic myelomonocytic leukemia in two molecular subtypes

Gene silencing by RNA interference (RNAi) has proven to be a powerful tool for investigating gene function in mammalian cells. Combination of several short interfering RNA (siRNA) targeting the same gene is commonly used to improve RNA interference. However, in contrary to the well-described mechanism of RNAi, efficiency of single siRNA compared to pool remains poorly documented. We addressed this issue using several active and inactive siRNA targeting Eg5, a kinesin-related motor involved in mitotic spindle assembly. These siRNA, used alone or in combination, were tested for their silencing efficiency in several cancer cell lines. Here we show that presence of inactive Eg5 siRNA in a pool dramatically decreases knockdown efficacy in a cell line- and dose-dependent manner. Lack of inhibition by unrelated siRNA suggests that a competition may occur during siRNA incorporation into RNA-induced silencing complexes (RISCs) along with the target mRNA. Altogether, our results, which need to be confirmed with additional inactive siRNA, indicate that combination of siRNA may not increase but instead decrease silencing efficiency.

Objective: To study the impact of antiretroviral therapy on the tropism of plasma HIV-1 virus populations during treatment response and virological escape. Design: To investigate whether the selective pressure exerted by antiretroviral treatment influences the tropism of the plasma virus populations, we retrospectively determined the co-receptor usage of viruses present in plasma samples obtained before and at varying intervals after starting antiviral therapy. Methods: The co-receptor usage of plasma virus was determined using our recently published tropism recombinant test (V. Trouplin et al., J Virol, 2001; 75:251–259). This assay relies on virus production by homologous recombination between a plasmid encoding the entire HIV genome except for a deletion of the major tropism determinant, and a polymerase chain reaction (PCR) product spanning this region and the adjoining flanking sequences, obtained by reverse transcriptase (RT)-PCR amplification of viral RNA from the patient's plasma. Results: Twenty-four of the 32 patients analysed harboured exclusively R5 virus in plasma before the initiation of treatment, whereas eight had mixed R5/X4 virus populations. In four of these eight patients, all of whom initially responded to treatment, the persistence of R5 virus in plasma was observed, whereas the X4 component of the virus population became undetectable. The suppression of the X4 virus component was a transient phenomenon and variants able to use CXCR4 re-emerged after a variable delay. Conclusions: The impact of therapy on virus populations differs according to virus tropism. Differences in target cell populations, tissue distribution and replication characteristics between R5 and X4 viruses could explain the preferential suppression of X4 virus.

We developed a recombinant virus technique to determine the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from plasma samples, the source expected to represent the most actively replicating virus population in infected subjects. This method is not subject to selective bias associated with virus isolation in culture, a step required for conventional tropism determination procedures. The addition of a simple subcloning step allowed semiquantitative evaluation of virus populations with a different coreceptor (CCR5 or CXCR4) usage specificity present in each plasma sample. This procedure detected mixtures of CCR5- and CXCR4-exclusive virus populations as well as dualtropic viral variants, in variable proportions. Sequence analysis of dualtropic clones indicated that changes in the V3 loop are necessary for the use of CXCR4 as a coreceptor, but the overall context of the V1-V3 region is important to preserve the capacity to use CCR5. This convenient technique can greatly assist the study of virus evolution and compartmentalization in infected individuals.