Pattern recognition analysis on heavy ion reaction data

One of the problems in the analysis of nucleus-nucleus collisions is to get information on the value of the impact parameter b. This work consists in the application of pattern recognition techniques aimed at associating values of b to groups of events. To this end, a support vec- tor machine (SVM) classifier is adopted to analyze multifragmentation reactions. This method allows to backtracing the values of b through a particular multidimensional analysis. The SVM classification con- sists of two main phase. In the first one, known as training phase, the classifier learns to discriminate the events that are generated by two different model:Classical Molecular Dynamics (CMD) and Heavy- Ion Phase-Space Exploration (HIPSE) for the reaction: 58Ni +48 Ca at 25 AMeV. To check the classification of events in the second one, known as test phase, what has been learned is tested on new events generated by the same models. These new results have been com- pared to the ones obtained through others techniques of backtracing the impact parameter. Our tests show that, following this approach, the central collisions and peripheral collisions, for the CMD events, are always better classified with respect to the classification by the others techniques of backtracing. We have finally performed the SVM classification on the experimental data measured by NUCL-EX col- laboration with CHIMERA apparatus for the previous reaction.

Costruzione di chimere molecolari dell'enzima Integrasi di HIV con attività idrolizzante delle LTR virali.

La Sindrome da Immunodeficienza Acquisita (AIDS o SIDA) causata da HIV-1 (Virus dell'Immunodeficienza umana) è caratterizzata dalla graduale compromissione del sistema immunitario del soggetto colpito. Le attuali terapie farmacologiche, purtroppo, non riescono a eliminare l'infezione a causa della comparsa di continui ceppi resistenti ai farmaci, e inoltre questi trattamenti non sono in grado di eliminare i reservoir virali latenti e permettere l'eradicazione definitiva del virus dall’organismo. E' in questo ambito che si colloca il progetto a cui ho lavorato principalmente in questi anni, cioè la creazione di una strategia per eradicare il provirus di HIV integrato nel genoma della cellula ospite. L'Integrasi di HIV-1 è un enzima che media l'integrazione del cDNA virale nel genoma della cellula ospite. La nostra idea è stata, quindi, quella di associare all'attività di legame dell'IN stessa, un'attività catalitica. A tal fine abbiamo creato una proteina chimerica costituita da un dominio DNA-binding, dato dall'Integrasi, e da un dominio con attività nucleasica fornito dall'enzima FokI. La chimera ottenuta è stata sottoposta a mutagenesi random mediante UV, ed è stata oggetto di selezione in vivo, al fine di ottenere una chimera capace di riconoscere, specificamente le LTR di HIV-1, e idrolizzare i siti di inserzione. Questo lavoro porterà a definire pertanto se l'IN di HIV può essere riprogrammata a catalizzare una nuova funzione mediante la sostituzione dell'attività del proprio dominio catalitico con quello di FokI.

Lysosomaler Transport kationischer Aminosäuren durch Mitglieder der SLC7-Familie

Lysosomaler Transport kationischer Aminosäuren (KAS) stellt einen Rettungsweg in der Cystinose-Therapie dar. Ein solches Transportsystem wurde in humanen Hautfibroblasten beschrieben und mit System c benannt. Des Weiteren stellt lysosomales Arginin eine Substratquelle für die endotheliale NO-Synthase (eNOS) dar. Das von der eNOS gebildete NO ist ein wichtiges vasoprotektiv wirkendes Signalmolekül. Ziel war es daher, herauszufinden, ob Mitglieder der SLC7-Unterfamilie hCAT möglicherweise System c repräsentieren.rnIn dieser Arbeit konnte ich die lysosomale Lokalisation verschiedener endogener, sowie als EGFP-Fusionsproteine überexprimierter CAT-Isoformen nachweisen. Mittels Fluoreszenz-mikroskopie wurde festgestellt, dass die in U373MG-Zellen überexprimierten Fusionsproteine hCAT-1.EGFP sowie SLC7A14.EGFP mit dem lysosomalen Fluoreszenz-Farbstoff LysoTracker co-lokalisieren. Eine Lokalisation in Mitochondrien oder dem endoplasmatischem Retikulum konnte mit entsprechenden Fluoreszenz-Farbstoffen ausgeschlossen werden. Zusätzlich reicherten sich die überexprimierten Proteine hCAT-1.EGFP, hCAT-2B.EGFP und SLC7A14.EGFP in der lysosomalen Fraktion C aus U373MG-Zellen zusammen mit den lysosomalen Markern LAMP-1 und Cathepsin D an. Gleiches galt für den endogenen hCAT-1 in der lysosomalen Fraktion C aus EA.hy926- und U373MG-Zellen sowie für den SLC7A14 in den humanen Hautfibroblasten FCys5. Mit dem im Rahmen dieser Arbeit generierte Antikörper gegen natives SLC7A14 konnte erstmals die endogene Expression und Lokalisation von SLC7A14 in verschiedenen Zelltypen analysiert werden.rnObwohl eine Herunterregulation des hCAT-1 in EA.hy926-Endothelzellen nicht zu einer Reduktion der Versorgung der eNOS mit lysosomalem Arginin führte, ist eine Funktion von hCAT-1 im Lysosom wahrscheinlich. Sowohl die [3H]Arginin- als auch die [3H]Lysin-Aufnahme der Fraktion C aus U373MG-hCAT-1.EGFP war signifikant höher als in die Fraktion C aus EGFP-Kontrollzellen. Dies konnte ebenfalls für den hCAT-2B.EGFP gezeigt werden. Zusätzlich zeigten lysosomale Proben aus U373MG-hCAT-2B.EGFP-Zellen in der SSM-basierten Elektrophysiologie eine elektrogene Transportaktivität für Arginin. Das Protein SLC7A14.EGFP zeigte in keiner der beiden durchgeführten Transportstudien eine Aktivität. Dies war unerwartet, da die aus der Diplomarbeit stammende und im Rahmen dieser Dissertation erweiterte Charakterisierung der hCAT-2/A14_BK-Chimäre, die die „funktionelle Domäne“ des SLC7A14 im Rückgrat des hCAT-2 trug, zuvor den Verdacht erhärtet hatte, dass SLC7A14 ein lysosomal lokalisierter Transporter für KAS sein könnte. Diese Studien zeigten allerding erstmals, dass die „funktionelle Domäne“ der hCATs die pH-Abhängigkeit vermittelt und eine Rolle in der Substraterkennung spielt.rnZukünftig soll weiter versucht werden auch endogen eine Transportaktivität der hCATs für KAS im Lysosom nachzuweisen und das Substrat für das intrazellulär lokalisierte Waisen-Protein SLC7A14 zu finden. Eine mögliche Rolle könnte SLC7A14 als Transporter für Neurotransmitter spielen, da eine sehr prominente Expression im ZNS festgestellt wurde.rn

von Willebrand factor-mediated platelet adhesion is critical for deep vein thrombosis in mouse models

Deep vein thrombosis (DVT) and its complication, pulmonary embolism, are frequent causes of disability and mortality. Although blood flow disturbance is considered an important triggering factor, the mechanism of DVT initiation remains elusive. Here we show that 48-hour flow restriction in the inferior vena cava (IVC) results in the development of thrombi structurally similar to human deep vein thrombi. von Willebrand factor (VWF)-deficient mice were protected from thrombosis induced by complete (stasis) or partial (stenosis) flow restriction in the IVC. Mice with half normal VWF levels were also protected in the stenosis model. Besides promoting platelet adhesion, VWF carries Factor VIII. Repeated infusions of recombinant Factor VIII did not rescue thrombosis in VWF(-/-) mice, indicating that impaired coagulation was not the primary reason for the absence of DVT in VWF(-/-) mice. Infusion of GPG-290, a mutant glycoprotein Ib?-immunoglobulin chimera that specifically inhibits interaction of the VWF A1 domain with platelets, prevented thrombosis in wild-type mice. Intravital microscopy showed that platelet and leukocyte recruitment in the early stages of DVT was dramatically higher in wild-type than in VWF(-/-) IVC. Our results demonstrate a pathogenetic role for VWF-platelet interaction in flow disturbance-induced venous thrombosis.

Estrogen Receptor α Signaling in T Lymphocytes Is Required for Estradiol-Mediated Inhibition of Th1 and Th17 Cell Differentiation and Protection against Experimental Autoimmune Encephalomyelitis

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17β-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.

Interferon γ-Dependent Migration of Microglial Cells in the Retina after Systemic Cytomegalovirus Infection

Microglial cells are the resident macrophages of the central nervous system and participate in both innate and adaptive immune responses but can also lead to exacerbation of neurodegenerative pathologies after viral infections. Microglia in the outer layers of the retina and the subretinal space are thought to be involved in retinal diseases where low-grade chronic inflammation and oxidative stress play a role. This study investigated the effect of systemic infection with murine cytomegalovirus on the distribution and dynamics of retinal microglia cells. Systemic infection with murine cytomegalovirus elicited a significant increase in the number of microglia in the subretinal space and an accumulation of iris macrophages, along with morphological signs of activation. Interferon γ (IFN-γ)-deficient mice failed to induce changes in microglia distribution. Bone marrow chimera experiments confirmed that microglial cells in the subretinal space were not recruited from the circulating monocyte pool, but rather represented an accumulation of resident microglial cells from within the retina. Our results demonstrate that a systemic viral infection can lead to IFN-γ-mediated accumulation of microglia into the outer retinal layers and offer proof of concept that systemic viral infections alter the ocular microenvironment and therefore, may influence the course of diseases such as macular degeneration, diabetic retinopathy, or autoimmune uveitis, where low-grade inflammation is implicated.

Eukaryotic translation elongation factor 1A (eEF1A) domain I from S. cerevisiae is required but not sufficient for inter-species complementation

Ethanolamine phosphoglycerol (EPG) is a protein modification attached exclusively to eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is linked to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote Trypanosoma brucei, only domain III is modified by a single EPG. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but nothing is known about the EPG modifying enzyme(s). By expressing human eEF1A in T. brucei, we now show that EPG attachment to eEF1A is evolutionarily conserved between T. brucei and Homo sapiens. In contrast, S. cerevisiae eEF1A, which has been shown to lack EPG is not modified in T. brucei. Furthermore, we show that eEF1A cannot functionally complement across species when using T. brucei and S. cerevisiae as model organisms. However, functional complementation in yeast can be obtained using eEF1A chimera containing domains II or III from other species. In contrast, yeast domain I is strictly required for functional complementation in S. cerevisiae.

SerpinB1 is critical for neutrophil survival through cell-autonomous inhibition of cathepsin G

Bone marrow (BM) holds a large reserve of polymorphonuclear neutrophils (PMNs) that are rapidly mobilized to the circulation and tissues in response to danger signals. SerpinB1 is a potent inhibitor of neutrophil serine proteases neutrophil elastase (NE) and cathepsin G (CG). SerpinB1 deficiency (sB1(-/-)) results in a severe reduction of the BM PMN reserve and failure to clear bacterial infection. Using BM chimera, we found that serpinB1 deficiency in BM cells was necessary and sufficient to reproduce the BM neutropenia of sB1(-/-) mice. Moreover, we showed that genetic deletion of CG, but not NE, fully rescued the BM neutropenia in sB1(-/-) mice. In mixed BM chimera and in vitro survival studies, we showed that CG modulates sB1(-/-) PMN survival through a cell-intrinsic pathway. In addition, membrane permeabilization by lysosomotropic agent l-leucyl-l-leucine methyl ester that allows cytosolic release of granule contents was sufficient to induce rapid PMN death through a CG-dependent pathway. CG-mediated PMN cytotoxicity was only partly blocked by caspase inhibition, suggesting that CG cleaves a distinct set of targets during apoptosis. In conclusion, we have unveiled a new cytotoxic function for the serine protease CG and showed that serpinB1 is critical for maintaining PMN survival by antagonizing intracellular CG activity.

Primary Hyperventilation in the Emergency Department: A First Overview.

BACKGROUND Primary hyperventilation is defined as a state of alveolar ventilation in excess of metabolic requirements, leading to decreased arterial partial pressure of carbon dioxide. The primary aim of this study was to characterise patients diagnosed with primary hyperventilation in the ED. METHODS Our retrospective cohort study comprised adult (≥16 years) patients admitted to our ED between 1 January 2006 and 31 December 2012 with the primary diagnosis of primary (=psychogenic) hyperventilation. RESULTS A total of 616 patients were eligible for study. Participants were predominantely female (341 [55.4%] female versus 275 [44.6%] male respectively, p <0.01). The mean age was 36.5 years (SD 15.52, range 16-85). Patients in their twenties were the most common age group (181, 29.4%), followed by patients in their thirties (121, 19.6%). Most patients presented at out-of-office hours (331 [53.7%]. The most common symptom was fear (586, 95.1%), followed by paraesthesia (379, 61.5%) and dizziness (306, 49.7%). Almost a third (187, 30.4%) of our patients had previously experienced an episode of hyperventilation and half (311, 50.5%) of patients had a psychiatric co-morbidity. CONCLUSION Hyperventilation is a diagnostic chimera with a wide spectrum of symptoms. Patients predominantly are of young age, female sex and often have psychiatric comorbidities. The severity of symptoms accompanied with primary hyperventilation most often needs further work-up to rule out other diagnosis in a mostly young population. In the future, further prospective multicentre studies are needed to evaluate and establish clear diagnostic criteria for primary hyperventilation and possible screening instruments.

The IL-33/ST2 pathway contributes to intestinal tumorigenesis in humans and mice.

Colorectal cancer (CRC) develops through a multistep process and is modulated by inflammation. However, the inflammatory pathways that support intestinal tumors at different stages remain incompletely understood. Interleukin (IL)-33 signaling plays a role in intestinal inflammation, yet its contribution to the pathogenesis of CRC is unknown. Using immunohistochemistry on 713 resected human CRC specimens, we show here that IL-33 and its receptor ST2 are expressed in low-grade and early-stage human CRCs, and to a lesser extent in higher-grade and more advanced-stage tumors. In a mouse model of CRC, ST2-deficiency protects from tumor development. Moreover, bone marrow (BM) chimera studies indicate that engagement of the IL-33/ST2 pathway on both the radio-resistant and radio-sensitive compartment is essential for CRC development. Mechanistically, activation of IL-33/ST2 signaling compromises the integrity of the intestinal barrier and triggers the production of pro-tumorigenic IL-6 by immune cells. Together, this data reveals a tumor-promoting role of IL-33/ST2 signaling in CRC.

The role of Lmx1b in the control of dorsoventral compartmentalization in the distal mesenchyme of the developing mouse limb

Lmx1b encodes a LIM-homeodomain transcription factor required for dorso-ventral (D-V) patterning in the mesenchyme of the vertebrate limb. In the absence of Lmx1b function, limbs exhibit a bi-ventral pattern indicating that Lmx1b is required for cells to adopt a dorsal cell fate. However, how Lmx1b specifies dorsal cell fates in the mesenchyme of the distal limb is unknown. Lmx1b is initially expressed throughout the dorsal and ventral limb bud mesenchyme, then becomes dorsally restricted around E10.5. At this stage, there is a sharp boundary between Lmx1b expressing and Lmx1b non-expressing cells. How the dorso-ventral Lmx1b expression boundary is formed and maintained is currently unknown. One mechanism that may contribute to establishing and/or maintaining the Lmx1b expression boundary is if the dorsal mesenchyme is a lineage-based compartment, where different groups of non-mingling cells are separated. Compartment formation has been proposed to rely on compartment-specific selector gene activity which functions to restrict cells to a compartment and specifies the identity of cells within that compartment. Based on the evidence that the dorsal limb identity relies on the expression of Lmx1b in the dorsal half of the limb mesenchyme, we hypothesized that Lmx1b might function as a dorsal limb bud mesenchyme selector gene to set up a dorsal compartment. To test this hypothesis, we developed an inducible CreERT2/ loxP based fate mapping approach that permanently marks Lmx1b wild-type and mutant cells and examined the distribution of their descendents in the developing limb. Our data is the first to show that dorso-ventral lineage compartments exist in the limb bud mesenchyme. Furthermore, Lmx1b is required for maintenance of the dorso-ventral compartment lineage boundary. The behavior of Lmx1b mutant cells that cross into the ventral mesenchyme, as well as previous chimera analysis in which mutant cells spread evenly in the ventral limb and form patches in the dorsal side, suggest that cell affinity differences prevent intermingling of dorsal and ventral cells. ^

Functional studies of a LIM -homeodomain transcription factor lmx1b in murine mid -hindbrain and limb development

This thesis is centered on applying molecular genetics to study pattern formation during animal development. More specifically, this thesis describes the functional studies of a LIM-homeodomain gene called lmx1b during murine embryogenesis. Lmx1b expression is restricted to the mid-hindbrain junction as well as to the dorsal mesenchyme of the limb, suggesting important functions during mid-hindbrain and limb development. To test these possibilities, lmx1b homozygous mutant mice were generated and their limb and CNS phenotypes examined. Lmx1b homozygous mutant mice exhibit a large reduction of mid-hindbrain structures, and that their limbs are symmetrical along the dorsal-ventral axis as the result of a dorsal to ventral transformation. Taken together, these studies define essential functions for lmx1b in mid-hindbrain patteming and in dorsal limb cell fate determination. However, the molecular mechanisms which accounts for these phenotypes are unknown, and whether lmx1b has same or distinctive functions during the mid-hindbrain and limb development is also unclear. ^ Recently, insight into molecular mechanisms of mid-hindbrain patterning and limb development has resulted from the identification of several factors with restricted expression patterns within these regions. These include the secreted factors wnt-1, fgf-8, wnt-7a and the transcription factors pax-2, and en-1. Targeted disruption of any of these genes in mice suggests that these genes might be involved in similar regulatory pathways. Analysis of the expression of these genes in lmx1b mutants demonstrates that lmxlb is not required for the initiation, but is required to maintain their expression at the mid-hindbrain junction. Thus, lmxlb is not required for specifying mid-hindbrain cell fates, rather, it functions to ensure the establishment or maintenance of a proper organizing center at the mid-hindbrain junction. Interestingly, lmxlb functions cell non-autonomously in chimera analysis, which indicates that lmx1b might regulate the expression of secreted factors such as wnt-1 and/or fgf-8 in the organizing center. In contrast, lmx1b functions cell autonomously in the dorsal limb to govern dorsal ventral limb development and its expression is regulated by with wnt-7a and en-1. However, single and double mutant analysis suggest that all three genes have partially overlapping functions as well as independent functions. The results point toward a complicated network of cross-talks among all three limb axes. ^

Developmental functions of XKaiso, a transcriptional repressor associating with the p120 catenin in Xenopus laevis

The Armadillo family catenin proteins function in multiple capacities including cadherin-mediated cell-cell adhesion and nuclear signaling. The newest catenin, p120 catenin, differs from the classical catenins and binds to the membrane-proximal domain of cadherins. Recently, a novel transcription factor Kaiso was found to interact with p120 catenin, suggesting that p120 catenin also possesses a nuclear function. We isolated the Xenopus homolog of Kaiso, XKaiso, from a Xenopus stage 17 cDNA library. XKaiso contains an amino-terminal BTB/POZ domain and three carboxyl-terminal zinc fingers. The XKaiso transcript was present maternally and expressed throughout early embryonic development. XKaiso's spatial expression was defined via in situ hybridization and was found localized to the brain, eye, ear, branchial arches, and spinal cord. Co-immunoprecipitation of Xenopus p120 catenin and XKaiso demonstrated their mutual association, while related experiments employing differentially epitope-tagged XKaiso constructs suggest that XKaiso also self-associates. On the functional level, reporter assays employing a chimera of XKaiso fused to the GAL4 DNA binding domain indicated that XKaiso is a transcriptional repressor. To better understand the significance of the Kaiso-p120 catenin complex in vertebrate development, Kaiso knock-down experiments were undertaken, and the modulatory role of p120 catenin in Kaiso function examined during Xenopus development. Using morpholino antisense oligonucleotides to block translation of XKaiso, XKaiso was found to be essential for Xenopus gastrulation, being required for correct morphogenetic movements in early embryogenesis. Molecular marker analyses indicated that one target gene of the Wnt/β-catenin pathway, Siamois, is significantly increased in embryos depleted for XKaiso, while other dorsal, ventral, and mesodermal cell fate markers were unaltered. In addition, the non-canonical Wnt-11, known to participate in planar cell polarity/convergent extension processes, was significantly upregulated following depletion of XKaiso. Such increased Wnt-11 expression likely contributed to the XKaiso depletion phenotype because a dominant negative form of Wnt-11 or of the downstream effector Dishevelled partially rescued the observed gastrulation defects. These results show that XKaiso is essential for proper gastrulation movements, resulting at least in part from its modulation of non-canonical Wnt signaling. The significance of the XKaiso-p120 catenin interaction has yet to be determined, but appears to include a role in modulating genes promoting canonical and non-canonical Wnt signals. ^

Metadata und statistic analysis of archael and bacterial sequences originating from sediments of the Håkon Mosby mud volcano (Z1-Z3, all habitats)

DNA extraction was carried out as described on the MICROBIS project pages (http://icomm.mbl.edu/microbis ) using a commercially available extraction kit. We amplified the hypervariable regions V4-V6 of archaeal and bacterial 16S rRNA genes using PCR and several sets of forward and reverse primers (http://vamps.mbl.edu/resources/primers.php). Massively parallel tag sequencing of the PCR products was carried out on a 454 Life Sciences GS FLX sequencer at Marine Biological Laboratory, Woods Hole, MA, following the same experimental conditions for all samples. Sequence reads were submitted to a rigorous quality control procedure based on mothur v30 (doi:10.1128/AEM.01541-09) including denoising of the flow grams using an algorithm based on PyroNoise (doi:10.1038/nmeth.1361), removal of PCR errors and a chimera check using uchime (doi:10.1093/bioinformatics/btr381). The reads were taxonomically assigned according to the SILVA taxonomy (SSURef v119, 07-2014; doi:10.1093/nar/gks1219) implemented in mothur and clustered at 98% ribosomal RNA gene V4-V6 sequence identity. V4-V6 amplicon sequence abundance tables were standardized to account for unequal sampling effort using 1000 (Archaea) and 2300 (Bacteria) randomly chosen sequences without replacement using mothur and then used to calculate inverse Simpson diversity indices and Chao1 richness (doi:10.2307/4615964). Bray-Curtis dissimilarities (doi:10.2307/1942268) between all samples were calculated and used for 2-dimensional non metric multidimensional scaling (NMDS) ordinations with 20 random starts (doi:10.1007/BF02289694). Stress values below 0.2 indicated that the multidimensional dataset was well represented by the 2D ordination. NMDS ordinations were compared and tested using Procrustes correlation analysis (doi:10.1007/BF02291478). All analyses were carried out with the R statistical environment and the packages vegan (available at: http://cran.r-project.org/package=vegan), labdsv (available at: http://cran.r-project.org/package=labdsv), as well as with custom R scripts. Operational taxonomic units at 98% sequence identity (OTU0.03) that occurred only once in the whole dataset were termed absolute single sequence OTUs (SSOabs; doi:10.1038/ismej.2011.132). OTU0.03 sequences that occurred only once in at least one sample, but may occur more often in other samples were termed relative single sequence OTUs (SSOrel). SSOrel are particularly interesting for community ecology, since they comprise rare organisms that might become abundant when conditions change.16S rRNA amplicons and metagenomic reads have been stored in the sequence read archive under SRA project accession number SRP042162.

The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop

Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.