Downregulation of miR-218 contributes to epithelial-mesenchymal transition and tumor metastasis in lung cancer by targeting Slug/ZEB2 signaling.

Epithelial-mesenchymal transition (EMT) has been recognized as a key element of cell migration and invasion in lung cancer; however, the underlying mechanisms are not fully elucidated. Recently, emerging evidence suggest that miRNAs have crucial roles in control of EMT and EMT-associated traits such as migration, invasion and chemoresistance. Here, we found that miR-218 expression levels were significantly downregulated in lung cancer tissues compared with adjacent non-cancerous tissues, and the levels of miR-218 were significantly associated with histological grades and lymph node metastasis. Overexpression of miR-218 inhibited cell migration and invasion as well as the EMT process. Of particular importance, miR-218 was involved in the metastatic process of lung cancer cells in vivo by suppressing local invasion and distant colonization. We identified Slug and ZEB2 as direct functional targets of miR-218. Inverse correlations were observed between miR-218 levels and Slug/ZEB2 levels in cancer tissue samples. In addition, overexpression of miR-218 in H1299 increased chemosensitivity of cells to cisplatin treatment through suppression of Slug and ZEB2. These findings highlight an important role of miR-218 in the regulation of EMT-related traits and metastasis of lung cancer in part by modulation of Slug/ZEB2 signaling, and provide a potential therapeutic strategy by targeting miR-218 in NSCLC.

Efficacy of DSP30-IL2/TPA for detection of cytogenetic abnormalities in chronic lymphocytic leukaemia/small lymphocytic lymphoma.

INTRODUCTION: Chronic lymphocytic leukaemia (CLL) is the most prevalent leukaemia in the Western Hemisphere. Cytogenetic abnormalities in CLL are used for diagnosis, prognosis and treatment. However, detecting these is difficult because mature B cells do not readily divide in culture. Here, we present data on two mitogen cocktails: CpG-oligonucleotide DSP30/Interleukin-2 (IL-2) and DSP30/IL-2 in combination with 12-O-tetradecanoylphorbol-13-acetate (TPA). METHODS: We analysed 165 cases of CLL with FISH and cytogenetics from January 2011 to June 2013. In 2011, three cultures were set-up: unstimulated, DSP30/IL-2-stimulated and TPA-stimulated. In 2012-2013, two cultures were set-up: unstimulated and stimulated with TPA/DSP30/IL-2. RESULTS: In 2011, FISH had a detection rate of 91% and cytogenetics using DSP30/IL2 had a detection rate of 91% (n = 22). In 2012-2013, FISH had a detection rate of 79% and cytogenetics using TPA/DSP30/IL-2 had a detection rate of 98% (n = 40). The percentage of cases with normal FISH but abnormal cytogenetics increased from 9% in 2011 to 21% in 2012-2013. The TPA/DSP30/IL-2 cultures in 2012-2013 detected more novel abnormalities (n = 5) as compared to DSP30/IL-2 alone (n = 3). CONCLUSIONS: TPA/DSP30/IL2 was as good as or better than DSP30/IL2 alone. TPA/DSP30/IL-2 offers a high detection rate for CLL abnormalities with a single stimulated culture and may increase detection of clinically significant abnormalities.

Regulatory circuit of PKM2/NF-κB/miR-148a/152-modulated tumor angiogenesis and cancer progression.

Upregulation of the embryonic M2 isoform of pyruvate kinase (PKM2) emerges as a critical player in the cancer development and metabolism, yet the underlying mechanism of PKM2 overexpression remains to be elucidated. Here we demonstrate that IGF-1/IGF-IR regulates PKM2 expression by enhancing HIF-1α-p65 complex binding to PKM2 promoter. PKM2 expression is regulated by miR-148a/152 suppression. PKM2 directly interacts with NF-κB p65 subunit to promote EGR1 expression for regulating miR-148a/152 feedback circuit in normal cells, but not in cancer cells because of the DNA hypermethylation of miR-148a and miR-152 gene promoters. The silencing of miR-148a/152 contributes to the overexpression of PKM2, NF-κB or/and IGF-IR in some cancer cells. We show that disruption of PKM2/NF-κB/miR-148a/152 feedback loop can regulate cancer cell growth and angiogenesis, and is also associated with triple-negative breast cancer (TNBC) phenotype, which may have clinical implication for providing novel biomarker(s) of TNBC and potential therapeutic target(s) in the future.

Distinct molecular signatures in pediatric infratentorial glioblastomas defined by aCGH.

Glioblastomas (GBM) are rare in children, but reportedly have more varied outcome which suggests differences in tumor etiology compared to typical GBM of adults. To investigate this we performed high resolution array comparative genomic hybridization (aCGH) analysis on three pediatric infratentorial GBM, ages 3.5, 7 and 14 years. Two of these tumors occurred in the brainstem and one in the spinal cord. While histologically typical, one brainstem tumor showed mainly pleomorphic astrocytic cells, whereas the other brainstem and spinal tumors showed a GFAP positive small cell component. Whole chromosomal gains (#1 and #2) and loss (#20) were seen only in the pleomorphic brainstem GBM, which also showed a high level of segmental genomic copy number changes. Segmental loss involving chromosome 8 was seen in all three tumors (Chr8;133039446-136869494, Chr8;pter-3581577, and Chr8;pter-30480019 respectively), whereas loss involving chromosome 16 was seen in only 2 cases with small cell components (Chr16;31827239-qter and Chr16;pter-29754532). Segmental gain of chromosome 7 was shared only between the 2 brainstem cases (Chr7;17187166-qter and Chr7;69824947-qter). Chromosome 17 showed segmental gain of 17q in the backdrop of loss of 17p only in case 1. Segmental gain of chromosome 1q was seen only in case 2. The spinal GBM showed a relatively stable karyotype with a unique loss of Chr19;32848902-qter. None of the frequent losses, gains and amplifications known to occur in adult GBM were identified, suggesting that pediatric infratentorial glioblastomas show a molecular karyotype that was more characteristic of pediatric embryonal tumors than adult GBM.

Gene amplification and associated loss of 5' regulatory sequences of CoAA in human cancers.

CoAA is an RRM-containing transcriptional coactivator that stimulates transcriptional activation and regulates alternative splicing. We show that the CoAA gene is amplified at the chromosome 11q13 locus in a subset of primary human cancers including non-small cell lung carcinoma, squamous cell skin carcinoma and lymphoma. Analysis of 42 primary tumors suggests that CoAA amplifies independently from the CCND1 locus. Detailed mapping of three CoAA amplicons reveals that the amplified CoAA gene is consistently located at the 5' boundaries of the amplicons. The CoAA coding and basal promoter sequences are retained within the amplicons but upstream silencing sequences are lost. CoAA protein is overexpressed in tumors containing the amplified CoAA gene. RNA dot blot analysis of 100 cases of primary tumors suggests elevated CoAA mRNA expression. CoAA positively regulates its own basal promoter in transfection assays. Thus, gene amplification, loss of silencing sequence and positive feedback regulation may lead to drastic upregulation of CoAA protein. CoAA has transforming activities when tested in soft agar assays, and CoAA is homologous to oncoproteins EWS and TLS, which regulate alternative splicing. These data imply that CoAA may share a similar oncogenic mechanism with oncogene EWS and that CoAA deregulation may alter the alternative splicing of target genes.

Unique monoclonal antibody recognizing the third extracellular loop of CXCR4 induces lymphocyte agglutination and enhances human immunodeficiency virus type 1-mediated syncytium formation and productive infection.

To increase insight into the structural basis of CXCR4 utilization in human immunodeficiency virus type 1 (HIV-1) infection, a new generation of three monoclonal antibodies (MAbs) was developed in WKA rats. The A80 MAb, which binds an epitope in the third extracellular loop (ECL3) of CXCR4, has unique biologic properties that provide novel insights into CXCR4 function. This agent enhanced syncytium formation in activated human peripheral blood mononuclear cells (PBMC) infected with X4 or R5 and CEM cells infected with X4 HIV-1 strains. Exposure to A80 increased the productive infection of activated CD4(+) T cells and CEM cells with R5 and X4 viruses, respectively. This antibody uniquely induced agglutination of PBMC and CEM cells but did not activate calcium mobilization. Agglutination induced by A80 was inhibited by stromal cell-derived factor 1, T22, and phorbol 12-myristate 13-acetate but was not significantly altered by pretreatment of cells with pertussis toxin, wortmannin, or MAbs to LFA-1, ICAM-1, ICAM-2, and ICAM-3. The binding of the A145 and A120 MAbs was mapped to the N-terminal extracellular domain and a conformational epitope involving ECL1 and ECL2, respectively. Both of these MAbs inhibited HIV-1 infection and lacked the novel properties of A80. These results suggest a new role for CXCR4 in homologous lymphocyte adhesion that is ligand independent and in HIV-1 infection.

Molecular anatomy of CCR5 engagement by physiologic and viral chemokines and HIV-1 envelope glycoproteins: differences in primary structural requirements for RANTES, MIP-1 alpha, and vMIP-II Binding.

Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has implicated the N-terminal extracellular domain (N-ter) and regions vicinal to the second extracellular loop (ECL2) in this activity. It was shown that residues in the N-ter are necessary for binding of the physiologic ligands, RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kaposi's sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for engagement by vMIP-II of CCR5 to its interaction with physiologic ligands. RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants of charged extracellular amino acids revealed that alteration of acidic residues in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II. Whereas mutation of residues in ECL2 of CCR5 dramatically reduced the binding of RANTES and MIP-1 alpha and their ability to induce signaling, interaction with vMIP-II was not altered by any mutation in the exoloops of the receptor. Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an affinity similar to that of the wild-type receptor. Engagement of CCR5 by vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal antibodies to the receptor, providing additional evidence for a distinct mechanism for viral chemokine binding. Analysis of the coreceptor activity of randomly generated mouse-human CCR5 chimeras implicated residues in ECL2 between H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to mutations in ECL2 provides a potential rationale to its inefficiency as an antagonist of CCR5 coreceptor activity. These findings suggest that the molecular anatomy of CCR5 binding plays a critical role in antagonism of coreceptor activity.

Conformational study of a highly specific CXCR4 inhibitor, T140, disclosing the close proximity of its intrinsic pharmacophores associated with strong anti-HIV activity.

We report the solution structure of T140, a truncated polyphemusin peptide analogue that efficiently inhibits infection of target cells by T-cell line-tropic strains of HIV-1 through its specific binding to a chemokine receptor, CXCR4. Nuclear magnetic resonance analysis and molecular dynamic calculations revealed that T140 has a rigidly structured conformation constituted by an antiparallel beta-sheet and a type II' beta-turn. A protuberance is formed on one side of the beta-sheet by the side-chain functional groups of the three amino acid residues (L-3-(2-naphthyl)alanine, Tyr5 and Arg14), each of which is indispensable for strong anti-HIV activity. These findings provide a rationale to dissect the structural basis for the ability of this compound to block the interaction between CXCR4 and envelope glycoproteins from T-tropic strains of HIV-1.

Gene expression profile of ARPE-19 during repair of the monolayer.

BACKGROUND: The purpose of this study was to identify the profile of gene expression during retinal pigment epithelial (RPE) wound repair. METHODS: ARPE-19 cells derived from a human RPE cell line were grown for 4 weeks and injured by creating multiple concentric wounds. Unwounded cultures served as controls. During the proliferative phase of wound repair, total RNA was extracted from control and wounded cultures, and a [32P]dATP-labeled cDNA probe was synthesized and hybridized to Atlas Arrays (Clontech, Palo Alto, Calif.) containing 588 cDNAs. The autoradiograms obtained were then analyzed using the Molecular Dynamics software program. Semiquantitative PCR was carried out to confirm up-regulation of four genes associated with wound repair. ELISA was performed to quantitate the secreted MCP-1. RESULTS: In wounded cultures prominent up-regulation (greater than fivefold) was seen for genes encoding DNA synthesis and DNA repair proteins. A greater than threefold increase was seen for genes encoding mitogen-activated protein kinase, CD44, MCP-1 (monocyte chemotactic protein), thymosin beta-10, and HDGF (hepatoma-derived growth factor), among others. Genes encoding tumor suppressors were downregulated three- to five-fold in the wounded compared with the unwounded cultures. Semiquantitative PCR confirmed up-regulation of transcripts for thymosin beta-10, HDGF, CD44, and MCP-1. ELISA showed a 20% increase in secreted MCP-1. CONCLUSIONS: Gene array analysis revealed a differentiation program that included increased expression of genes involved in wound repair (adhesion molecules, cytokines, signal transducers), along with increased MCP-1 secretion. The RPE may be an early participant in the inflammatory response that occurs with proliferative vitreoretinopathy.

Pharmacophore identification of a specific CXCR4 inhibitor, T140, leads to development of effective anti-HIV agents with very high selectivity indexes.

A polyphemusin peptide analogue, T22 ([Tyr(5,12), Lys7]-polyphemusin II), and its shortened potent analogues, T134 (des-[Cys(8,13), Tyr(9,12)]-[D-Lys10, Pro11, L-citrulline16]-T22 without C-terminal amide) and T140 [[L-3-(2-naphthyl)alanine3]-T134], strongly inhibit the T-cell line-tropic (T-tropic) HIV-1 infection through their specific binding to a chemokine receptor, CXCR4. T22 is an extremely basic peptide possessing five Arg and three Lys residues in the molecule. In our previous study, we found that there is an apparent correlation in the T22-related peptides between the number of total positive charges and anti-HIV activity or cytotoxicity. Here, we have conducted the conventional Ala-scanning study in order to define the anti-HIV activity pharmacophore of T140 (the strongest analogue among our compounds) and identified four indispensable amino acid residues (Arg2, Nal3, Tyr5, and Arg14). Based on this result, a series of L-citrulline (Cit)-substituted analogues of T140 with decreased net positive charges have been synthesized and evaluated in terms of anti-HIV activity and cytotoxicity. As a result, novel effective inhibitors, TC14003 and TC14005, possessing higher selectivity indexes (SIs, 50% cytotoxic concentration/50% effective concentration) than that of T140 have been developed.

Evolution of the human immunodeficiency virus type 1 envelope during infection reveals molecular corollaries of specificity for coreceptor utilization and AIDS pathogenesis.

The evolution of human immunodeficiency virus type 1 infection is associated with a shift in the target cell population, driven by variability in coreceptor utilization resulting from diversity in env. To elucidate the potential consequences of these changes for Env-mediated fusion over the course of AIDS, we examined the biological properties of serial viral isolates and determined coreceptor utilization by the products of env cloned from two individuals, followed from the detection of seroconversion throughout the course of their infection. One had a typical course, and the other had an accelerated progression. Early isolates were non-syncytium inducing, and the corresponding Env exclusively utilized CCR5, whereas Env from late phases of infection showed restricted utilization of CXCR4 in both patients. Env from subject SC24, who had a standard progression, demonstrated multitropism, manifested by utilization of CCR3, CXCR4, and CCR5 in the intervening period. In contrast, Env from patient SC51, who experienced early conversion to the syncytium-inducing phenotype, developed dualtropic coreceptor utilization of CCR5 and CXCR4. Genetic analysis of env from each isolate revealed that those with an X4 phenotype formed a distinct subcluster within each subject. Analysis of chimeras constructed from R5 and multispecific env from patient SC24 demonstrated that while the V3 domain played a dominant role in determining coreceptor utilization, sequences in the V4-V5 region also contributed to the latter phenotype. Immunoprecipitation experiments confirmed that the hybrid Env proteins were expressed at similar levels. These experiments demonstrate that progression from the R5 to X4 phenotype may occur through a multi- or dual-tropic intermediate and that multiple domains contribute to this process.

Comparison of the structure of vMIP-II with eotaxin-1, RANTES, and MCP-3 suggests a unique mechanism for CCR3 activation.

Herpesvirus-8 macrophage inflammatory protein-II (vMIP-II) binds a uniquely wide spectrum of chemokine receptors. We report the X-ray structure of vMIP-II determined to 2.1 A resolution. Like RANTES, vMIP-II crystallizes as a dimer and displays the conventional chemokine tertiary fold. We have compared the surface topology and electrostatic potential of vMIP-II to those of eotaxin-1, RANTES, and MCP-3, three CCR3 physiological agonists with known three-dimensional structures. Surface epitopes identified on RANTES to be involved in binding to CCR3 are mimicked on the eotaxin-1 and MCP-3 surface. However, the surface topology of vMIP-II in these regions is markedly different. The results presented here indicate that the structural basis for interaction with the chemokine receptor CCR3 by vMIP-II is different from that for the physiological agonists eotaxin-1, RANTES, and MCP-3. These differences on vMIP-II may be a consequence of its broad-range receptor recognition capabilities.

Identification of ENV determinants in V3 that influence the molecular anatomy of CCR5 utilization.

The V3 loop of the ENV glycoprotein exerts a dominant influence on the interaction of gp120 with coreceptors. Primary env genes cloned from sequential isolates from two seroconverters revealed Pro-->Ala conversion in the conserved GPG motif of the V3 crown in seven of 17 R5 ENV. ENV containing the GPG motif in the V3 crown had fusogenic activity with chimeric receptors containing either the N terminus or loops of CCR5, whereas those with the GAG variant utilized only the former. Site-directed mutagenesis of multiple primary and prototypic R5 env genes demonstrated that the GPG motif was necessary for dual utilization of the N terminus and body of CCR5 in both gain and loss-of-function experiments. All ENV containing the GPG V3 crown showed CCR5 binding in the presence of soluble CD4, whereas it was not detected with the GAG variants. Molecular dynamic simulations of a V3 peptide predicts that the Pro-->Ala substitution results in a conformational change with loss of the crown structure. These studies demonstrate that sequences in the third hypervariable region determine the specificity of coreceptor utilization for fusion, and that a conserved motif in the crown directly influences the molecular anatomy of the interaction between gp120 and CCR5.

Exaggerated response to endotoxin in mice lacking the Duffy antigen/receptor for chemokines (DARC).

Duffy antigen/receptor for chemokines (DARC) is a promiscuous receptor for chemokines that is required for Plasmodium vivax infection of erythroid cells. This receptor is expressed by subsets of endothelial, as well as erythroid cells. Selection for protection from malaria infection resulted in an erythroid-specific defect, suggesting that DARC may play a critical role in endothelial biology. Mice with targeted disruption of this gene were generated, and the function of DARC in inflammation was explored. RNA from spleens of homozygous mutant mice lacked DARC transcripts, which were abundant in wild-type (+/+) and heterozygote (+/-) mice. DARC(-/-) mice lacked developmental abnormalities and were healthy at 1 year. Whereas hematologic parameters were within normal ranges, erythrocytes from nullizygous mice lacked CXC and CC chemokine-binding activity. Challenge with lipopolysaccharide resulted in significantly increased inflammatory infiltrates in lung and liver of nullizygous mice. These results suggest that DARC modulates the intensity of inflammatory reactions as a sink for chemokines. (Blood. 2000;96:1681-1684)

Expression of the chemokine receptor CXCR2 in normal and neoplastic neuroendocrine cells.

BACKGROUND: Chemokines effect their proinflammatory and growth regulatory roles through interaction with serpentine receptors. One such receptor, CXCR2, binds multiple CXC chemokines, including interleukin 8, GRO-alpha, GRO-beta, GRO-gamma, and NAP-2. We have previously identified CXCR2 expression on myeloid cells, notably mature granulocytes, and projection neurons. OBJECTIVE: To determine the expression of CXCR2 by cells of the neuroendocrine system. DESIGN: Archival specimens from normal neuroendocrine tissues and their malignant counterparts were analyzed by immunohistochemistry with monoclonal antibodies specific for CXCR1 and CXCR2. RESULTS: Immunohistochemical analysis revealed high-level expression of CXCR2 by cells in the pituitary, adrenal medulla, pancreatic islets, thyroid C cells, scattered Kulchitsky cells in the bronchi, and counterpart neuroendocrine cells in the stomach, small bowel, colon, and appendix. Neuroendocrine neoplasms that demonstrated high-level CXCR2 expression included (1) primary carcinoids localized to the stomach, small bowel, colon, appendix, fallopian tube, ovary, and lung; (2) atypical carcinoids of the lung; (3) metastatic carcinoids; (4) pituitary adenomas; (5) pheochromocytomas; and (6) medullary carcinomas of the thyroid. Small cell lung carcinomas, large cell neuroendocrine carcinomas of the lung, small cell carcinoma of the cervix, Merkel cell carcinomas, neuroblastomas, and malignant melanomas lacked evidence of CXCR2 expression. CONCLUSIONS: The expression of CXCR2 by normal neuroendocrine cells and neoplastic counterparts that have retained phenotypic features of this differentiation program suggests that chemokines may play an important role in functions that are characteristic of this cell type. In addition, this raises the possibility that chemokines may modulate secretion of biologically active products of these cells and their neoplastic counterparts.

The solution structure of the anti-HIV chemokine vMIP-II.

We report the solution structure of the chemotactic cytokine (chemokine) vMIP-II. This protein has unique biological activities in that it blocks infection by several different human immunodeficiency virus type 1 (HIV-1) strains. This occurs because vMIP-II binds to a wide range of chemokine receptors, some of which are used by HJV to gain cell entry. vMIP-II is a monomeric protein, unlike most members of the chemokine family, and its structure consists of a disordered N-terminus, followed by a helical turn (Gln25-Leu27), which leads into the first strand of a three-stranded antiparallel beta-sheet (Ser29-Thr34; Gly42-Thr47; Gln52-Asp56). Following the sheet is a C-terminal alpha-helix, which extends from residue Asp60 until Gln68. The final five residues beyond the C-terminal helix (Pro70-Arg74) are in an extended conformation, but several of these C-terminal residues contact the first beta-strand. The structure of vMIP-II is compared to other chemokines that also block infection by HIV-1, and the structural basis of its lack of ability to form a dimer is discussed.

Localization of chromogranin, synaptophysin, serotonin, and CXCR2 in neuroendocrine cells of the minor vestibular glands: an immunohistochemical study.

Sections of vulvar tissue containing minor vestibular glands (15 sections from 14 women with vulvar vestibulitis syndrome and six control sections from five asymptomatic women) were immunohistochemically stained with antibodies against chromogranin, synaptophysin, serotonin, and CXCR2. The degree of inflammation in vestibular tissue from women with and without vulvar vestibulitis syndrome was not significantly different. All vulvar minor vestibular glands contained neuroendocrine cells that expressed chromogranin to some degree. Only one case failed to express synaptophysin. The number of cells expressing chromogranin and synaptophysin was the same regardless of the degree of inflammation. However, moderate to severe inflammation was associated with a statistically significant increase in the number of cells expressing serotonin (p < 0.001) and CXCR2 (p < 0.02). It is concluded that neuroendocrine cells are present within minor vestibular glands of the vulva. The number of cells expressing the inflammatory mediator serotonin and CXCR2, the shared interleukin-8 receptor, are upregulated with inflammation. Chronic inflammation is a normal finding in vestibular tissue and does not serve as a histologic marker for vulvar vestibulitis.

CCR5 HIV-1 coreceptor activity. Role of cooperativity between residues in N-terminal extracellular and intracellular domains.

Human (H-) CCR5 is the primary coreceptor for ENV-mediated fusion by R5 strains of human immunodeficiency virus type 1, whereas mouse (M-) CCR5 lacks this function. An array of 23 H/M-CCR5 hybrids containing increasing amounts of H-CCR5 extending from the N terminus generated by random chimeragenesis had a biphasic pattern of coreceptor activity with JRFL and 89.6, revealing active regions in the N-terminal extracellular domain (N-ED) and at the junction of cytoplasmic loop 3. The M-CCR5 mutant in which divergent residues were replaced with the corresponding H-CCR5 N-ED sequence (NyYTsE) gained coreceptor function in fusion but not infection experiments. A M-CCR5 double mutant with substitution of human sequences for divergent residues from the N-ED and cytoplasmic loop 3 had augmented coreceptor activity in fusion assays and gain of function in infection experiments. The SIV-251 ENV utilized H- and M-CCR5 and variants. Flow cytometric analysis of M-CCR5 mutants and bifunctional receptors composed of CD4 domains fused to M-CCR5 mutants excluded the possibility that differences in coreceptor activity resulted from variations in cell surface expression. These results demonstrate that the coreceptor activity of the H-CCR5 N-ED is modulated by intracellular residues, illustrating the complexity of CCR5 requirements for interaction with ENV.