No description available.

Viral appropriation of chemokine receptors is an effective way to prevent a host immune response against the invading virus. Many viruses, including poxviruses, subvert the host immune response by encoding several chemokine receptor homologues, capable of binding to and thereby precluding chemokines from activating their cognate cell surface receptors. All poxviruses employ strategies to modulate chemokine activity, including virus-encoded chemokine-binding proteins, receptor homologues and ligand mimics. The potential for the involvement of certain chemokine receptors in poxviral infection was suggested in studies utilizing the rabbit poxvirus, myxoma. Specifically, CCR5 was implicated in mediating cell target susceptibility to infection. Our data suggest virus-CCR5 interactions may lead to the selective activation of distinct signaling pathways that are advantageous for the virus. VACV, a member of the poxvirus family, produces two structurally distinct forms of virions, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), for which the immediate events following cell entry are ill-defined. Using confocal microscopy, we provided evidence that IMV and EEV enter both permissive and non-permissive cells, and that introduction of CCR5 into non-permissive cells – mouse fibroblasts and human PM1 T cells - renders them permissive for VACV replication. We showed that virus activation of CCR5 leads to the selective activation of distinct signaling pathways that are advantageous for the virus. We demonstrated that VACV infection in permissive cells is inhibited by siRNA knockdown of cell surface CCR5 expression and by the CCR5 antagonist, TAK-779. The importance of tyrosine phosphorylation of CCR5 was suggested by the observation that introduction of a CCR5 mutant, in which all the intracellular tyrosines are replaced by phenylalanines, effectively reduces VACV infection in permissive cells. Moreover, tyrosine-339 was implicated in CCR5 as the critical residue for mediating viral infection, since cells expressing CCR5.Y339F do not support viral replication. The cascade of events that leads to permissive phenotype of these cells includes phosphorylation activation of multiple signaling effectors: Jak-2, IRS-2, ERK1/ 2 and Grb2. These data were supported by findings that viral replication in permissive CCR5 expressing cells is blocked by Herbimycin A, and the Jak2 inhibitor, tyrophostin AG490, but not pertussis toxin. Viewed altogether, a critical role of post-entry events, specifically intracellular tyrosine phosphorylation events, was established in determining permissiveness of cells to VACV replication. Furthermore, evidence was provided that introduction of CCR5 in primary human T cells renders them permissive to VACV replication. Since permissive infection of T cells might represent a mechanism for VACV dissemination throughout the lymphatic system, we hypothesized that the absence of CCR5 may be protective against VACV infection in vivo. To test this hypothesis, wild-type and CCR5 null mice were challenged with VACV by intranasal inoculation. In time course studies we identified aggressive viral replication in the lungs and spleens of CCR5+/+ mice, with no evidence of infection in the CCR5-/- mice. Moreover, associated with VACV infection, we provided evidence for CD4+ and CD8+ T as well as CD11c+ and F4/80+ cell infiltration into the lungs of CCR5+/+ but not CCR5-/- mice, and showed that CCR5-expressing T cells harbor replicating virus. We showed that this CCR5-dependence is VACV-specific, since CCR5-/- mice were as susceptible to intranasal influenza (A/WSN/33) infection as CCR5+/+ mice. In a final series of experiments we provided evidence that adoptive transfer of CCR5+/+ bone marrow into CCR5-/- mice restored VACV permissiveness, with evidence of lung and spleen infection. Taken together, our data showed a critical and novel role for CCR5 in VACV infection and dissemination in vivo. Moreover, our confocal studies suggested a possible physical interaction between cellular proteins and the VACV in cytosole. Using mass spectrometry-based proteomics, glomulin was identified as a host cell protein that interacts with VACV. Knockdown of glomulin expression in human PM1.CCR5 T cells reduced VACV infection. We demonstrate that treatment of PM1.CCR5 T cells with a c-Met phosphorylation inhibitor led to a significant reduction in VACV infectivity. The data indicated that inhibition of c-Met phosphorylation, reduces the cytosolic availability of activated glomulin, thus leading to a decrease in VACV infectivity. These data identify glomulin as a permissivity factor for VACV infection, and as a potential therapeutic target for VACV.

Viral appropriation of chemokine receptors is an effective way to prevent a host immune response against the invading virus. Many viruses, including poxviruses, subvert the host immune response by encoding several chemokine receptor homologues, capable of binding to and thereby precluding chemokines from activating their cognate cell surface receptors. All poxviruses employ strategies to modulate chemokine activity, including virus-encoded chemokine-binding proteins, receptor homologues and ligand mimics. The potential for the involvement of certain chemokine receptors in poxviral infection was suggested in studies utilizing the rabbit poxvirus, myxoma. Specifically, CCR5 was implicated in mediating cell target susceptibility to infection. Our data suggest virus-CCR5 interactions may lead to the selective activation of distinct signaling pathways that are advantageous for the virus. VACV, a member of the poxvirus family, produces two structurally distinct forms of virions, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), for which the immediate events following cell entry are ill-defined. Using confocal microscopy, we provided evidence that IMV and EEV enter both permissive and non-permissive cells, and that introduction of CCR5 into non-permissive cells – mouse fibroblasts and human PM1 T cells - renders them permissive for VACV replication. We showed that virus activation of CCR5 leads to the selective activation of distinct signaling pathways that are advantageous for the virus. We demonstrated that VACV infection in permissive cells is inhibited by siRNA knockdown of cell surface CCR5 expression and by the CCR5 antagonist, TAK-779. The importance of tyrosine phosphorylation of CCR5 was suggested by the observation that introduction of a CCR5 mutant, in which all the intracellular tyrosines are replaced by phenylalanines, effectively reduces VACV infection in permissive cells. Moreover, tyrosine-339 was implicated in CCR5 as the critical residue for mediating viral infection, since cells expressing CCR5.Y339F do not support viral replication. The cascade of events that leads to permissive phenotype of these cells includes phosphorylation activation of multiple signaling effectors: Jak-2, IRS-2, ERK1/ 2 and Grb2. These data were supported by findings that viral replication in permissive CCR5 expressing cells is blocked by Herbimycin A, and the Jak2 inhibitor, tyrophostin AG490, but not pertussis toxin. Viewed altogether, a critical role of post-entry events, specifically intracellular tyrosine phosphorylation events, was established in determining permissiveness of cells to VACV replication. Furthermore, evidence was provided that introduction of CCR5 in primary human T cells renders them permissive to VACV replication. Since permissive infection of T cells might represent a mechanism for VACV dissemination throughout the lymphatic system, we hypothesized that the absence of CCR5 may be protective against VACV infection in vivo. To test this hypothesis, wild-type and CCR5 null mice were challenged with VACV by intranasal inoculation. In time course studies we identified aggressive viral replication in the lungs and spleens of CCR5+/+ mice, with no evidence of infection in the CCR5-/- mice. Moreover, associated with VACV infection, we provided evidence for CD4+ and CD8+ T as well as CD11c+ and F4/80+ cell infiltration into the lungs of CCR5+/+ but not CCR5-/- mice, and showed that CCR5-expressing T cells harbor replicating virus. We showed that this CCR5-dependence is VACV-specific, since CCR5-/- mice were as susceptible to intranasal influenza (A/WSN/33) infection as CCR5+/+ mice. In a final series of experiments we provided evidence that adoptive transfer of CCR5+/+ bone marrow into CCR5-/- mice restored VACV permissiveness, with evidence of lung and spleen infection. Taken together, our data showed a critical and novel role for CCR5 in VACV infection and dissemination in vivo. Moreover, our confocal studies suggested a possible physical interaction between cellular proteins and the VACV in cytosole. Using mass spectrometry-based proteomics, glomulin was identified as a host cell protein that interacts with VACV. Knockdown of glomulin expression in human PM1.CCR5 T cells reduced VACV infection. We demonstrate that treatment of PM1.CCR5 T cells with a c-Met phosphorylation inhibitor led to a significant reduction in VACV infectivity. The data indicated that inhibition of c-Met phosphorylation, reduces the cytosolic availability of activated glomulin, thus leading to a decrease in VACV infectivity. These data identify glomulin as a permissivity factor for VACV infection, and as a potential therapeutic target for VACV.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Metastasis is a multi-step process during which cancer cells disseminate from the primary tumour and establish secondary tumours in distant sites. The mechanisms for organ-specific metastasis are poorly understood, although recent findings suggest the role of a number of chemokine receptors on various cancer cells such as breast CXCR4 as well as CCR7, are a protein-coupled chemokine receptor (apCR) that have proven to be of considerable biological significance, since their expression has been shown on various malignant breast cancer cell lines, tumours and metastases. In this study the expression and function of CXCR4 and CCR7 was examined in a range of human breast cancer cell lines covering a spectrum of malignant and non-malignant phenotypes. The data revealed that while surface levels of CXCR4 and CCR7 were uniform across the entire panel of breast cancer cell lines, only highly invasive cells, metastatic in immunocompromised mice, expressed functional chemokine receptors. Moreover, multiple signalling pathways downstream of a proteins in the highly invasive cells were found to be activated however chemokine treatment failed to activate any of the downstream kinase cascades examined in non-invasive cell lines. For the first time, to the best of our knowledge, chemokine receptor function was demonstrated to be subject to complex and tightly-controlled regulation in epithelial breast cancer cells via differential a protein-receptor complex formation and that this regulation might significantly contribute to the transition from non-metastatic to malignant tumours. Finally, the role of CXCR4 and CCR7 during breast cancer metastasis was verified using a humanised breast cancer metastasis mouse modeL By modulating the expression of chemokine receptors using siRNA-mediated knockdown, metastasis of breast cancer cells to the lungs of SCID mice was dramatically inhibited. In summary, the data point to distinct molecular mechanisms of chemokine receptor activation used by transformed invasive breast epithelial cells which leads to the metastatic spread of these cancer cells to distant sites. Improved understanding of the role of chemokine receptor/ligand interaction in metastasis may lead to novel approaches in the treatment and management of breast cancer as well as other solid tumour malignancies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1289342 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2007

Erlangen, Nürnberg, Universiẗat, Diss., 2006. / Enth. 1 Sonderabdr. aus: International journal of cancer ; 116. 2005. - Beitr. teilw. dt., teilw. engl.

Universiẗat, Diss.--Hannover, 1999.

Stuttgart, Univ., Diss., 2001.

Universiẗat, Diss., 2005--Würzburg. / Erscheinungsjahr an der Haupttitelstelle: 2004.

CXCR4 is a chemokine receptor that has been linked to several disease related pathways including: HIV-1 proliferation, autoimmune disorders, inflammatory disease and cancer metastasis. The interaction of the C-X-C chemokine receptor type 4 (CXCR4) with C-X-C chemokine ligand 12 (CXCL12) plays a key role in triggering these disease related pathways. Various antagonists for these receptors have been synthesized and tested, but many are not useful clinically either because of toxicity or poor pharmacokinetics. Some of the most extensive CXCR4 antagonist libraries stem from a class of compounds, p-xylyl-enediamines, which all feature a benzene ring as the core of the compound. This work focuses on the design and synthesis of a new class of compounds that show potential as CXCR4 antagonists by using heterocyclic aromatic rings (2,6-pyridine, 2,5-furan, 2,5-pyrazine and 3,4-thiophene) as the core of the scaffold. After synthesis, these analogues were probed through a variety of assays and techniques by our collaborators in the Shim lab at Emory University including: preliminary binding assays, Matrigel invasion assays, carrageenan mouse paw edema tests, and in silico analysis. In silico analysis also probed 2,5-thiophene-based analogues previously synthesized. This work has produced the beginnings of a new library of CXCR4 antagonists and has identified fifteen hit compounds that are promising leads for further testing and modification.

No description available.