Jennifer Vomaske

Human cytomegalovirus chemokine receptor US28-induced smooth muscle cell migration is mediated by focal adhesion kinase and Src

The human cytomegalovirus-encoded chemokine receptor US28 induces arterial smooth muscle cell (SMC) migration; however, the underlying mechanisms involved in this process are unclear. We have previously shown that US28-mediated SMC migration occurs by a ligand-dependent process that is sensitive to protein-tyrosine kinase inhibitors. We demonstrate here that US28 signals through the non-receptor protein-tyrosine kinases Src and focal adhesion kinase (FAK) and that this activity is necessary for US28-mediated SMC migration. In the presence of RANTES (regulated on activation normal T cell expressed and secreted), US28 stimulates the production of a FAK·Src kinase complex. Interestingly, Src co-immunoprecipitates with US28 in a ligand-dependent manner. This association occurs earlier than the formation of the FAK·Src kinase complex, suggesting that US28 activates Src before FAK. US28 binding to RANTES also promotes the formation of a Grb2·FAK complex, which is sensitive to treatment with the Src inhibitor PP2, further highlighting the critical role of Src in US28 activation of FAK. Human cytomegalovirus US28-mediated SMC migration is inhibited by treatment with PP2 and through the expression of either of two dominant negative inhibitors of FAK (F397Y and NH2-terminal amino acids 1–401). These findings demonstrate that activation of FAK and Src plays a critical role in US28-mediated signaling and SMC migration.

Chikungunya Virus Infection Results in Higher and Persistent Viral Replication in Aged Rhesus Macaques Due to Defects in Anti-Viral Immunity

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne Alphavirus that causes a clinical disease involving fever, myalgia, nausea and rash. The distinguishing feature of CHIKV infection is the severe debilitating poly-arthralgia that may persist for several months after viral clearance. Since its re-emergence in 2004, CHIKV has spread from the Indian Ocean region to new locations including metropolitan Europe, Japan, and even the United States. The risk of importing CHIKV to new areas of the world is increasing due to high levels of viremia in infected individuals as well as the recent adaptation of the virus to the mosquito species Aedes albopictus. CHIKV re-emergence is also associated with new clinical complications including severe morbidity and, for the first time, mortality. In this study, we characterized disease progression and host immune responses in adult and aged Rhesus macaques infected with either the recent CHIKV outbreak strain La Reunion (LR) or the West African strain 37997. Our results indicate that following intravenous infection and regardless of the virus used, Rhesus macaques become viremic between days 1–5 post infection. While adult animals are able to control viral infection, aged animals show persistent virus in the spleen. Virus-specific T cell responses in the aged animals were reduced compared to adult animals and the B cell responses were also delayed and reduced in aged animals. Interestingly, regardless of age, T cell and antibody responses were more robust in animals infected with LR compared to 37997 CHIKV strain. Taken together these data suggest that the reduced immune responses in the aged animals promotes long-term virus persistence in CHIKV-LR infected Rhesus monkeys.

Mouse Cytomegalovirus M33 Is Necessary and Sufficient in Virus-Induced Vascular Smooth Muscle Cell Migration

ABSTRACT Mouse cytomegalovirus (MCMV) encodes two potential seven-transmembrane-spanning proteins with homologies to cellular chemokine receptors, M33 and M78. While these virus-encoded chemokine receptors are necessary for the in vivo pathogenesis of MCMV, the function of these proteins is unknown. Since vascular smooth muscle cell (SMC) migration is of critical importance for the development of atherosclerosis and other vascular diseases, the ability of M33 to promote SMC motility was assessed. Similar to human CMV, MCMV induced the migration of mouse aortic SMCs but not mouse fibroblasts. To demonstrate whether M33 was required for MCMV-induced SMC migration, we employed interfering-RNA technology to specifically knock down M33 expression in the context of viral infection. The knockdown of M33 resulted in the specific reduction of M33 protein expression and ablation of MCMV-mediated SMC migration but failed to reduce viral growth in cultured cells. Adenovirus vector expression of M33 was sufficient to promote SMC migration, which was enhanced in the presence of recombinant mouse RANTES (mRANTES). In addition, M33 promoted the activation of Rac1 and extracellular signal-related kinase 1/2 upon stimulation with mRANTES. These findings demonstrate that mRANTES is a ligand for this chemokine receptor and that the activation of M33 occurs in a ligand-dependent manner. Thus, M33 is a functional homologue of US28 that is required for MCMV-induced vascular SMC migration.

Differential Ligand Binding to a Human Cytomegalovirus Chemokine Receptor Determines Cell Type-Specific Motility

While most chemokine receptors fail to cross the chemokine class boundary with respect to the ligands that they bind, the human cytomegalovirus (HCMV)-encoded chemokine receptor US28 binds multiple CC-chemokines and the CX3C-chemokine Fractalkine. US28 binding to CC-chemokines is both necessary and sufficient to induce vascular smooth muscle cell (SMC) migration in response to HCMV infection. However, the function of Fractalkine binding to US28 is unknown. In this report, we demonstrate that Fractalkine binding to US28 not only induces migration of macrophages but also acts to inhibit RANTES-mediated SMC migration. Similarly, RANTES inhibits Fractalkine-mediated US28 migration in macrophages. While US28 binding of both RANTES and Fractalkine activate FAK and ERK-1/2, RANTES signals through Gα12 and Fractalkine through Gαq. These findings represent the first example of differential chemotactic signaling via a multiple chemokine family binding receptor that results in migration of two different cell types. Additionally, the demonstration that US28-mediated chemotaxis is both ligand-specific and cell type–specific has important implications in the role of US28 in HCMV pathogenesis.

Human Cytomegalovirus US28: A Functionally Selective Chemokine Binding Receptor

Chemokines are small cytokines that are part of a large family of molecules that bind to G-protein coupled receptors, which, as a family, are the most widely targeted group of molecules in the treatment of disease. Chemokines are critical for recruiting and activating the cells of the immune system during inflammation especially during viral infections. However, a number of viruses including the large herpes virus human cytomegalovirus (HCMV) encode mechanisms to impede the effects of chemokines or has gained the ability to use these molecules to its own advantage. The Human Cytomegalovirus (HCMV)-encoded chemokine receptor US28 is the best characterized of the four unique chemokine receptor-like molecules found in the HCMV genome. US28 has been studied as an important virulence factor for HCMV-mediated vascular disease and, more recently, in models of HCMV-associated malignancy. US28 is a rare multi-chemokine family binding receptor with the ability to bind ligands from two distinct chemokine classes. Ligand binding to US28 activates cell-type and ligand-specific signaling pathways leading to cellular migration, which is an important example of receptor functional selectivity. Additionally, US28 has been demonstrated to constitutively activate phospholipase C (PLC) and NF-kB signaling pathways. Understanding the structure/function relationships between US28, its ligands and intracellular signaling molecules will provide essential clues for effective pharmacological targeting of this multifunctional chemokine receptor.

Cytomegalovirus CC Chemokine Promotes Immune Cell Migration

ABSTRACT Cytomegaloviruses manipulate the host chemokine/receptor axis by altering cellular chemokine expression and by encoding multiple chemokines and chemokine receptors. Similar to human cytomegalovirus (HCMV), rat cytomegalovirus (RCMV) encodes multiple CC chemokine-analogous proteins, including r129 (HCMV UL128 homologue) and r131 (HCMV UL130 and MCMV m129/130 homologues). Although these proteins play a role in CMV entry, their function as chemotactic cytokines remains unknown. In the current study, we examined the role of the RCMV chemokine r129 in promoting cellular migration and in accelerating transplant vascular sclerosis (TVS) in our rat heart transplant model. We determined that r129 protein is released into culture supernatants of infected cells and is expressed with late viral gene kinetics during RCMV infection and highly expressed in heart and salivary glands during in vivo rat infections. Using the recombinant r129 protein, we demonstrated that r129 induces migration of lymphocytes isolated from rat peripheral blood, spleen, and bone marrow and from a rat macrophage cell line. Using antibody-mediated cell sorting of rat splenocytes, we demonstrated that r129 induces migration of naïve/central memory CD4+ T cells. Through ligand-binding assays, we determined that r129 binds rat CC chemokine receptors CCR3, CCR4, CCR5, and CCR7. In addition, mutational analyses identified functional domains of r129 resulting in recombinant proteins that fail to induce migration (r129-ΔNT and -C31A) or alter the chemotactic ability of the chemokine (r129-F43A). Two of the mutant proteins (r129-C31A and -ΔNT) also act as dominant negatives by inhibiting migration induced by wild-type r129. Furthermore, infection of rat heart transplant recipients with RCMV containing the r129-ΔNT mutation prevented CMV-induced acceleration of TVS. Together our findings indicate that RCMV r129 is highly chemotactic, which has important implications during RCMV infection and reactivation and acceleration of TVS.

HCMV pUS28 Initiates Pro-Migratory Signaling via Activation of Pyk2 Kinase

BackgroundHuman Cytomegalovirus (HCMV) has been implicated in the acceleration of vascular disease and chronic allograft rejection. Recently, the virus has been associated with glioblastoma and other tumors. We have previously shown that the HCMV-encoded chemokine receptor pUS28 mediates smooth muscle cell (SMC) and macrophage motility and this activity has been implicated in the acceleration of vascular disease. pUS28 induced SMC migration involves the activation of the protein tyrosine kinases (PTKs) Src and Focal adhesion kinase as well as the small GTPase RhoA. The PTK Pyk2 has been shown to play a role in cellular migration and formation of cancer, especially glioblastoma. The role of Pyk2 in pUS28 signaling and migration are unknown.MethodsIn the current study, we examined the involvement of the PTK Pyk2 in pUS28-induced cellular motility. We utilized in vitro migration of SMC to determine the requirements for Pyk2 in pUS28 pro-migratory signaling. We performed biochemical analysis of Pyk2 signaling in response to pUS28 activation to determine the mechanisms involved in pUS28 migration. We performed mass spectrometric analysis of Pyk2 complexes to identify novel Pyk2 binding partners.ResultsExpression of a mutant form of Pyk2 lacking the autophosphorylation site (Tyr-402) blocks pUS28-mediated SMC migration in response to CCL5, while the kinase-inactive Pyk2 mutant failed to elicit the same negative effect on migration. pUS28 stimulation with CCL5 results in ligand-dependent and calcium-dependent phosphorylation of Pyk2 Tyr-402 and induced the formation of an active Pyk2 kinase complex containing several novel Pyk2 binding proteins. Expression of the autophosphorylation null mutant Pyk2 F402Y did not abrogate the formation of an active Pyk2 kinase complex, but instead prevented pUS28-mediated activation of RhoA. Additionally, pUS28 activated RhoA via Pyk2 in the U373 glioblastoma cells. Interestingly, the Pyk2 kinase complex in U373 contained several proteins known to participate in glioma tumorigenesis.ConclusionsThese findings represent the first demonstration that pUS28 signals through Pyk2 and that this PTK participates in pUS28-mediated cellular motility via activation of RhoA. Furthermore, these results provide a potential mechanistic link between HCMV-pUS28 and glioblastoma cell activation.

Rat cytomegalovirus infection depletes MHC II in bone marrow derived dendritic cells.

While cytomegalovirus (CMV) infects and replicates in a multitude of cell types, the ability of the virus to replicate in antigen presenting cells (APCs) is believed to play a critical role in the viral dissemination and latency. CMV infection of APCs and manipulation of their function are important areas of investigation. CMV down regulation of MHC II is reportedly mediated by the HCMV proteins US2, US3, UL83, UL111a (vIL10) or through the induction of cellular IL10. In this study, we demonstrate that rat CMV (RCMV) significantly reduces MHC II expression neither by mechanisms that do not involve orthologues of the known HCMV genes nor by an increase in cellular IL10. Rat bone marrow derived dendritic cells (BMDC) were highly susceptible to infection with RCMV and a recombinant RCMV expressing eGFP. RCMV infection of BMDCs depleted both surface and intracellular MHC II to nearly undetectable levels as well as reduced surface expression of MHC I. The effect on MHC II only occurred in the infected GFP positive cells and is mediated by an immediate early or early viral gene product. Furthermore, treatment of uninfected immature DCs with virus-free conditioned supernatants from infected cells failed to down regulate MHC II. RCMV depletion of MHC II was sensitive to treatment with lysosomal inhibitors but not proteasomal inhibitors suggesting that the mechanism of RCMV-mediated down regulation of MHC II occurs through endocytic degradation. Since RCMV does not encode homologues of US2, US3, UL83 or UL111a, these data indicate a novel mechanism for RCMV depletion of MHC II.

KSHV infection of endothelial cells manipulates CXCR7-mediated signaling: implications for Kaposi’s Sarcoma progression and intervention

CXCR7 was recently characterized as an alternative receptor for the chemokine CXCL12/SDF-1, previously thought to bind and signal exclusively through CXCR4. We recently identified CXCR7 as a key cellular factor in the endothelial cell (EC) dysfunction associated with KSHV infection. CXCL12 signaling is critically associated with tumor growth, angiogenesis and metastasis in several diverse tumors and is one of the most studied chemokine/chemokine receptor interactions in cancer systems. The tumorigenic activity of the CXCL12 signaling axis offers an attractive target for therapeutic intervention in multiple cancers including Kaposi’ sS arcoma (KS). However, most of the research to date was based on the assumption that CXCR4 was the sole CXCL12 receptor, and thus focused on the development of CXCR4-targeted treatments. CXCR4 participates in important homeostatic functions including hematopoiesis and mucosal immunity, while CXCR7 is rarely expressed in normal adult cells. As a result, CXCR7 may be a more specific chemotherapeutic target for tumor cells and tumor-associated vasculature with fewer adverse effects than treatments targeting CXCR4. CXCR7 is poorly studied throughout the cancer literature and although CXCR7 expression has been found in tumor-associated vasculature, no studies comprehensively examine the biology of CXCR7 in EC and its implications for tumor biology. We seek to define th er ole of CXCR7-mediated CXCL12 signaling in EC biology, and in the context of KSHV infection, in order to determine potential contributions of CXCR7 signaling to KSHV-mediated EC transformation and KS tumorigenesis. We demonstrate that CXCR7 is strongly expressed on LANA+ spindle cells in KS biopsy tissue at all stages of tumor progression. We further demonstrate that CXCR7 induction by KSHV in vitro is specific to lymphatic EC lineages and occurs coincident with the acquisition of spindle morphology. Detailed examination of CXCR7 functions in EC biology reveals multiple roles for CXCR7 that could impact KS tumorigenesis, including effects on cellular proliferation, junctional integrity, cell survival and metastatic capacity. Specifically, we determine that CXCR7 expression results in a loss of PECAM/CD31 expression, perturbing the formation and maintenance of EC monolayers. Moreover, CXCR7+ EC display significant SDF-1 dependent hypermotility, as measured via Electrical Cell-Substrate Impedence Sensing (ECIS). We also demonstrate that SDF-1 signaling through CXCR7 expression is enhanced in EC undergoing anchorage-deprivation, affecting EC cell survival and invasion into SDF-1 rich niches. Taken together, these results demonstrate that CXCR7 is a novel KSHV-induced oncogene with the capacity to influence multiple aspects of KS pathogenesis including tumor growth, seeding and metastasis.