Identification of novel allosteric modulators for the G-protein coupled US28 receptor of human cytomegalovirus.

The highly constitutively active G-protein coupled receptor US28 of human cytomegalovirus (HCMV) is an interesting pharmacological target because of its implication on viral dissemination, cardiovascular diseases and tumorigenesis. We found that dihydroisoquinolinone and tetrahydroisoquinoline scaffolds may be promising lead structures for novel US28 allosteric inverse agonists. These scaffolds were rapidly synthesized by radical carboamination reactions followed by non-radical transformations. Our novel US28 allosteric modulators provide valuable scaffolds for further ligand optimization and may be helpful chemical tools to investigate molecular mechanisms of US28 constitutive signaling and its role in pathogenesis.

Murine cytomegalovirus infection leads to increased levels of transplant arteriosclerosis in a murine aortic allograft model.

INTRODUCTION: Cytomegalovirus infection after heart transplantation is considered as risk factor for the development of transplant arteriosclerosis. Therefore, the aim of this study was to investigate the effect of murine cytomegalovirus (MCMV) as a single risk factor on transplant arteriosclerosis in an experimental aortic allograft model. METHODS: Major histocompatibility complex class I-mismatched aortas of C.B10-H2(b)/LilMcdJ donor were transplanted into BALB/c recipients, which were either mock-infected or infected with MCMV (strain Smith) on day 7 and harvested 37 days after transplantation. In one experimental group animals received a daily dose of everolimus to increase the viral load of recipients. Grafts were analyzed by histology, morphometry, and immunofluorescence. Intragraft cytokine mRNA production was analyzed by real-time polymerase chain reaction (PCR), and persistence of cytomegalovirus infection was confirmed by TaqMan PCR. RESULTS: After infection with MCMV, there was significantly more intimal proliferation when compared with uninfected controls (intimal proliferation 83.5%+/-9.6% [MCMV] vs. 43.9%+/-5.1% [MCMV]), indicating that MCMV plays a role in the development of transplant arteriosclerosis. Even after treatment with everolimus, MCMV infection pronounced significantly more intimal proliferation (intimal proliferation 52.5%+/-7.3% [MCMV] vs. 20.2%+/-1.7% [MCMV]). Intragraft mRNA expression showed significantly higher production of CD62E, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 after infection with MCMV. Cellular infiltration revealed significantly more CD4, CD8, and dendritic cells. We could also confirm the presence of MCMV for the duration of the experimental protocol by PCR within the spleen, liver, salivary glands, lung, and the aortic transplant. CONCLUSION: These data suggest that MCMV infection plays an important role in the development of transplant arteriosclerosis.

Rag2-/- gamma-chain-/- mice as hosts for human vessel transplantation and allogeneic human leukocyte reconstitution.

BACKGROUND: Rodent models are a very helpful tool to investigate immunological mechanisms in allograft rejection. The aim of this study was to compare two different immunodeficient recipients in a humanized mouse model of arterial xenotransplantation in terms of reconstitution of the human immune system and rejection of the arterial graft. METHODS: Side branches of human mammary artery were transplanted as infrarenal aortic interposition grafts into C.B-17-SCID beige and C57BL/6-Rag2(-/-)gammac(-/-) recipients. 7days after surgery mice were reconstituted with 5x10(7) human peripheral blood mononuclear cells (hu PBMCs) and 30days after reconstitution mice were sacrificed and histologic analysis was performed. Peripheral blood and splenocytes were investigated by FACS and ELISA analysis to ensure engraftment of human CD45(+) cells. RESULTS: Transplant arteriosclerosis developed in non-PBMC-reconstituted C.B-17-SCID beige mice (intimal proliferation: 36.31+/-4.37%), but significantly less in C57BL/6-Rag2(-/-) gammac(-/-) recipients (intimal proliferation: 12.26+/-5.21%). After reconstitution with 5x10(7) unfractionated human PBMCs both mouse strains showed intima proliferation 30days after reconstitution (C.B-17-SCID beige: 28.49+/-7.95% and C57BL/6-Rag2(-/-) gammac(-/-): 44.58+/-11.08%). Whereas only very few human CD45(+) cells were found in mouse blood and spleen of C.B-17-SCID beige mice, C57BL/6-Rag2(-/-) gammac(-/-) mice revealed a reliable reconstitution. In addition, levels of human IgG and IgM within the peripheral blood were markedly higher in C57BL/6-Rag2(-/-) gammac(-/-) recipients. CONCLUSION: In this study we can show, that the use of C57BL/6-Rag2(-/-) gammac(-/-) mice may be advantageous compared to C.B-17-SCID beige recipients in a humanized mouse model of vessel transplantation.

Influenza A virus proteins PB1 and NS1 are subject to functionally important phosphorylation by protein kinase C.

The virulence of influenza A viruses depends on the activity of the viral RNA polymerase complex and viral regulatory phosphoproteins. We identified that the protein kinase C (PKC) inhibitor Gö6976 had a post-entry anti-influenza viral effect, by using a polymerase activity-based reporter assay. This inhibitory effect was observed for influenza virus-infected cells as well as for cells transiently transfected with constructs for the RNA polymerase complex. Importantly, the in vitro analysis of viral protein phosphorylation identified PKCalpha as a kinase phosphorylating PB1 and NS1, but not PB2, PA or NP. Gö6976 was able to block PKC-specific phosphorylation in vitro. Thus, our data suggest that PKC contributes to the phosphorylation of influenza PB1 and NS1 proteins which appears to be functionally relevant for both viral RNA polymerase activity and efficient viral replication.