Replication of human cytomegalovirus is limited at the level of nucleocytoplasmic transport of viral capsids, a process that requires the disassembly of the nuclear lamina. Deletion of the protein kinase gene UL97 from the viral genome showed that the activity of pUL97 plays an important role for viral capsid egress. Here, we report that p32, a novel cellular interactor of the viral kinase pUL97, promotes the accumulation of pUL97 at the nuclear membrane by recruiting the p32-pUL97 complex to the lamin B receptor. Transfection of active pUL97, but not a catalytically inactive mutant, induced a redistribution of lamina components as demonstrated for recombinant lamin B receptor-green fluorescent protein and endogenous lamins A and C. Consistent with this, p32 itself and lamins were phosphorylated by pUL97. Importantly, overexpression of p32 in human cytomegalovirus-infected cells resulted in increased efficiency of viral replication and release of viral particles. Thus, it is highly suggestive that the cellular protein p32 recruits pUL97 to induce a dissolution of the nuclear lamina thereby facilitating the nuclear export of viral capsids.The transport of macromolecules in eukaryotic cells is subject to a strict compartmentalization into nucleus and cytoplasm. Exchange reactions between the two compartments are mediated through the nuclear pore complex, and thus the integrity of the nuclear envelope, composed of membrane and lamina constituents, is crucial for intracellular transport pathways. The nuclear lamina, underlining the inner nuclear membrane, contains a variable number of lamin isoforms (which are members of the intermediate filament family of cytoskeletal proteins) and forms a rigid, proteinaceous meshwork. During infection with herpesviruses, the nuclear lamina represents a barrier to the nucleocytoplasmic transport of viral capsids (1). Because of the large size of herpesviral capsids (ϳ120 nm), which does not allow their direct cytoplasmic release through nuclear pores, the structural destabilization of the nuclear lamina is an important prerequisite of virus budding. Lamina destabilization requires site-specific phosphorylation of lamins and lamin-binding membrane proteins. Phosphorylation leads to lamin depolymerization and may also permit their release from lamin-binding membrane proteins, including the lamin B receptor (LBR) 2 (2, 3). Protein kinase C and Cdc2 have been identified as kinases phosphorylating lamins during mitosis (3, 4). Interestingly, protein kinase C is involved in the dissolution of the nuclear lamina in cells infected with murine cytomegalovirus (5). In addition to cellular protein kinases, the activity of virus-encoded protein kinases has been suspected as an important additional critical factor for nuclear export of herpesviruses, such as herpes simplex virus type 1 (HSV-1) and pseudorabies virus (6, 7). Concerning the replication of human cytomegalovirus (HCMV), which is a major human pathogenic herpesvirus, little information has been published on destabilization of the n...

Background: U3-1565 is a fully human monoclonal antibody directed against human heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family of ligands that bind to and activate the EGFR and HER4. HB-EGF-mediated activation of its target receptors results in oncogenic signaling. Methods: To determine inhibition of basal EGFR phosphorylation, human tumor cell lines were treated with U3-1565 or IgG2 and pEGFR levels were analyzed by ELISA. Inhibition of in vivo angiogenesis was analyzed by implanting HUVEC-based spheroids under the skin of SCID mice, which were treated with U3-1565, bevacizumab or PBS and analyzed for human neovasculature. To determine in vivo efficacy, SCID mice bearing EFO27-CL58 ovarian cancer xenografts were treated weekly with U3-1565, Cisplatin or with a combination of U3-1565 and Cisplatin. To perform xenograft analysis tumor-bearing mice were treated with U3-1565, cetuximab and erlotinib and primary xenograft tissue was collected, lyzed and pEGFR and pERK levels were analyzed by Western blotting. To determine expression of HB-EGF and pEGFR in human tumor tissue, Immunohistochemistry (IHC) stainings of human carcinoma samples were performed. Results: Inhibition of basal pEGFR levels by U3-1565 in comparison to control IgG2 treatment was demonstrated in various cancer cell lines. U3-1565 showed dose-dependent inhibition of HB-EGF-stimulated pHER4 and pERK activation in vitro (data not shown). Using a HUVEC-based spheroid in vivo system, inhibition of human endothelial cell sprouting by U3-1565 was shown. In a HB-EGF overexpressing ovarian cancer xenograft model U3-1565 demonstrated in vivo tumor cell growth inhibition as a single agent. Combination treatment of U3-1565 with Cisplatin resulted in tumor regression during the treatment phase and prevented re-growth of xenograft tumors after treatment stop. Analysis of EFO27-CL58 tumor xenograft tissue demonstrated strong reduction of pEGFR and partially reduced pERK levels after U3-1565 in vivo treatments. IHC staining of fresh human tumor samples revealed an overlapping expression pattern of HB-EGF and pEGFR. Conclusions: Our results demonstrate that U3-1565 inhibits basal activation of EGFR activation in vitro and reduces neo-vasculature formation (angiogenesis) in vivo. In a tumor xenograft model U3-1565 demonstrated tumor cell growth arrest as a single agent or in combination with Cisplatin or erlotinib. Analysis of xenograft material after in vivo exposure to U3-1565 strongly suggest that U3-1565's anti-tumor activity is based on inhibition of EGFR activation and prevention of oncogenic MAPK signaling. These data together with the evidence that human HB-EGF and activated EGFR are coexpressed in human tumor samples provide preclinical rational to develop U3-1565 as a clinical mAb candidate to treat cancer in human patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2519. doi:1538-7445.AM2012-2519