Lysophosphatidic acid (LPA) is a serum-borne phospholipid with hormone and growth factor-like properties. LPA has been shown to modulate tumor cell invasion and malignant cell growth. Here, we report that two human pancreatic carcinoma cell lines, PANC-1 and BxPC-3, express functionally active LPA receptors coupled to pertussis toxin-sensitive Gi/o-proteins. In contrast to other cell types, LPA does not act as a mitogen, but is an efficacious stimulator of cell migration of these tumor cells. LPA-induced chemotaxis is markedly dependent on activation of PTX-sensitive heterotrimeric G-proteins, on activation of the small GTPases Ras, Rac and RhoA, and on GTPase-dependent activation of ERK. LPA-induced ERK activation results in a transient translocation of the phosphorylated ERK to newly forming focal contact sites at the leading edge of the migrating cells. Inhibition of ERK activation and its subsequent translocation impaired LPA-induced chemotaxis and LPA-induced actin reorganization. Thus, pancreatic tumor cell migration in response to LPA is essentially controlled by activation of a Gi/o-ERK pathway and requires the LPA-induced activation of Ras, Rac1 and RhoA.

The tegument protein ppUL82 (pp71) of human cytomegalovirus (HCMV) has previously been shown to activate the immediate-early transcription of HCMV and to enhance the infectivity of viral DNA. This is concordant with its localization adjacent to promyelocytic leukemia oncogenic domains (PODs) immediately after infection. In a yeast two-hybrid screen, we identified the tegument protein ppUL35 as an interacting partner of ppUL82. The interaction could be confirmed in transfected and infected cells. The domain responsible for interaction was narrowed down to amino acids 447 to 516 within ppUL35, thus allowing both forms of ppUL35 to interact with ppUL82. Immunofluorescence experiments showed a relocalization of ppUL35 from a diffuse nuclear pattern when expressed alone to PODs when expressed together with ppUL82. In accordance with this observation and the role of ppUL82 as a transactivator, we observed a cooperative activation of the HCMV major immediate-early enhancer but not of heterologous viral enhancer elements. These results suggest an important role for this interaction in the stimulation of viral immediate-early gene expression and viral infection.

No abstract

The tegument proteins ppUL35 and ppUL82 (pp71) of human cytomegalovirus (HCMV) physically interact and cooperatively activate the major immediate-early transcription. While an HCMV mutant lacking UL82 displayed a multiplicity of infection (MOI)-dependent growth, the biological significance of ppUL35 has not been addressed so far. We generated a mutant virus with a deletion of the UL35 gene. Using an MOI of 0.1, the progeny virus yield of this mutant was reduced by a factor of 1,000; however, when infected at a low MOI (0.01), the gene was essential. Characterization of the replication cycle showed that the mutant virus had two defects: when virus inoculum was standardized by the amount of viral DNA, a reduced immediate-early gene expression was observed, leading to a strongly delayed expression of lytic genes. A second defect was apparent in the virus assembly, as fewer enveloped particles and no dense bodies were present in cells infected with the mutant virus. However, the particles produced by wild-type and mutant viruses did not show significant ultrastructural differences. These results suggest an important role for ppUL35 in immediate-early gene expression and virus assembly.Human cytomegalovirus (HCMV) is an exclusively human pathogen causing considerable morbidity and mortality in individuals with immature or compromised immune systems (34). HCMV is classified as a herpesvirus belonging to the Betaherpesvirinae subfamily. The genome of HCMV, comprising 235 kbp, is one of the largest genomes of animal viruses, encoding 165 to 190 genes, depending on the method of prediction (12, 30). A substantial part of the coding potential is devoted to virion proteins.The virion of HCMV is composed of at least 40 proteins and has a tripartite structure, consisting of an envelope, a tegument, and a capsid (16, 43). The tegument is a proteinaceous structure, interspersed between the envelope and the capsid, and is characteristic of herpesviruses. Whereas the envelope and capsid are relatively well defined, characterization of the tegument has lagged behind. Long regarded as an unstructured repository for proteins, the tegument is now thought to have an ordered architecture and multiple functions (27).At present, about 20 proteins have been localized to the tegument of HCMV, and they have been implicated in virion formation (3, 28), virus transport (41), immunomodulation (8, 10, 17) and transactivation. The first tegument protein which was shown to be involved in the activation of the major immediate-early enhancer-promoter (MIEP) of HCMV was ppUL82 (pp71) (23). Later, another regulatory protein, pUL69, was found in the tegument and was shown to transactivate the MIEP cooperatively with ppUL82 (47, 48). These observations were reproduced and extended, with the HCMV TRS1/IRS1 and UL35 genes added to the list (24, 36). Recently, we were able to show a physical interaction between two of these cooperatively trans-activating proteins, ppUL82 and ppUL35 (38). The UL35 gene encodes two proteins, of which the shorter form, ppUL...

Our data suggest that HCMV directly activates the PDGF system, which could promote atherogenesis and restenosis by activation of smooth muscle cell proliferation and neointima formation.