Inactivation of p120 catenin in mice disturbs intrahepatic bile duct development and aggravates liver carcinogenesis.

p120 catenin (p120ctn) is required for the stability of classic cadherins at the cell surface and is thought to play a central role in modulating cell-cell adhesion. Cytoplasmic p120ctn promotes cell motility, and probably other activities, by modulating the activities of RhoA, Rac and Cdc42. E-cadherin is expressed in periportal but not in perivenous hepatocytes. In contrast, all hepatocytes of normal mouse liver express N-cadherin. Cholangiocytes express exclusively E-cadherin. Mice with p120ctn ablation in hepatocytes and cholangiocytes (p120LiKO mice) were generated by Cre-loxP technology. Livers were examined by histological, immunohistochemical, ultrastructural and serum analysis to determine the effect of the p120ctn ablation on liver structure and function. Mouse hepatocyte differentiation and homeostasis were not impaired. However, hepatoblasts differentiated abnormally into hybrid hepato-biliary cells, ductal plate structures were irregular in p120LiKO newborns, and further development of intrahepatic bile ducts was severely impaired. In adults, enrichment of ductular structures was accompanied by portal inflammation and fibrosis. p120LiKO mice did not spontaneously develop hepatocellular carcinoma but initiation of hepatocarcinogenesis by diethylnitrosamine was accelerated. In summary: p120ctn has a critical role in biliary differentiation and is a potent suppressor of liver tumor growth.

Pseudorabies virus US3 leads to filamentous actin disassembly and contributes to viral genome delivery to the nucleus.

The conserved alphaherpesvirus US3 tegument protein induces rearrangements of the actin cytoskeleton, consisting of protrusion formation and stress fiber breakdown. Although US3 does not affect levels of total actin protein, it remains unclear whether US3 modulates the total levels of filamentous (F) actin. In this report, we show that the pseudorabies virus (PRV) US3 protein, via its kinase activity, leads to disassembly of F-actin in porcine ST cells. F-actin disassembly has been reported before to contribute to host cell entry of HIV. In line with this, in the current study, we report that US3 has a previously uncharacterized role in viral genome delivery to the nucleus, since quantitative polymerase chain reaction (qPCR) assays on nuclear fractions demonstrated a reduced nuclear delivery of US3null PRV compared to wild type PRV genomes. Treatment of cells with the actin depolymerizing drug cytochalasin D enhanced virus genome delivery to the nucleus, particularly of US3null PRV, supporting a role for F-actin disassembly during certain aspects of viral entry. In conclusion, the US3 kinase of PRV leads to F-actin depolymerization, and US3 and F-actin disassembly contribute to viral genome delivery to the nucleus.

Rho'ing in and out of cells: viral interactions with Rho GTPase signaling.

Rho GTPases are key regulators of actin and microtubule dynamics and organization. Increasing evidence shows that many viruses have evolved diverse interactions with Rho GTPase signaling and manipulate them for their own benefit. In this review, we discuss how Rho GTPase signaling interferes with many steps in the viral replication cycle, especially entry, replication, and spread. Seen the diversity between viruses, it is not surprising that there is considerable variability in viral interactions with Rho GTPase signaling. However, several largely common effects on Rho GTPases and actin architecture and microtubule dynamics have been reported. For some of these processes, the molecular signaling and biological consequences are well documented while for others we just begin to understand them. A better knowledge and identification of common threads in the different viral interactions with Rho GTPase signaling and their ultimate consequences for virus and host may pave the way toward the development of new antiviral drugs that may target different viruses.

Alphaherpesviral US3 kinase induces cofilin dephosphorylation to reorganize the actin cytoskeleton.

The conserved alphaherpesviral serine/threonine kinase US3 causes dramatic actin rearrangements, associated with increased viral spread. Here, we show that US3 of pseudorabies virus (PRV) leads to activation (dephosphorylation) of the central actin regulator cofilin. A mutation that impairs US3 kinase activity and the group I p21-activated kinase inhibitor IPA-3 inhibited US3-mediated cofilin activation. Additionally, expression of phosphomimetic S3D cofilin significantly suppressed the ability of US3 to cause cell projections and cell rounding. In conclusion, the US3 kinase of PRV leads to activation (dephosphorylation) of cofilin, and cofilin contributes to US3-mediated actin rearrangements.

Actin' up: herpesvirus interactions with Rho GTPase signaling.

Herpesviruses constitute a very large and diverse family of DNA viruses, which can generally be subdivided in alpha-, beta- and gammaherpesvirus subfamilies. Increasing evidence indicates that many herpesviruses interact with cytoskeleton-regulating Rho GTPase signaling pathways during different phases of their replication cycle. Because of the large differences between herpesvirus subfamilies, the molecular mechanisms and specific consequences of individual herpesvirus interactions with Rho GTPase signaling may differ. However, some evolutionary distinct but similar general effects on Rho GTPase signaling and the cytoskeleton have also been reported. Examples of these include Rho GTPase-mediated nuclear translocation of virus during entry in a host cell and Rho GTPase-mediated viral cell-to-cell spread during later stages of infection. The current review gives an overview of both general and individual interactions of herpesviruses with Rho GTPase signaling.

Effect of the US3 protein of bovine herpesvirus 5 on the actin cytoskeleton and apoptosis.

The US3 protein is a unique protein kinase only present in the Alphaherpesvirinae subfamily of the herpesviruses. Studies performed with several alphaherpesviruses demonstrated that the US3 protein is involved in cytoskeleton modifications during viral infection and displays anti-apoptotic activity. However, the US3 protein of BoHV-5 has not been studied up to now. As reported for other alphaherpesviruses, our results showed that BoHV-5 US3 confers resistance against apoptosis and induces cytoskeletal reorganization leading to cell rounding, actin stress fiber breakdown and cell projections that interconnect cells. The expression of a kinase-dead version of BoHV-5 US3 showed that the anti-apoptotic activity and the induction of cell projections are kinase-dependent whereas kinase activity is not absolutely required for actin stress fiber breakdown. Besides, the kinase-dead version of US3, but not the wild type protein, was found excluded from the nucleus. These results constitute the first report on the BoHV-5 US3 functions, and highlight that there are functional differences and similarities among US3 proteins of different alphaherpesviruses.

Viral serine/threonine protein kinases.

Phosphorylation represents one the most abundant and important posttranslational modifications of proteins, including viral proteins. Virus-encoded serine/threonine protein kinases appear to be a feature that is unique to large DNA viruses. Although the importance of these kinases for virus replication in cell culture is variable, they invariably play important roles in virus virulence. The current review provides an overview of the different viral serine/threonine protein kinases of several large DNA viruses and discusses their function, importance, and potential as antiviral drug targets.

Alphaherpesvirus use and misuse of cellular actin and cholesterol.

Two major structural elements of a cell are the cytoskeleton and the lipid membranes. Actin and cholesterol are key components of the cytoskeleton and membranes, respectively, and are involved in a plethora of different cellular processes. This review summarizes and discusses the interaction of alphaherpesviruses with actin and cholesterol during different stages of the replication cycle: virus entry, replication and assembly in the nucleus, and virus egress. Elucidating these interactions not only yields novel insights into the biology of these important pathogens, but may also shed new light on cell biological aspects of actin and cholesterol, and lead to novel avenues in the design of antiviral strategies.

An emerging role for p21-activated kinases (Paks) in viral infections.

p21-activated protein kinases (Paks) are cytosolic serine/threonine protein kinases that act as effectors for small (p21) GTPases of the Cdc42 and Rac families. It has long been established that Paks play a major role in a host of vital cellular functions such as proliferation, survival and motility, and abnormal Pak function is associated with a number of human diseases. Here, we discuss emerging evidence that these enzymes also play a major role in the entry, replication and spread of many important pathogenic human viruses, including HIV. Careful assessment of the potential role of Paks in antiviral immunity will be pivotal to evaluate thoroughly the potential of agents that inhibit Pak as a new class of anti-viral therapeutics.

Pseudorabies virus US3- and UL49.5-dependent and -independent downregulation of MHC I cell surface expression in different cell types.

Many herpesviruses interfere with the MHC I antigen-processing pathway in order to limit elimination by cytotoxic T-lymphocytes. For varicelloviruses, the largest subgroup of alphaherpesviruses, two viral proteins have been reported to downregulate MHC I cell surface expression: UL49.5 for BoHV-1, PRV, and EHV-1 and the US3 orthologue for VZV. Here, we report that PRV reduces MHC I cell surface expression during infection in a cell-type-dependent manner. In ST cells, a kinase-active US3 was necessary but not sufficient to downregulate cell surface MHC I expression, whereas US3 was not required in PK-15 cells and porcine alveolar macrophages (PAM). MHC I downregulation was not (PAM, ST) or only partly (PK-15) dependent on UL49.5. In conclusion, we show that the mechanism(s) of PRV-mediated cell surface MHC I downregulation are cell-type-dependent, with variable roles for US3, UL49.5, and additional, yet unidentified early viral proteins.

Alphaherpesvirus US3-mediated reorganization of the actin cytoskeleton is mediated by group A p21-activated kinases.

The US3 protein is a viral serine/threonine kinase that is conserved among all members of the Alphaherpesvirinae. The US3 protein of different alphaherpesviruses causes dramatic alterations in the actin cytoskeleton, such as the disassembly of actin stress fibers and formation of cell projections, which have been associated with increased intercellular virus spread. Here, we find that inhibiting group A p21-activated kinases (PAKs), which are key regulators in Cdc42/Rac1 Rho GTPase signaling pathways, impairs US3-mediated actin alterations. By using PAK1(-/-) and PAK2(-/-) mouse embryo fibroblasts (MEFs), we show that US3-mediated stress fiber disassembly requires PAK2, whereas US3-mediated cell projection formation mainly is mediated by PAK1, also indicating that PAK1 and PAK2 can have different biological effects on the organization of the actin cytoskeleton. In addition, US3 was found to bind and phosphorylate group A PAKs. Lack of group A PAKs in MEFs was correlated with inefficient virus spread. Thus, US3 induces its effect on the actin cytoskeleton via group A PAKs.

The kinase activity of pseudorabies virus US3 is required for modulation of the actin cytoskeleton.

Different viruses exploit the host cytoskeleton to facilitate replication and spread. The conserved US3 protein of the alphaherpesvirus pseudorabies virus induces actin stress fiber disassembly and formation of actin-containing cell projections, which are associated with enhanced intercellular virus spread. Proteins of members of other virus families, notably vaccinia virus F11L protein and human immunodeficiency virus Nef protein, induce actin rearrangements that are very similar to those induced by US3. Interestingly, unlike F11L and Nef, the US3 protein displays serine/threonine kinase activity. Here, we report that the kinase activity of pseudorabies virus US3 is absolutely required for its actin modulating activity. These data show that different viruses have developed independent mechanisms to induce very similar actin rearrangements.

Vitamin D binding protein, bone status and body composition in community-dwelling elderly men.

The vitamin D binding protein (DBP) is the major carrier protein for vitamin D metabolites in plasma. Polymorphisms in DBP have been described to be associated with an increased bone fracture risk and diabetes. The present study investigates the influence of both phenotypic and (TAAA)(n)-Alu repeat DBP-polymorphism and DBP-concentration on bone mineral density, body composition, bone turnover- and metabolic markers in a cohort of ambulatory elderly men. We included 211 men (>70 years) in this study. Bone mineral density (BMD) was determined by dual energy X-ray absorptiometry. Bone turnover was assessed by measurement of serum osteocalcin, serum and urinary C-terminal telopeptides of type I collagen and urinary deoxypyridinoline, together with 25(OH)-vitamin D and 1,25(OH)(2)-vitamin D concentrations. DBP-phenotypes were determined electrophoretically and the (TAAA)(n)-Alu repeat polymorphism was determined by polymerase chain reaction. Body composition was estimated using bioelectrical impedance analysis, together with handgrip and arm strength, fasting serum glucose and leptin concentrations. No differences in BMD or bone turnover markers among DBP-phenotypes or (TAAA)(n)-genotypes were observed in this study. Serum 25(OH)-vitamin D was comparable among DBP-variants and did not relate to DBP-concentrations, whereas 1,25(OH)(2)-vitamin D was different among DBP-phenotypes and was correlated positively with DBP-concentrations. DBP-concentrations related positively to body mass index, fat mass, leptin and glucose concentration. The correlation with leptin remained significant after correction for fat mass. Fasting glucose concentrations were different among DBP-phenotypes, whereas no difference was observed between (TAAA)(n)-genotypes. In conclusion, serum 1,25(OH)(2)-vitamin D concentrations are codetermined by DBP-phenotypes and DBP-concentrations. No major effect of DBP-polymorphism was demonstrated on BMD, bone turnover markers or body composition.