Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMVspecific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/ gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC. Molecular & Cellular

The uptake of polyelectrolyte multilayer coated colloids into cells, subsequent defoliation and plasmid delivery was studied by means of confocal microscopy and flow cytometry. Silica particles coated layer-wise with protamine and dextran sulfate were given to HEK 293T cells. Optimum uptake was found with protamine as the top layer. The particle uptake likely follows an non-receptor-mediated endocytotic pathway. Defoliation of polyelectrolyte multilayer coated particles within cells was demonstrated by the release of incorporated plasmids as indicated by the expression of plasmid encoded proteins using the enhanced green fluorescence proteine (pEGFP-C1) plasmid and a red fluorescence protein (pDsRed1-N1) plasmid. This proves, together with the direct observation of fluorescent layer debris, the defoliation of coated particles and the release of layer components into the cytoplasm. Particle uptake and GFP expression.

Cover: Silica particles can be coated layerwise with polyelectrolytes. Using the plasmid encoding the green fluorescent protein (pEGFP-C1) as a layer constituent particle uptake, release of particles into cytoplasm and defoliation of the polyelectrolyte multilayer are demonstrated by GFP expression in HEK 293T cells. Further details can be found in the Full Paper by U. Reibetanz, C. Claus, E. Typlt, J. Hofmann, and E. Donath* on page 153.

The interaction of the rubella virus (RV) capsid (C) protein and the mitochondrial p32 protein is believed to participate in virus replication. In this study, the physiological significance of the association of RV with mitochondria was investigated by silencing p32 through RNA interference. It was demonstrated that downregulation of p32 interferes with microtubule-directed redistribution of mitochondria in RV-infected cells. However, the association of the viral C protein with mitochondria was not affected. When cell lines either pretreated with respiratory chain inhibitors or cultivated under (mild) hypoxic conditions were infected with RV, viral replication was reduced in a time-dependent fashion. Additionally, RV infection induces increased activity of mitochondrial electron transport chain complex III, which was associated with an increase in the mitochondrial membrane potential. These effects are outstanding among the examples of mitochondrial alterations caused by viruses. In contrast to the preferential localization of p32 to the mitochondrial matrix in most cell lines, RV-permissive cell lines were characterized by an almost exclusive membrane association of p32. Conceivably, this contributes to p32 function(s) during RV replication. The data presented suggest that p32 fulfills an essential function for RV replication in directing trafficking of mitochondria near sites of viral replication to meet the energy demands of the virus.Rubella virus (RV), a single-stranded RNA virus, is the sole member of the genus Rubivirus in the family Togaviridae and causes a generally mild exanthematous childhood disease. However, severe malformations known as congenital rubella syndrome may result from the infection of seronegative women, especially during the first trimester of pregnancy. The mechanisms contributing to RV teratogenesis remain largely unknown. The 5Ј-proximal open reading frame (ORF) of the genome encodes the two replicase proteins P150 and P90, while the 3Ј ORF encodes the structural proteins, the capsid (C) protein and two envelope glycoproteins (E1 and E2). Viral RNA synthesis occurs on replication complexes, which are membrane bound to a structure called the cytopathic vacuole (CPV). CPVs are of endolysosomal origin and surrounded by rough endoplasmic reticulum (RER) cisternae, the Golgi apparatus, and mitochondria (13,14). CPVs are replication factories and provide a protected environment for virus replication and assembly.The C protein of RV represents one of the few RNA virusencoded structural proteins that interact with mitochondria and is so far the only known viral protein that impairs protein transport into mitochondria (17). Additionally, the C protein participates in viral RNA synthesis (42), which is emphasized by its accumulation around CPVs (14). The C protein is also involved in the process of mitochondrial redistribution to a perinuclear region in proximity to CPVs (3,28) and interacts with the p32 protein (3). Besides its predominant localization to the matrix of mitochondria, p32 is a...

bMitochondria are important for the viral life cycle, mainly by providing the energy required for viral replication and assembly. A highly complex interaction with mitochondria is exerted by rubella virus (RV), which includes an increase in the mitochondrial membrane potential as a general marker for mitochondrial activity. We aimed in this study to provide a more comprehensive picture of the activity of mitochondrial respiratory chain complexes I to IV. Their activities were compared among three different cell lines. A strong and significant increase in the activity of mitochondrial respiratory enzyme succinate:ubiquinone oxidoreductase (complex II) and a moderate increase of ubiquinol:cytochrome c oxidoreductase (complex III) were detected in all cell lines. In contrast, the activity of mitochondrial respiratory enzyme cytochrome c oxidase (complex IV) was significantly decreased. The effects on mitochondrial functions appear to be RV specific, as they were absent in control infections with measles virus. Additionally, these alterations of the respiratory chain activity were not associated with an elevated transcription of oxidative stress proteins, and reactive oxygen species (ROS) were induced only marginally. Moreover, protein and/or mRNA levels of markers for mitochondrial biogenesis and structure were elevated, such as nuclear respiratory factors (NRFs) and mitofusin 2 (Mfn2). Together, these results establish a novel view on the regulation of mitochondrial functions by viruses. Mitochondria are required for the maintenance of cell function and integrity. Their most important role lies in energy production, but they are also at the intersection of regulatory pathways that coordinate metabolic processes (e.g., calcium homeostasis and cellular proliferation), cellular fate (apoptosis and necrosis), and antiviral defense (1, 2). Even a participation of mitochondria in the innate immune response was identified (2). There are a number of viruses that interfere with the important role of mitochondria in cellular antiviral response pathways, mainly with the regulation of apoptosis (1). Additionally, as the powerhouses of the cell, mitochondria provide most of the energy for viral replication and assembly. Up to 90% of the cellular ATP is produced in the inner mitochondrial membrane (IMM) by oxidative phosphorylation (OXPHOS), (3). OXPHOS comprises a series of redox reactions carried out by four multisubunit enzyme complexes (complexes I to IV) of the electron transport chain (ETC). Electrons are transferred in a stepwise manner through this series of electron carriers from NADH (and FADH 2 ) as reducing equivalents to the final acceptor molecular oxygen. A small percentage of electrons that are transported through the respiratory complexes leaks out, which results in generation of reactive oxygen species (ROS). The main ROS species is hydrogen peroxide, which is converted to water by enzymes such as catalase, peroxiredoxin, or glutathione peroxidase as components of the cellular antioxidant system. Respiratory c...

Mitochondria fulfil several key functions within cellular metabolic and antiviral signalling pathways, including their central role in ATP generation. Viruses, as intracellular parasites, require from their cellular host the building blocks for generation of their viral progeny and the energy that drives viral replication and assembly. While some viruses have adopted ways to manipulate the infected cell such that cellular metabolism supports optimal virus production, other viruses simply exhaust cellular resources. The association of viruses with mitochondria is influenced by several important factors such as speed of the viral replication cycle and viral dependence on cellular enzymes and metabolites. This review will highlight the complex interconnectivity of viral life cycles with the three main mitochondrial metabolic pathways, namely β-oxidation, the tricarboxylic (TCA) cycle, and oxidative phosphorylation. This interconnectivity has the potential to reveal interesting points for antiviral therapy with either prometabolites or antimetabolites and highlights the importance of the viral association with mitochondrial metabolism.

Human induced pluripotent stem cell (iPSC) lines are a promising model for the early phase of human embryonic development. Here, their contribution to the still incompletely understood pathogenesis of congenital virus infections was evaluated. The infection of iPSC lines with miscarriage-associated coxsackievirus B3 (CVB3) and measles virus (MV) was compared to the efficient teratogen rubella virus (RV). While CVB3 and MV were found to be cytopathogenic on iPSC lines, RV replicated without impairment of iPSC colony morphology and integrity. This so far outstanding course of infection enabled maintenance of RV-infected iPSC cultures over several passages and their subsequent differentiation to ectoderm, endoderm, and mesoderm. A modification of the metabolic profile of infected iPSC lines was the only common aspect for all three viruses. This study points toward two important aspects. First, iPSC lines represent a suitable cell culture model for early embryonic virus infection. Second, metabolic activity represents an important means for evaluation of pathogen-associated alterations in iPSC lines.

The flexible regulation of cellular metabolic pathways enables cellular adaptation to changes in energy demand under conditions of stress such as posed by a virus infection. To analyze such an impact on cellular metabolism, rubella virus (RV) was used in this study. RV replication under selected substrate supplementation with glucose, pyruvate, and glutamine as essential nutrients for mammalian cells revealed its requirement for glutamine. The assessment of the mitochondrial respiratory (based on the oxygen consumption rate) and glycolytic (based on the extracellular acidification rate) rate and capacity by respective stress tests through Seahorse technology enabled determination of the bioenergetic phenotype of RV-infected cells. Irrespective of the cellular metabolic background, RV infection induced a shift of the bioenergetic state of epithelial cells (Vero and A549) and human umbilical vein endothelial cells to a higher oxidative and glycolytic level. Interestingly there was a RV strain-specific, but genotype-independent demand for glutamine to induce a significant increase in metabolic activity. While glutaminolysis appeared to be rather negligible for RV replication, glutamine could serve as donor of its amide nitrogen in biosynthesis pathways for important metabolites. This study suggests that the capacity of RVs to induce metabolic alterations could evolve differently during natural infection. Thus, changes in cellular bioenergetics represent an important component of virus-host interactions and could complement our understanding of the viral preference for a distinct host cell population. RV pathologies, especially during embryonal development, could be connected with its impact on mitochondrial metabolism. With bioenergetic phenotyping we pursued a rather novel approach in virology. For the first time it was shown that a virus infection could shift the bioenergetics of its infected host cell to a higher energetic state. Notably, the capacity to induce such alterations varied among different RV isolates. Thus, our data add viral adaptation of cellular metabolic activity to its specific needs as a novel aspect to virus-host evolution. In addition, this study emphasizes the implementation of different viral strains in the study of virus-host interactions and the use of bioenergetic phenotyping of infected cells as a biomarker for virus-induced pathological alterations.