Christian Sinzger

Publications of Christian Sinzger

• Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner.

  Authors: Fabienne Haspot, Amélie Lavault, Christian Sinzger, Kerstin Laib Sampaio, York-Dieter Stierhof, Paul Pilet, Céline Bressolette-Bodin, Franck Halary

  PloS one. 01/2012; 7(4):e34795.

  Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to infect fibroblastic, epithelial, endothelial and hematopoietic cells. Over the past ten years, several groups have providedHuman cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to infect fibroblastic, epithelial, endothelial and hematopoietic cells. Over the past ten years, several groups have provided direct evidence that dendritic cells (DCs) fully support the HCMV lytic cycle. We previously demonstrated that the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) has a prominent role in the docking of HCMV on monocyte-derived DCs (MDDCs). The DC-SIGN/HCMV interaction was demonstrated to be a crucial and early event that substantially enhanced infection in trans, i.e., from one CMV-bearing cell to another non-infected cell (or trans-infection), and rendered susceptible cells fully permissive to HCMV infection. Nevertheless, nothing is yet known about how HCMV enters MDDCs. In this study, we demonstrated that VHL/E HCMV virions (an endothelio/dendrotropic strain) are first internalized into MDDCs by a macropinocytosis-like process in an actin- and cholesterol-dependent, but pH-independent, manner. We observed the accumulation of virions in large uncoated vesicles with endosomal features, and the virions remained as intact particles that retained infectious potential for several hours. This trans-infection property was specific to MDDCs because monocyte-derived macrophages or monocytes from the same donor were unable to allow the accumulation of and the subsequent transmission of the virus. Together, these data allowed us to delineate the early mechanisms of the internalization and entry of an endothelio/dendrotropic HCMV strain into human MDDCs and to propose that DCs can serve as a "Trojan horse" to convey CMV from entry sites to other locations that may favor the occurrence of either latency or acute infection.

• Mutational mapping of pUL131A of human cytomegalovirus emphasizes its central role for endothelial cell tropism.

  Authors: Andrea Schuessler, Kerstin Laib Sampaio, Sarah Straschewski, Christian Sinzger

  Journal of virology. 01/2012; 86(1):504-12.

  The UL131A protein is part of a pentameric variant of the gcIII complex in the virion envelope of human cytomegalovirus (HCMV), which has been found essential for efficient entry into endothelialThe UL131A protein is part of a pentameric variant of the gcIII complex in the virion envelope of human cytomegalovirus (HCMV), which has been found essential for efficient entry into endothelial cells (ECs). Using a systematic mutational scanning approach, we aimed to define peptide motifs within the UL131A protein that contribute to EC infection. Mutant viruses were generated in which charged amino acids within frames of 2 to 6 amino acids were replaced with alanines. The resulting viruses were evaluated with regard to their potential to infect EC cultures. Four clusters of charged amino acids essential for EC infection were identified (amino acids 22 to 27, 32 to 35, 64 to 69, and 116 to 121). Mutations of individual charge clusters within amino acids 72 to 104 caused minor reductions of EC tropism, but these effects were additive in a combined mutation, showing that this region also contributes to EC tropism. Only charge clusters within amino acids 46 to 58 were found irrelevant for EC infection. In conclusion, the unusual sensitivity to mutations, together with the remarkable conservation of the UL131A protein, emphasizes its particular role for EC tropism of HCMV.

• UL74 of human cytomegalovirus reduces the inhibitory effect of gH-specific and gB-specific antibodies.

  Authors: Xiao Jing Jiang, Kerstin Laib Sampaio, Nicole Ettischer, York-Dieter Stierhof, Gerhard Jahn, Barbara Kropff, Michael Mach, Christian Sinzger

  Archives of virology. 09/2011; 156(12):2145-55.

  The human cytomegalovirus (HCMV) glycoproteins gH (UL75) and gL (UL115) can form complexes with gO (UL74) or with proteins of the UL128-UL131A locus. Deletion of gO abolishes cell-free virusThe human cytomegalovirus (HCMV) glycoproteins gH (UL75) and gL (UL115) can form complexes with gO (UL74) or with proteins of the UL128-UL131A locus. Deletion of gO abolishes cell-free virus transmission and renders cell-associated virus transmission in fibroblasts more sensitive to inhibition by human anti-HCMV serum. To test whether the latter effect is specific for gO, we compared mutants with deletions in UL74, UL99 and the UL128-131A locus regarding their sensitivity to anti-HCMV antibodies. UL74 deletion mutants were more sensitive to a further restriction by polyspecific or gH-specific antibodies than control mutants, showing that gO specifically protects focal growth against inhibitory antibodies. This effect was not confined to gH-specific antibodies, as UL74 deletion mutants were also inhibited by an anti-gB antibody. In conclusion, gO specifically promotes focal spread in the presence of gH and gB antibodies, thus contributing to the ability of HCMV to resist the host's immune response.

• The polymorphic HCMV glycoprotein UL20 is targeted for lysosomal degradation by multiple cytoplasmic dileucine motifs.

  Authors: Ivan Jelcic, Johanna Reichel, Christoph Schlude, Eva Treutler, Christian Sinzger, Alexander Steinle

  Traffic (Copenhagen, Denmark). 06/2011; 12(10):1444-56.

  Human cytomegalovirus (HCMV) is a widespread and persistent beta-herpesvirus. The large DNA genome of HCMV encodes many proteins that are non-essential for viral replication including numerousHuman cytomegalovirus (HCMV) is a widespread and persistent beta-herpesvirus. The large DNA genome of HCMV encodes many proteins that are non-essential for viral replication including numerous proteins subverting host immunosurveillance. One of them is the barely characterized UL20, which is encoded adjacent to the well-defined immunoevasins UL16 and UL18. UL20 is a type I transmembrane glycoprotein with an immunoglobulin-like ectodomain that is highly polymorphic among HCMV strains. Here, we show that the homodimeric UL20, by virtue of its cytoplasmic domain, does not reach the cell surface but is targeted to endosomes and lysosomes. Accordingly, UL20 exhibits a short half-life because of rapid lysosomal degradation. Trafficking of UL20 to lysosomes is determined by several, independently functioning dileucine-based sorting motifs in the cytoplasmic domain of UL20 and involves the adaptor protein (AP) complex AP-1. Combined substitution of three dileucine motifs allowed strong cell surface expression of UL20 comparable to UL20 mutants lacking the cytoplasmic tail. Finally, we show that the intracellularly located UL20 also is subject to lysosomal degradation in the context of viral infection. Altogether, from these data, we hypothesize that UL20 is destined to efficiently sequester yet-to-be defined cellular proteins for degradation in lysosomes.

• HCMV spread and cell tropism are determined by distinct virus populations.

  Authors: Laura Scrivano, Christian Sinzger, Hans Nitschko, Ulrich H Koszinowski, Barbara Adler

  PLoS pathogens. 01/2011; 7(1):e1001256.

  Human cytomegalovirus (HCMV) can infect many different cell types in vivo. Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereasHuman cytomegalovirus (HCMV) can infect many different cell types in vivo. Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts. Here, we describe that depending on the cell type in which virus replication takes place, virus carrying the gH/gL/pUL(128,130,131A) complex is either released or retained cell-associated. We observed that virus spread in fibroblast cultures was predominantly supernatant-driven, whereas spread in endothelial cell (EC) cultures was predominantly focal. This was due to properties of virus released from fibroblasts and EC. Fibroblasts released virus which could infect both fibroblasts and EC. In contrast, EC released virus which readily infected fibroblasts, but was barely able to infect EC. The EC infection capacities of virus released from fibroblasts or EC correlated with respectively high or low amounts of gH/gL/pUL(128,130,131A) in virus particles. Moreover, we found that focal spread in EC cultures could be attributed to EC-tropic virus tightly associated with EC and not released into the supernatant. Preincubation of fibroblast-derived virus progeny with EC or beads coated with pUL131A-specific antibodies depleted the fraction that could infect EC, and left a fraction that could predominantly infect fibroblasts. These data strongly suggest that HCMV progeny is composed of distinct virus populations. EC specifically retain the EC-tropic population, whereas fibroblasts release EC-tropic and non EC-tropic virus. Our findings offer completely new views on how HCMV spread may be controlled by its host cells.

• Mutational mapping of UL130 of human cytomegalovirus defines peptide motifs within the C-terminal third as essential for endothelial cell infection.

  Authors: Andrea Schuessler, Kerstin Laib Sampaio, Laura Scrivano, Christian Sinzger

  Journal of virology. 09/2010; 84(18):9019-26.

  The UL130 gene is one of the major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV). In order to define functionally important peptides within this protein, we haveThe UL130 gene is one of the major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV). In order to define functionally important peptides within this protein, we have performed a charge-cluster-to-alanine (CCTA) mutational scanning of UL130 in the genetic background of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain. A total of 10 charge clusters were defined, and in each of them two or three charged amino acids were replaced with alanines. While the six N-terminal clusters were phenotypically irrelevant, mutation of the four C-terminal clusters each caused a reduction of EC tropism. The importance of this protein domain was further emphasized by the fact that the C-terminal pentapeptide PNLIV was essential for infection of ECs, and the cell tropism could not be rescued by a scrambled version of this sequence. We conclude that the C terminus of the UL130 protein serves an important function for infection of ECs by HCMV. This makes UL130 a promising molecular target for antiviral strategies, e.g., the development of antiviral peptides.

• Improvement of a quantitative cell tropism assay for rapid and reliable characterization of human cytomegalovirus mutants.

  Authors: Andrea Schuessler, Kerstin Laib Sampaio, Christian Sinzger

  Journal of virological methods. 08/2010; 167(2):218-22.

  Endothelial cell tropism is an inherent property of isolates of human cytomegalovirus (HCMV), and has been proposed as a pathogenicity factor of HCMV. Mutational approaches can be applied forEndothelial cell tropism is an inherent property of isolates of human cytomegalovirus (HCMV), and has been proposed as a pathogenicity factor of HCMV. Mutational approaches can be applied for analysis of the molecular determinants of endothelial cell tropism, but the evaluation of mutants is limited by the low reliability of the widely employed cell tropism assay. The aim of this study was to improve the assay conditions in order to allow for accurate discrimination of phenotypic differences between HCMV mutants. Target cell density and input virus titres were identified to account for most of the variation in the apparent endothelial cell tropism of a given cytomegalovirus strain. Coating of culture dishes, cell attachment at ambient temperature, and standardization of virus input titres together with automated counting of immunofluorescence signals enabled the discrimination of differences of as little as 25% in endothelial cell tropism of HCMV strains. This improvement will facilitate rapid and reliable quantitation of cell tropism of HCMV, and is particularly suitable for the analysis of large numbers of mutants with only minor changes in tropism.

• Endothelial cells in human cytomegalovirus infection: one host cell out of many or a crucial target for virus spread?

  Authors: Barbara Adler, Christian Sinzger

  Thrombosis and haemostasis. 12/2009; 102(6):1057-63.

  Endothelial cells (EC) are assumed to play a central role in the spread of human cytomegalovirus (HCMV) throughout the body. Results from in-situ analyses of infected tissues and data from cellEndothelial cells (EC) are assumed to play a central role in the spread of human cytomegalovirus (HCMV) throughout the body. Results from in-situ analyses of infected tissues and data from cell culture systems together strongly suggest that vascular EC can support productive replication of HCMV and thus contribute to its haematogeneous dissemination. By inducing an angiogenic response, HCMV may even promote growth of its own habitat. The particular role of EC is further supported by the fact that entry of HCMV into EC is dependent on a complex of the envelope glycoproteins gH and gL with a set of proteins (UL128-131A) which is dispensable for HCMV entry into most other cell types. These molecular requirements may also be reflected by cell type-dependent differences in entry routes, i.e. endocytosis versus fusion at the plasma membrane. An animal model with trackable murine CMV is now available to clarify the pathogenetic role of EC during haematogeneous dissemination of this virus.

• Charge-cluster-to-alanine scanning of UL128 for fine tuning of the endothelial cell tropism of human cytomegalovirus.

  Authors: Andrea Schuessler, Kerstin Laib Sampaio, Christian Sinzger

  Journal of virology. 10/2008;

  The viral genes UL128, UL130 and UL131A have been identified as major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV), deletion of either gene causing a null phenotype.The viral genes UL128, UL130 and UL131A have been identified as major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV), deletion of either gene causing a null phenotype. We hypothesized that a functional scanning of these genes by minor genetic modifications would allow for the generation of mutants with an intermediate phenotype. By combining charge-cluster-to-alanine (CCTA) mutagenesis with markerless mutagenesis of a BAC-cloned endotheliotropic HCMV-strain, we analyzed UL128 in order to identify functional sites and hence enable targeted modulation of the EC tropism of HCMV. A total of 9 mutations in 8 charge clusters were tested. Three of the CCTA mutations severely reduced EC tropism, three were irrelevant, two had a weak effect on cell tropism, and one mutation in the most C-terminal cluster caused an intermediate phenotype. All of the highly effective mutations were located in a core region (amino acids 72 to 106) which appears to be particularly crucial for EC tropism. The intermediate effect of mutations in the C-terminal cluster could be modulated by varying the number of amino acids replaced with alanine. This study provides a rational approach for targeted modulation of HCMV cell tropism, which may aid in the development of HCMV strains with a desired degree of attenuation.

• hDectin-1 is involved in uptake and cross-presentation of cellular antigens.

  Authors: Markus M Weck, Silke Appel, Daniela Werth, Christian Sinzger, Anita Bringmann, Frank Grünebach, Peter Brossart

  Blood. 05/2008; 111(8):4264-72.

  Human Dectin-1 (hDectin-1) is a member of the C-type lectin-like receptor family that was shown to be the major receptor for fungal beta-glucans and to play an important role in the cellularHuman Dectin-1 (hDectin-1) is a member of the C-type lectin-like receptor family that was shown to be the major receptor for fungal beta-glucans and to play an important role in the cellular responses mediated by these carbohydrates. In this study, we demonstrate that hDectin-1 is involved in the uptake and cross-presentation of cellular antigens. Furthermore, activation of monocyte-derived dendritic cells (MDCs) with toll-like receptor 3 (TLR3) ligand but not with TLR2 ligand or TLR7 ligand resulted in down-regulation of hDectin-1 expression and reduced phagocytosis of apoptotic tumor cells as well as presentation of pp65-derived T-cell epitopes upon engulfment of cytomegalovirus (CMV)-infected human foreskin fibroblasts.

• UL74 of human cytomegalovirus contributes to virus release by promoting secondary envelopment of virions.

  Authors: Xiao Jing Jiang, Barbara Adler, Kerstin Laib Sampaio, Margarete Digel, Gerhard Jahn, Nicole Ettischer, York-Dieter Stierhof, Laura Scrivano, Ulrich Koszinowski, Michael Mach, Christian Sinzger

  Journal of virology. 04/2008; 82(6):2802-12.

  The glycoprotein (g) complex gH/gL represents an essential part of the herpesvirus fusion machinery mediating entry of cell-free virions and cell-associated viral spread. In some herpesvirusesThe glycoprotein (g) complex gH/gL represents an essential part of the herpesvirus fusion machinery mediating entry of cell-free virions and cell-associated viral spread. In some herpesviruses additional proteins are associated with gH/gL contributing to the cell tropism of the respective virus. Human cytomegalovirus (HCMV) gH/gL forms complexes with either gO (UL74) or proteins of the UL128-131A gene locus. While a contribution of UL128-131A to endothelial cell tropism is known, the role of gO is less clear. We studied the role of gH/gL-associated proteins in HCMV replication in human foreskin fibroblasts (HFF) and human umbilical vein endothelial cells (HUVEC). Deletions of UL74 alone or in combination with mutations of the UL128-131A gene region were introduced into bacterial artificial chromosome vectors derived from the endotheliotropic strain TB40/E. Deletion of UL74 caused a profound defect regarding virus release from infected HFF and HUVEC. Large numbers of capsids accumulated in the cytoplasm of infected HFF but failed to acquire an envelope. Clear cell type differences were observed in the cell-associated spread of the UL74-defective virus. In HFF, focal growth was severely impaired, whereas it was normal in HUVEC. Deletion of UL131A abolished focal growth in endothelial cells. UL74/UL128-131A dual mutants showed severely impaired reconstitution efficiency. Our data suggest that gO plays a critical role in secondary envelopment and release of cell-free virions independent of the cell type but affects cell-associated growth specifically in HFF, whereas UL128-131A contributes to cell-associated spread in HFF and HUVEC.

• Entry route of HCMV into endothelial cells.

  Authors: Christian Sinzger

  Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology. 04/2008; 41(3):174-9.

  Human cytomegalovirus (HCMV) is generally assumed to enter its target cells by direct fusion at the plasma membrane while sequestration and lysis of viral particles within endocytic vesicles has beenHuman cytomegalovirus (HCMV) is generally assumed to enter its target cells by direct fusion at the plasma membrane while sequestration and lysis of viral particles within endocytic vesicles has been suggested as an abortive entry pathway of fibroblast-adapted HCMV strains in endothelial cells. However, the finding that virions of a highly endotheliotropic strain are also found within endocytic vesicles at 10min after penetration together with the fact that the majority of these particles is translocated towards the nucleus indicates that endocytosis can be an effective entry route for HCMV in endothelial cells. HCMV-containing vesicles resemble endocytic vesicles reported for other herpesviruses with regard to size and structure suggesting a similar entry route for various herpesviruses in endocytically active cells.

• Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E.

  Authors: Christian Sinzger, Gabriele Hahn, Margarete Digel, Ruth Katona, Kerstin Laib Sampaio, Martin Messerle, Hartmut Hengel, Ulrich Koszinowski, Wolfram Brune, Barbara Adler

  The Journal of general virology. 03/2008; 89(Pt 2):359-68.

  Human cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not beenHuman cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not been available in a phenotypically homogeneous form compatible with genetic analysis. To overcome this problem, we cloned the TB40/E strain into a bacterial artificial chromosome (BAC) vector. Both highly endotheliotropic and poorly endotheliotropic virus clones, representing three distinct restriction fragment patterns, were reconstituted after transfection of BAC clones derived from previously plaque-purified strain TB40/E. For one of the highly endotheliotropic clones, TB40-BAC4, we provide the genome sequence. Two BACs with identical restriction fragment patterns but different cell tropism were further analysed in the UL128-UL131A gene region. Sequence analysis revealed one coding-relevant adenine insertion at position 332 of UL128 in the BAC of the poorly endotheliotropic virus, which caused a frameshift in the C-terminal part of the coding sequence. Removal of this insertion by markerless mutagenesis restored the highly endotheliotropic phenotype, indicating that the loss of endothelial cell tropism was caused by this insertion. In conclusion, HCMV strain TB40/E, which combines the high endothelial cell tropism of a clinical isolate with the high titre growth of a cell culture adapted strain, is now available as a BAC clone suitable for genetic engineering. The results also suggest BAC cloning as a suitable method for selection of genetically defined virus clones.

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• TLR ligands differentially affect uptake and presentation of cellular antigens.

  Authors: Markus Michael Weck, Frank Grünebach, Daniela Werth, Christian Sinzger, Anita Bringmann, Peter Brossart

  Blood. 06/2007; 109(9):3890-4.

  Dendritic cells (DCs) have the unique ability to efficiently present T-cell epitopes from exogenous antigens on MHC class I molecules, a process called cross-presentation. In our study we demonstrateDendritic cells (DCs) have the unique ability to efficiently present T-cell epitopes from exogenous antigens on MHC class I molecules, a process called cross-presentation. In our study we demonstrate that stimulation of monocyte-derived DCs with Toll-like receptor (TLR) ligands differentially affects the uptake and cross-presentation of cellular antigens. Activation of DCs with TLR3 or TLR4 but not with TLR2 or TLR7/8 ligands inhibited phagocytosis of apoptotic tumor cells and resulted in a reduced cross-presentation of pp65-derived T-cell epitopes on MHC class I molecules upon engulfment of cytomegalovirus (CMV)-infected fibroblasts. These results have an important impact on the understanding of the interactions between the immune system and pathogens and the development of vaccination strategies to treat malignant diseases.

• Effect of serum and CTL on focal growth of human cytomegalovirus.

  Authors: Christian Sinzger, Martina Mangin, Christof Weinstock, Max S Topp, Holger Hebart, Hermann Einsele, Gerhard Jahn

  Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology. 03/2007; 38(2):112-9.

  BACKGROUND: In immunocompromised patients only cytotoxic T-lymphocytes (CTL) but not antiviral antibodies appear to be efficient in control of human cytomegalovirus (HCMV) infection. This isBACKGROUND: In immunocompromised patients only cytotoxic T-lymphocytes (CTL) but not antiviral antibodies appear to be efficient in control of human cytomegalovirus (HCMV) infection. This is contrasted by the well-documented neutralising activity of patient sera against standard HCMV strains. OBJECTIVE: We tested the hypothesis that a cell-culture model based on a recent clinical HCMV isolate would more accurately approximate the clinical situation and provide an explanation for the failure of neutralising antibodies in efficient restriction of HCMV infection. METHODS: Sera from five bone marrow transplant recipients with or without prolonged HCMV replication were analysed by an enzyme-linked immunoassay for their capacity to neutralise cell-free HCMV preparations. The inhibitory effect of these sera on viral cell-to-cell-spread was then quantified by focus expansion assays using a recent clinical HCMV-isolate and was finally compared to the inhibitory effect of HCMV-specific CTL lines. RESULTS: Prolonged HCMV replication occurred in three patients despite high titres of neutralising antibodies. In contrast to the strong inhibitory effect on cell-free HCMV, their sera could not inhibit the focal growth of a recent cell-associated HCMV isolate, whereas CTL clones directed against pUL123 or pUL83 of HCMV effectively limited focal expansion of the clinical isolate in fibroblast culture. CONCLUSIONS: Focus expansion assays based on a cell-associated clinical HCMV isolate provide a model for the in vivo effectiveness of virus-specific CTL and neutralising antibodies. Our data support the assumption that due to their strict cell-association clinical HCMV strains are withdrawn from neutralising antibodies.

• Human cytomegalovirus (HCMV) infection of endothelial cells promotes naive monocyte extravasation and transfer of productive virus to enhance hematogenous dissemination of HCMV.

  Authors: Gretchen L Bentz, Marta Jarquin-Pardo, Gary Chan, M Shane Smith, Christian Sinzger, Andrew D Yurochko

  Journal of virology. 01/2007; 80(23):11539-55.

  Human cytomegalovirus (HCMV) pathogenesis is dependent on the hematogenous spread of the virus to host tissue. While data suggest that infected monocytes are required for viral dissemination from theHuman cytomegalovirus (HCMV) pathogenesis is dependent on the hematogenous spread of the virus to host tissue. While data suggest that infected monocytes are required for viral dissemination from the blood to the host organs, infected endothelial cells are also thought to contribute to this key step in viral pathogenesis. We show here that HCMV infection of endothelial cells increased the recruitment and transendothelial migration of monocytes. Infection of endothelial cells promoted the increased surface expression of cell adhesion molecules (intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and platelet endothelial cell adhesion molecule 1), which were necessary for the recruitment of naïve monocytes to the apical surface of the endothelium and for the migration of these monocytes through the endothelial cell layer. As a mechanism to account for the increased monocyte migration, we showed that HCMV infection of endothelial cells increased the permeability of the endothelium. The cellular changes contributing to the increased permeability and increased naïve monocyte transendothelial migration include the disruption of actin stress fiber formation and the decreased expression of lateral junction proteins (occludin and vascular endothelial cadherin). Finally, we showed that the migrating monocytes were productively infected with the virus, documenting that the virus was transferred to the migrating monocyte during passage through the lateral junctions. Together, our results provide evidence for an active role of the infected endothelium in HCMV dissemination and pathogenesis.

• hCMV induced IL-6 release in trophoblast and trophoblast like cells.

  Authors: Gabriele Halwachs-Baumann, Gisela Weihrauch, Hans-Jürgen Gruber, Gernot Desoye, Christian Sinzger

  Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology. 11/2006; 37(2):91-7.

  BACKGROUND: Human cytomegalovirus (hCMV) infection of the trophoblasts is a crucial event in virus transmission from mother to child, being one responsible factor for intrauterine infection of theBACKGROUND: Human cytomegalovirus (hCMV) infection of the trophoblasts is a crucial event in virus transmission from mother to child, being one responsible factor for intrauterine infection of the unborn. Differences of virus replication in trophoblasts depending on time point of pregnancy and degree of differentiation of trophoblasts might influence this transmission. Furthermore, immunological reactions of the trophoblasts to hCMV infection might be important defence mechanisms too. OBJECTIVES: hCMV replication and interleukin-6 release in trophoblasts and trophoblast like cells (choriocarcinoma cells) was investigated. STUDY DESIGN: Trophoblasts from term and 1st trimester placentas were isolated and infected with hCMV. hCMV production and release to the supernatant as well as interleukin-6 release and interleukin-6 mRNA production by these infected cells was measured. Choriocarcinoma cell lines (JEG-3, JAR) were treated the same. Non-infected trophoblasts were used as controls. RESULTS: In 1st trimester trophoblast, term trophoblasts and JEG-3 permissive hCMV replication was observed, although with different kinetics and efficiency. In JAR no complete virus replication was seen. High levels of interleukin-6 were measured in the supernatants of all hCMV infected cells immediately after infection. IL-6 mRNA upregulation was seen 48 h after infection in those cell types replication of hCMV occurred (1st trimester trophoblasts, term trophoblasts, JEG-3). At that time-point hCMV immediate early proteins appeared. In JARs no virus production and no IL-6 mRNA upregulation was seen, and IL-6 levels in the supernatant of these hCMV infected cells declined significantly until day 6 after infection compared to mock infected cells. CONCLUSION: These observations show that hCMV replication is influenced by the degree of trophoblast differentiation. Interleukin-6 is upregulated by hCMV infection, but is independent of complete virus replication.

• Role of human cytomegalovirus UL131A in cell type-specific virus entry and release.

  Authors: Barbara Adler, Laura Scrivano, Zsolt Ruzcics, Brigitte Rupp, Christian Sinzger, Ulrich Koszinowski

  The Journal of general virology. 10/2006; 87(Pt 9):2451-60.

  The human cytomegalovirus (HCMV) genes UL128, UL130 and UL131A are essential for endothelial cell infection. Complementation of the defective UL131A gene of the non-endotheliotropic HCMV strain AD169The human cytomegalovirus (HCMV) genes UL128, UL130 and UL131A are essential for endothelial cell infection. Complementation of the defective UL131A gene of the non-endotheliotropic HCMV strain AD169 with wild-type UL131A in cis in an ectopic position restored endothelial cell tropism. The UL131A protein was found in virions in a complex with gH. Coinfection of fibroblasts with UL131A-negative and -positive viruses restored the endothelial cell tropism of UL131A-negative virions by complementing the virions with UL131A protein. Virus entry into endothelial cells, but not into fibroblasts, was blocked by an antipeptide antiserum to pUL131A. AD169, cis-complemented with wild-type UL131A, showed an impaired release of infectious particles from fibroblasts. A comparable defect in virus release was observed when UL131A was expressed ectopically in a virus background already expressing an intact copy of UL131A. In contrast, virus release from infected endothelial cells was not affected by UL131A. These data suggest a dual role for pUL131A in virus entry and virus exit from infected cells.

• Evidence for direct transfer of cytoplasmic material from infected to uninfected cells during cell-associated spread of human cytomegalovirus.

  Authors: Margarete Digel, Kerstin Laib Sampaio, Gerhard Jahn, Christian Sinzger

  Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology. 10/2006; 37(1):10-20.

  Cell-associated spread is assumed to be the predominant mode of human cytomegalovirus (HCMV) dissemination in infected patients, however the underlying mechanisms are poorly understood. We tested theCell-associated spread is assumed to be the predominant mode of human cytomegalovirus (HCMV) dissemination in infected patients, however the underlying mechanisms are poorly understood. We tested the hypothesis that cell-to-cell spread of HCMV may be associated with direct transfer of cytoplasmic material by analyzing focal growth of green fluorescent HCMVDeltaUL16GFP. In this recombinant virus, UL16 was partially replaced by the green fluorescent protein (EGFP). The resulting HCMVDeltaUL16GFP showed unrestricted growth and expressed EGFP from the early UL16 promoter. EGFP transmission was then investigated in relation to viral spread from productively infected cells to cocultured uninfected cells. Alternatively, microinjection of fluorescent dextrane allowed for direct visualization of inter-cell-connections. Within 5h of coculture, 8% of cells neighbouring productively infected cells had acquired EGFP. Detection of EGFP in the absence of IE antigen and during cycloheximide block excluded the possibility of de novo synthesis. Immediate distribution of microinjected fluorescent dyes from infected cells to adjacent cells proved the existence of cell-cell-fusions. These data demonstrate that focal spread of HCMV is associated with direct transfer of cytoplasmic material, most likely through cell-cell-fusions. This would withdraw the virus from the control of neutralizing antibodies and thus provide an explanation for the limited antiviral effect of the humoral immune response.

• Macrophage cultures are susceptible to lytic productive infection by endothelial-cell-propagated human cytomegalovirus strains and present viral IE1 protein to CD4+ T cells despite late downregulation of MHC class II molecules.

  Authors: Christian Sinzger, Kathrin Eberhardt, Yolaine Cavignac, Christof Weinstock, Tobias Kessler, Gerhard Jahn, Jean-Luc Davignon

  The Journal of general virology. 08/2006; 87(Pt 7):1853-62.

  The contribution of CD4(+) T cells to control of human cytomegalovirus (HCMV) has been shown and infected tissue macrophages might contribute to this response by antigen presentation. As shownThe contribution of CD4(+) T cells to control of human cytomegalovirus (HCMV) has been shown and infected tissue macrophages might contribute to this response by antigen presentation. As shown previously, CD4(+) T cells recognize HCMV immediate-early antigen IE1 on glioblastoma cells manipulated to express MHC class II molecules. Here, the possible interference of virus-induced MHC class II downmodulation with the presentation of IE1 by natural target cells was analysed. The capacity of IE1-specific CD4(+) T-cell clones to recognize HCMV-infected monocyte-derived macrophages was tested. Various HCMV strains were used to achieve efficient infection of macrophages. Activation of CD4(+) T cells by infected macrophages was evaluated at different time points after infection. Endothelial-cell-adapted HCMV strains efficiently infected cultured human macrophages. However, the immediate-early and early phases of replication were prolonged. Infected cells entered the late replication phase only after 3 days of infection, which was associated with downmodulation of MHC class II molecules at the surface of infected cells. Strong stimulation of IE1-specific CD4(+) T cells resulted from endogenous de novo antigen production and presentation by infected macrophages during the first 3 days of virus replication, despite MHC class II downmodulation in the late replication phase. Therefore, infected macrophages are assumed to contribute to the antiviral immune response in infected organs.