Kerstin Laib Sampaio

Publications of Kerstin Laib Sampaio

• 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.

• Deletion mutant of human cytomegalovirus lacking US2-US6 and US11 maintains MHC class I expression and antigen presentation by infected dendritic cells.

  Authors: Susanne Schempp, Max Topp, Tobias Kessler, Kerstin Laib Sampaio, Kevin M Dennehy, Hermann Einsele, Gabriele Hahn, Götz Ulrich Grigoleit, Gerhard Jahn

  Virus research. 02/2011; 155(2):446-54.

  A HCMV mutant of endothelial- and DC-tropic strain TB40/E lacking the described MHC downregulating genes US2-6 and US11 (RVTB40/E(4)ΔUS11) was generated. We analyzed the susceptibility of DC toA HCMV mutant of endothelial- and DC-tropic strain TB40/E lacking the described MHC downregulating genes US2-6 and US11 (RVTB40/E(4)ΔUS11) was generated. We analyzed the susceptibility of DC to RVTB40/E(4)ΔUS11 and subsequently studied antigen presentation and T-cell stimulation. Wildtype TB40/E- and RVTB40/E(4)ΔUS11 showed no significant difference in the efficiency of infection of DC. Whereas infection with TB40/E induced downregulation of MHC I, no significant MHC I downregulation was observed on RVTB40/E(4)ΔUS11-infected DC, indicating that the US2-6, US11 region encodes for the major genes relevant for MHC I downregulation. However, both viruses induced downregulation of MHC II, as well as CD40, CD80, CD86 and CD83 to the same levels. Stimulation of IFN-γ production by HCMV-specific CD8+ T-cells by infected autologous DC correlated with the modulation of MHC expression. While TB40/E-infected DC did not efficiently stimulate IFN-γ production, RVTB40/E(4)ΔUS11-infected DC efficiently stimulated CD8+ T-cells to produce IFN-γ.

• 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.

• 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.

• 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.

• 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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• 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.

• Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements.

  Authors: Kerstin Laib Sampaio, Yolaine Cavignac, York-Dieter Stierhof, Christian Sinzger

  Journal of virology. 04/2005; 79(5):2754-67.

  Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. ToHuman cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.

• Selective intracellular retention of virally induced NKG2D ligands by the human cytomegalovirus UL16 glycoprotein.

  Authors: Stefan A Welte, Christian Sinzger, Stefan Z Lutz, Harpreet Singh-Jasuja, Kerstin Laib Sampaio, Ute Eknigk, Hans-Georg Rammensee, Alexander Steinle

  European journal of immunology. 02/2003; 33(1):194-203.

  Human cytomegalovirus (HCMV) has evolved a multitude of molecular mechanisms to evade the antiviral immune defense of the host. Recently, using soluble recombinant molecules, the HCMV UL16Human cytomegalovirus (HCMV) has evolved a multitude of molecular mechanisms to evade the antiviral immune defense of the host. Recently, using soluble recombinant molecules, the HCMV UL16 glycoprotein was shown to interact with some ligands of the activating immunoreceptor NKG2D and, therefore, may also function as a viral immunomodulator. However, the role of UL16 during the course of HCMV infection remained unclear. Here, we demonstrate that HCMV infection of fibroblasts induces expression of all known NKG2D ligands (NKG2DL). However, solely MICA and ULBP3 reach the cellular surface to engage NKG2D, whereas MICB, ULBP1 and ULBP2 are selectively retained in the endoplasmic reticulum by UL16. UL16-mediated reduction of NKG2DL cell surface density diminished NK cytotoxicity. Thus, UL16 functions by capturing activating ligands for cytotoxic lymphocytes that are synthesized in response to HCMV infection.

• Human cytomegalovirus induces a direct inhibitory effect on antigen presentation by monocyte-derived immature dendritic cells.

  Authors: Ulrich Grigoleit, Susanne Riegler, Hermann Einsele, Kerstin Laib Sampaio, Gerhard Jahn, Holger Hebart, Peter Brossart, Friderike Frank, Christian Sinzger

  British journal of haematology. 11/2002; 119(1):189-98.

  The hypothesis that productive infection of monocyte-derived immature dendritic cells (DCs) by the human cytomegalovirus (HCMV) is associated with decreased immunostimulatory capacity was tested inThe hypothesis that productive infection of monocyte-derived immature dendritic cells (DCs) by the human cytomegalovirus (HCMV) is associated with decreased immunostimulatory capacity was tested in this study. DCs were infected with 60-80% efficiency by HCMV strain TB40/E. Infected versus uninfected cells were analysed by fluorescence-activated cell sorting and by immunocytochemistry for surface expression of major histocompatibility complex (MHC) and co-stimulatory molecules as well as cytokine secretion during the 3 d after infection. The immunostimulatory capacity of these cells was measured by mixed leucocyte reaction. In spite of the fact that HCMV infection of DCs induced an increased release of tumour necrosis factor-alpha (TNF-alpha) and a decreased interleukin 10 (IL-10) production, expression of MHC class I and II, as well as CD40 and CD80 molecules, were downregulated on infected DCs. The mixed leucocyte reaction showed significantly reduced immunostimulatory capacity of infected DC cultures. Simultaneous detection of MHC antigens and virus antigens by double immunofluorescence revealed that downregulation occurred only on infected cells, but not on uninfected bystander cells. These findings demonstrate on a single cell level, together with the marked downregulation of MHC and co-stimulatory molecules in the presence of high TNF-alpha and low IL-10 levels, a direct inhibitory effect of HCMV on antigen presentation by immature DCs independent of soluble mediators.