CD147 Promotes Entry of Pentamer-Expressing Human Cytomegalovirus into Epithelial and Endothelial Cells

ABSTRACTHuman cytomegalovirus (HCMV) replicates in many diverse cell typesin vivo, and entry into different cells involves distinct entry mechanisms and different envelope glycoproteins. HCMV glycoprotein gB is thought to act as the virus fusogen, apparently after being triggered by different gH/gL proteins that bind distinct cellular receptors or entry mediators. A trimer of gH/gL/gO is required for entry into all cell types, and entry into fibroblasts involves trimer binding to platelet-derived growth factor receptor alpha (PDGFRα). HCMV entry into biologically relevant epithelial and endothelial cells and monocyte-macrophages also requires a pentamer, gH/gL complexed with UL128, UL130, and UL131, and there is evidence that the pentamer binds unidentified receptors. We screened an epithelial cell cDNA library and identified the cell surface protein CD147, which increased entry of pentamer-expressing HCMV into HeLa cells but not entry of HCMV that lacked the pentamer. A panel of CD147-specific monoclonal antibodies inhibited HCMV entry into epithelial and endothelial cells, but not entry into fibroblasts. shRNA silencing of CD147 in endothelial cells inhibited HCMV entry but not entry into fibroblasts. CD147 colocalized with HCMV particles on cell surfaces and in endosomes. CD147 also promoted cell-cell fusion induced by expression of pentamer and gB in epithelial cells. However, soluble CD147 did not block HCMV entry and trimer and pentamer did not bind directly to CD147, supporting the hypothesis that CD147 acts indirectly through other proteins. CD147 represents the first HCMV entry mediator that specifically functions to promote entry of pentamer-expressing HCMV into epithelial and endothelial cells.IMPORTANCEHuman cytomegalovirus infects nearly 80% of the world’s population and causes significant morbidity and mortality. The current method of treatment involves the use of antiviral agents that are prone to resistance and can be highly toxic to patients; currently, there is no vaccine against HCMV available. HCMV infections involve virus dissemination throughout the body, infecting a wide variety of tissues; however, the mechanism of spread is not well understood, particularly with regard to which cellular proteins are utilized by HCMV to establish infection. This report describes the characterization of a newly identified cellular molecule that affects HCMV entry into epithelial and endothelial cells. These results will lead to a better understanding of HCMV pathogenesis and have implications for the development of future therapeutics.

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Murine cytomegaloviruses m139 targets DDX3 to curtail interferon production and promote viral replication

10.1101/2020.04.17.046516 ◽

2020 ◽

Cited By ~ 1

Author(s):

Olha Puhach ◽

Eleonore Ostermann ◽

Christoph Krisp ◽

Giada Frascaroli ◽

Hartmut Schlüter ◽

...

Keyword(s):

Endothelial Cells ◽

Viral Replication ◽

Human Cytomegalovirus ◽

Antiviral Treatment ◽

Cell Types ◽

The Body ◽

Productive Infection ◽

Murine Cytomegalovirus ◽

Efficient Viral Replication

AbstractCytomegaloviruses (CMV) infect many different cell types and tissues in their respective hosts. Monocytes and macrophages play an important role in CMV dissemination from the site of infection to target organs. Moreover, macrophages are specialized in pathogen sensing and respond to infection by secreting cytokines and interferons. In murine cytomegalovirus (MCMV), a model for human cytomegalovirus, several genes required for efficient replication in macrophages have been identified, but their specific functions remain poorly understood. Here we show that MCMV m139, a gene of the conserved US22 gene family, encodes a protein that interacts with the DEAD box helicase DDX3, a protein involved in pathogen sensing and interferon (IFN) induction, and the E3 ubiquitin ligase UBR5. DDX3 and UBR5 also participate in the transcription, processing, and translation of a subset of cellular mRNAs. We show that m139 inhibits DDX3-mediated IFN-β induction and is necessary for efficient viral replication in bone-marrow derived macrophages. In vivo, m139 is crucial for viral dissemination to local lymph nodes and to the salivary glands. An m139-deficient MCMV also replicated to lower titers in SVEC4-10 endothelial cells. This replication defect was not accompanied by increased IFN-β transcription, but was rescued by knockout of either DDX3 or UBR5. Moreover, m139 co-localized with DDX3 and UBR5 in viral replication compartments in the cell nucleus. These results suggest that m139 inhibits DDX3-mediated IFN-β production in macrophages and antagonizes DDX3 and UBR5-dependent functions related to RNA metabolism in endothelial cells.Author SummaryHuman cytomegalovirus is an opportunistic pathogen that causes severe infections in immunocompromised individuals. The virus infects certain types, such as macrophages and endothelial cells, to ensure its dissemination within the body. Little is known about the viral factors that promote a productive infection of these cell types. The identification of critical viral factors and the molecular pathways they target can lead to the development of novel antiviral treatment strategies. Using the mouse cytomegalovirus as a model, we studied the viral m139 gene, which is important for virus replication in macrophages and endothelial cells and for dissemination in the mouse. This gene encodes a protein that interacts with the host proteins DDX3 and UBR5. Both proteins are involved in gene expression, and the RNA helicase DDX3 also participates in mounting an innate antiviral response. By interacting with DDX3 and UBR5, m139 ensures efficient viral replication in endothelial cells. Importantly, we identify m139 as a new viral DDX3 inhibitor, which curtails the production of interferon in macrophages.

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A synthetic polypeptide based on human E-cadherin inhibits invasion of human intestinal and liver cell lines by Listeria monocytogenes

Journal of Medical Microbiology ◽

10.1099/jmm.0.47194-0 ◽

2007 ◽

Vol 56 (8) ◽

pp. 1011-1016 ◽

Cited By ~ 6

Author(s):

Saura C. Sahu ◽

Dennis W. Gaines ◽

Kristina M. Williams ◽

Richard B. Raybourne

Keyword(s):

Listeria Monocytogenes ◽

Cell Lines ◽

Surface Protein ◽

Cell Types ◽

Single Amino Acid ◽

Cell Protein ◽

Biologically Relevant ◽

E Cadherin

Internalin A is a surface protein of the facultative intracellular pathogen Listeria monocytogenes that interacts with the human host cell protein E-cadherin to facilitate invasion of epithelial cells. A single amino acid substitution at position 16 in mouse E-cadherin prevents this interaction. Synthetic polypeptides of 30 aa encompassing position 16 of human and mouse E-cadherin were tested for their ability to inhibit in vitro invasion of Caco-2, HepG2 and TIB73 cell lines by L. monocytogenes. Only the human-derived peptide was capable of inhibiting invasion in the human-origin Caco-2 and HepG2 cell lines. These findings demonstrate that small polypeptides can inhibit invasion of biologically relevant cell types by L. monocytogenes in vitro and may be potentially useful as therapeutic agents in vivo.

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

Thrombosis and Haemostasis ◽

10.1160/th09-04-0213 ◽

2009 ◽

Vol 102 (12) ◽

pp. 1057-1063 ◽

Cited By ~ 35

Author(s):

Christian Sinzger ◽

Barbara Adler

Keyword(s):

Endothelial Cells ◽

Human Cytomegalovirus ◽

Cell Types ◽

The Body ◽

Cell Type ◽

Cell Culture Systems ◽

Human Cytomegalovirus Infection ◽

Productive Replication

SummaryEndothelial 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.

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Regenerating vascular mural cells in zebrafish fin blood vessels are not derived from pre-existing ones and differentially require pdgfrb signaling for their development

10.1101/2021.03.27.437334 ◽

2021 ◽

Author(s):

Arndt F Siekmann ◽

Elvin Vincent Leonard ◽

Ricardo Figueroa ◽

Jeroen Bussmann ◽

Julio D Amigo ◽

...

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Tissue Regeneration ◽

Growth Factor Receptor ◽

Cell Types ◽

Lineage Tracing ◽

The Body ◽

Mural Cell ◽

Caudal Fin ◽

Mural Cells

Vascular networks are comprised of endothelial cells and mural cells, which include pericytes and smooth muscle cells. It is well established that new endothelial cells are derived from pre-existing ones during the angiogenic phase of blood vessel growth. By contrast, mural cell ontogeny is less clear with an ongoing debate whether mural cells possess mesenchymal stem cell properties. To elucidate the mechanisms controlling mural cell recruitment during development and tissue regeneration, we studied the formation of zebrafish caudal fin arteries. Mural cells showed morphological heterogeneity: cells colonizing arteries proximal to the body wrapped around them, while those in more distal regions extended protrusions along the proximo-distal vascular axis. Despite these differences, both cell populations expressed platelet-derived growth factor receptor beta (Pdgfrb) and the smooth muscle cell marker myosin heavy chain 11a (Myh11a). Loss of Pdgfrb signalling during development or tissue regeneration resulted in a substantial decrease in mural cells at the vascular front, while those proximal to the body were less affected. Using lineage tracing, we demonstrate that precursor cells located in periarterial regions of the caudal fin and expressing Pgdfrb can give rise to mural cells, while in regeneration newly formed mural cells were not derived from pre-existing ones. Together, our findings reveal conserved roles for pdgfrb signalling in development and regeneration, while at the same time illustrating a limited capacity of mural cells to self-renew or contribute to other cell types during tissue regeneration.

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The Human Cytomegalovirus Trimer and Pentamer Promote Sequential Steps in Entry into Epithelial and Endothelial Cells at Cell Surfaces and Endosomes

Journal of Virology ◽

10.1128/jvi.01336-18 ◽

2018 ◽

Vol 92 (21) ◽

Cited By ~ 21

Author(s):

Jing Liu ◽

Ted S. Jardetzky ◽

Andrea L. Chin ◽

David C. Johnson ◽

Adam L. Vanarsdall

Keyword(s):

Endothelial Cells ◽

Epithelial Cells ◽

Cell Surface ◽

Human Cytomegalovirus ◽

Antiviral Agents ◽

Cell Types ◽

Surface Binding ◽

Surface Receptors ◽

Virus Particles ◽

Early Endosomes

ABSTRACTHuman cytomegalovirus (HCMV) infects a wide variety of human cell types by different entry pathways that involve distinct envelope glycoprotein complexes that include gH/gL, a trimer complex consisting of gHgL/gO, and a pentamer complex consisting of gH/gL/UL128/UL130/UL131. We characterized the effects of soluble forms of these proteins on HCMV entry. Soluble trimer and pentamer blocked entry of HCMV into epithelial and endothelial cells, whereas soluble gH/gL did not. Trimer inhibited HCMV entry into fibroblast cells, but pentamer and gH/gL did not. Both trimer and pentamer bound to the surfaces of fibroblasts and epithelial cells, whereas gH/gL did not bind to either cell type. Cell surface binding of trimer and pentamer did not involve heparin sulfate moieties. The ability of soluble trimer to block entry of HCMV into epithelial cells did not involve platelet-derived growth factor PDGFRα, which has been reported as a trimer receptor for fibroblasts. Soluble trimer reduced the amount of virus particles that could be adsorbed onto the surface of epithelial cells, whereas soluble pentamer had no effect on virus adsorption. However, soluble pentamer reduced the ability of virus particles to exit from early endosomes into the cytoplasm and then travel to the nucleus. These studies support a model in which both the trimer and pentamer are required for HCMV entry into epithelial and endothelial cells, with trimer interacting with cell surface receptors other than PDGFR and pentamer acting later in the entry pathway to promote egress from endosomes.IMPORTANCEHCMV infects nearly 80% of the world's population and causes significant morbidity and mortality. The current antiviral agents used to treat HCMV infections are prone to resistance and can be toxic to patients, and there is no current vaccine against HCMV available. The data in this report will lead to a better understanding of how essential HCMV envelope glycoproteins function during infection of biologically important cell types and will have significant implications for understanding HCMV pathogenesis for developing new therapeutics.

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Analyses of the pericyte transcriptome in ischemic skeletal muscles

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02247-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yuan-chi Teng ◽

Alfredo Leonardo Porfírio-Sousa ◽

Giulia Magri Ribeiro ◽

Marcela Corso Arend ◽

Lindolfo da Silva Meirelles ◽

...

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Limb Ischemia ◽

Inflammatory Cells ◽

Cell Types ◽

Arterial Disease ◽

The Body ◽

Time Points

Abstract Background Peripheral arterial disease (PAD) affects millions of people and compromises quality of life. Critical limb ischemia (CLI), which is the most advanced stage of PAD, can cause nonhealing ulcers and strong chronic pain, and it shortens the patients’ life expectancy. Cell-based angiogenic therapies are becoming a real therapeutic approach to treat CLI. Pericytes are cells that surround vascular endothelial cells to reinforce vessel integrity and regulate local blood pressure and metabolism. In the past decade, researchers also found that pericytes may function as stem or progenitor cells in the body, showing the potential to differentiate into several cell types. We investigated the gene expression profiles of pericytes during the early stages of limb ischemia, as well as the alterations in pericyte subpopulations to better understand the behavior of pericytes under ischemic conditions. Methods In this study, we used a hindlimb ischemia model to mimic CLI in C57/BL6 mice and explore the role of pericytes in regeneration. To this end, muscle pericytes were isolated at different time points after the induction of ischemia. The phenotypes and transcriptomic profiles of the pericytes isolated at these discrete time points were assessed using flow cytometry and RNA sequencing. Results Ischemia triggered proliferation and migration and upregulated the expression of myogenesis-related transcripts in pericytes. Furthermore, the transcriptomic analysis also revealed that pericytes induce or upregulate the expression of a number of cytokines with effects on endothelial cells, leukocyte chemoattraction, or the activation of inflammatory cells. Conclusions Our findings provide a database that will improve our understanding of skeletal muscle pericyte biology under ischemic conditions, which may be useful for the development of novel pericyte-based cell and gene therapies.

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Clock gene-independent daily regulation of haemoglobin oxidation in red blood cells

10.1101/2021.10.11.463714 ◽

2021 ◽

Author(s):

Andrew D. Beale ◽

Priya Crosby ◽

Utham K. Valekunja ◽

Rachel S. Edgar ◽

Johanna E. Chesham ◽

...

Keyword(s):

Circadian Rhythms ◽

Red Blood Cells ◽

Blood Cells ◽

Human Subjects ◽

Developmental Regulation ◽

Physiological Role ◽

Cell Types ◽

The Body

AbstractCellular circadian rhythms confer daily temporal organisation upon behaviour and physiology that is fundamental to human health and disease. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body. Being naturally anucleate, RBC circadian rhythms share key elements of post-translational, but not transcriptional, regulation with other cell types. The physiological function and developmental regulation of RBC circadian rhythms is poorly understood, however, partly due to the small number of appropriate techniques available. Here, we extend the RBC circadian toolkit with a novel biochemical assay for haemoglobin oxidation status, termed “Bloody Blotting”. Our approach relies on a redox-sensitive covalent haem-haemoglobin linkage that forms during cell lysis. Formation of this linkage exhibits daily rhythms in vitro, which are unaffected by mutations that affect the timing of circadian rhythms in nucleated cells. In vivo, haemoglobin oxidation rhythms demonstrate daily variation in the oxygen-carrying and nitrite reductase capacity of the blood, and are seen in human subjects under controlled laboratory conditions as well as in freely-behaving humans. These results extend our molecular understanding of RBC circadian rhythms and suggest they serve an important physiological role in gas transport.

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Mast Cell-Specific Expression of Human Siglec-8 in Conditional Knock-in Mice

International Journal of Molecular Sciences ◽

10.3390/ijms20010019 ◽

2018 ◽

Vol 20 (1) ◽

pp. 19 ◽

Cited By ~ 13

Author(s):

Yadong Wei ◽

Krishan Chhiba ◽

Fengrui Zhang ◽

Xujun Ye ◽

Lihui Wang ◽

...

Keyword(s):

Flow Cytometry ◽

Mast Cell ◽

Mast Cells ◽

Es Cells ◽

Embryonic Stem ◽

Surface Protein ◽

Cell Types ◽

Mouse Strains ◽

Specific Expression

Sialic acid-binding Ig-like lectin 8 (Siglec-8) is expressed on the surface of human eosinophils, mast cells, and basophils—cells that participate in allergic and other diseases. Ligation of Siglec-8 by specific glycan ligands or antibodies triggers eosinophil death and inhibits mast cell degranulation; consequences that could be leveraged as treatment. However, Siglec-8 is not expressed in murine and most other species, thus limiting preclinical studies in vivo. Based on a ROSA26 knock-in vector, a construct was generated that contains the CAG promoter, a LoxP-floxed-Neo-STOP fragment, and full-length Siglec-8 cDNA. Through homologous recombination, this Siglec-8 construct was targeted into the mouse genome of C57BL/6 embryonic stem (ES) cells, and chimeric mice carrying the ROSA26-Siglec-8 gene were generated. After cross-breeding to mast cell-selective Cre-recombinase transgenic lines (CPA3-Cre, and Mcpt5-Cre), the expression of Siglec-8 in different cell types was determined by RT-PCR and flow cytometry. Peritoneal mast cells (dual FcεRI+ and c-Kit+) showed the strongest levels of surface Siglec-8 expression by multicolor flow cytometry compared to expression levels on tissue-derived mast cells. Siglec-8 was seen on a small percentage of peritoneal basophils, but not other leukocytes from CPA3-Siglec-8 mice. Siglec-8 mRNA and surface protein were also detected on bone marrow-derived mast cells. Transgenic expression of Siglec-8 in mice did not affect endogenous numbers of mast cells when quantified from multiple tissues. Thus, we generated two novel mouse strains, in which human Siglec-8 is selectively expressed on mast cells. These mice may enable the study of Siglec-8 biology in mast cells and its therapeutic targeting in vivo.

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Human Cytomegalovirus Replication and Infection-Induced Syncytia Formation in Labial, Foreskin, and Fetal Lung Fibroblasts

Viruses ◽

10.3390/v13122355 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2355

Author(s):

Alexis Aguiar ◽

Melissa Galinato ◽

Maite’ Bradley Silva ◽

Bryant Toth ◽

Michael A. McVoy ◽

...

Keyword(s):

Endothelial Cells ◽

Human Cytomegalovirus ◽

Fetal Lung ◽

Lung Fibroblasts ◽

Anatomical Site ◽

Cmv Infection ◽

Viral Spread ◽

Syncytia Formation

Only a handful of cell types, including fibroblasts, epithelial, and endothelial cells, can support human cytomegalovirus (CMV) replication in vitro, in striking contrast to the situation in vivo. While the susceptibility of epithelial and endothelial cells to CMV infection is strongly modulated by their anatomical site of origin, multiple CMV strains have been successfully isolated and propagated on fibroblasts derived from different organs. As oral mucosal cells are likely involved in CMV acquisition, we sought to evaluate the ability of infant labial fibroblasts to support CMV replication, compared to that of commonly used foreskin and fetal lung fibroblasts. No differences were found in the proportion of cells initiating infection, or in the amounts of viral progeny produced after exposure to the fibroblast-adapted CMV strain AD169 or to the endothelial cell-adapted strain TB40/E. Syncytia formation was, however, significantly enhanced in infected labial and lung fibroblasts compared to foreskin-derived cells, and did not occur after infection with AD169. Together, these data indicate that fibroblast populations derived from different tissues are uniformly permissive to CMV infection but retain phenotypic differences of potential importance for infection-induced cell–cell fusion, and ensuing viral spread and pathogenesis in different organs.

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Cell type-specific biogenesis of novel vesicles containing viral products in human cytomegalovirus infection

10.1101/2020.12.10.420711 ◽

2020 ◽

Cited By ~ 1

Author(s):

Samina Momtaz ◽

Belen Molina ◽

Luwanika Mlera ◽

Felicia Goodrum ◽

Jean M. Wilson

Keyword(s):

Endothelial Cells ◽

Human Cytomegalovirus ◽

Biosynthetic Pathway ◽

Cell Types ◽

Endocytic Pathway ◽

Specific Cell ◽

Cell Type ◽

Subcellular Compartment ◽

Increased Risk ◽

Cell Type Specific

AbstractHuman cytomegalovirus (HCMV), while highly restricted for the human species, infects an unlimited array of cell types in the host. Patterns of infection are dictated by the cell type infected, but cell type-specific factors and how they impact tropism for specific cell types is poorly understood. Previous studies in primary endothelial cells showed that HCMV infection induces large multivesicular-like bodies that incorporate viral products including dense bodies and virions. Here we define the nature of these large vesicles using a recombinant virus where UL32, encoding the pp150 tegument protein, is fused in frame with green fluorescent protein (GFP, TB40/E-UL32-GFP). Cells were fixed and labeled with antibodies against subcellular compartment markers and imaged using confocal and super-resolution microscopy. In fibroblasts, UL32-GFP-positive vesicles were marked with classical markers of MVBs, including CD63 and lysobisphosphatidic acid (LBPA), both classical MVB markers, as well as the clathrin and LAMP1. Unexpectedly, UL32-GFP-positive vesicles in endothelial cells were not labeled by CD63, and LBPA was completely lost from infected cells. We defined these UL32-positive vesicles in endothelial cells using markers for the cis-Golgi (GM130), lysosome (LAMP1), and autophagy (LC3B). These findings suggest that virus-containing MVBs in fibroblasts are derived from the canonical endocytic pathway and takeover classical exosomal release pathway. Virus containing MVBs in HMVECs are derived from the early biosynthetic pathway and exploit a less characterized early Golgi-LAMP1-associated non-canonical secretory autophagy pathway. These results reveal striking cell-type specific membrane trafficking differences in host pathways that are exploited by HCMV.ImportanceHuman cytomegalovirus (HCMV) is a herpesvirus that, like all herpesvirus, that establishes a life long infection. HCMV remains a significant cause of morbidity and mortality in the immunocompromised and HCMV seropositivity is associated with increased risk vascular disease. HCMV infects many cells in the human and the biology underlying the different patterns of infection in different cell types is poorly understood. Endothelial cells are important target of infection that contribute to hematogenous spread of the virus to tissues. Here we define striking differences in the biogenesis of large vesicles that incorporate virions in fibroblasts and endothelial cells. In fibroblasts, HCMV is incorporated into canonical MVBs derived from an endocytic pathway, whereas HCMV matures through vesicles derived from the biosynthetic pathway in endothelial cells. This work defines basic biological differences between these cell types that may impact the outcome of infection.

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