Dendritic-cell infection by human cytomegalovirus is restricted to strains carrying functional UL131–128 genes and mediates efficient viral antigen presentation to CD8+ T cells

Human cytomegalovirus (HCMV) genetic determinants of endothelial-cell tropism and virus transfer to leukocytes (both polymorphonuclear and monocyte) have been recently identified in the UL131–128 genes. Here it is documented that the same genetic determinants of HCMV are responsible for monocyte-derived dendritic-cell (DC) tropism, i.e. all endotheliotropic and leukotropic strains of HCMV are also DC-tropic (or dendrotropic). In fact, all recent clinical HCMV isolates and deletion mutants sparing the UL131–128 locus as well as the endotheliotropic revertants AD169 and Towne were able to productively infect DC following co-culture with infected endothelial cells. On the contrary, the same clinical isolates extensively propagated in human fibroblasts, the UL131–128 deletion mutants and the reference laboratory strains were not. Peak extracellular virus titres in DC were reached 4–7 days post-infection (p.i.). Viral proteins pp65 and p72 were detected 1–3 h p.i., involving the great majority of DC 24 h p.i., while gB was abundantly detected 96 h p.i., when a cytopathic effect first appeared. Infection of DC with cell-free virus released into the medium could only be achieved with HCMV strains extensively adapted to growth in endothelial cells, reaching the peak titres 10 days p.i. DC infected for 24 h with cell-free virus and incubated for 16 h with autologous peripheral blood mononuclear cells were found to act as a potent stimulator of both HCMV-specific CD4+- and CD8+-mediated immune responses, as determined by cytokine flow cytometry. DC incubated with inactivated crude whole viral antigen preparations were only capable of eliciting a significant CD4+-mediated immune response.

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Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions

Journal of Virology ◽

10.1128/jvi.02256-09 ◽

2009 ◽

Vol 84 (5) ◽

pp. 2597-2609 ◽

Cited By ~ 38

Author(s):

Brent J. Ryckman ◽

Marie C. Chase ◽

David C. Johnson

Keyword(s):

Endothelial Cells ◽

Human Cytomegalovirus ◽

Null Mutant ◽

Virus Particles ◽

Extracellular Virus ◽

Biochemical Studies ◽

Infected Cells ◽

Low Passage ◽

Er Export ◽

Strain Ad169

ABSTRACT Human cytomegalovirus (HCMV) produces the following two gH/gL complexes: gH/gL/gO and gH/gL/UL128-131. Entry into epithelial and endothelial cells requires gH/gL/UL128-131, and we have provided evidence that gH/gL/UL128-131 binds saturable epithelial cell receptors to mediate entry. HCMV does not require gH/gL/UL128-131 to enter fibroblasts, and laboratory adaptation to fibroblasts results in mutations in the UL128-131 genes, abolishing infection of epithelial and endothelial cells. HCMV gO-null mutants produce very small plaques on fibroblasts yet can spread on endothelial cells. Thus, one prevailing model suggests that gH/gL/gO mediates infection of fibroblasts, while gH/gL/UL128-131 mediates entry into epithelial/endothelial cells. Most biochemical studies of gO have involved the HCMV lab strain AD169, which does not assemble gH/gL/UL128-131 complexes. We examined gO produced by the low-passage clinical HCMV strain TR. Surprisingly, TR gO was not detected in purified extracellular virus particles. In TR-infected cells, gO remained sensitive to endoglycosidase H, suggesting that the protein was not exported from the endoplasmic reticulum (ER). However, TR gO interacted with gH/gL in the ER and promoted export of gH/gL from the ER to the Golgi apparatus. Pulse-chase experiments showed that a fraction of gO remained bound to gH/gL for relatively long periods, but gO eventually dissociated or was degraded and was not found in extracellular virions or secreted from cells. The accompanying report by P. T. Wille et al. (J. Virol., 84:2585-2596, 2010) showed that a TR gO-null mutant failed to incorporate gH/gL into virions and that the mutant was unable to enter fibroblasts and epithelial and endothelial cells. We concluded that gO acts as a molecular chaperone, increasing gH/gL ER export and incorporation into virions. It appears that gO competes with UL128-131 for binding onto gH/gL but is released from gH/gL, so that gH/gL (lacking UL128-131) is incorporated into virions. Thus, our revised model suggests that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.

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The Human Cytomegalovirus Ribonucleotide Reductase Homolog UL45 Is Dispensable for Growth in Endothelial Cells, as Determined by a BAC-Cloned Clinical Isolate of Human Cytomegalovirus with Preserved Wild-Type Characteristics

Journal of Virology ◽

10.1128/jvi.76.18.9551-9555.2002 ◽

2002 ◽

Vol 76 (18) ◽

pp. 9551-9555 ◽

Cited By ~ 94

Author(s):

Gabriele Hahn ◽

Hanna Khan ◽

Fausto Baldanti ◽

Ulrich H. Koszinowski ◽

M. Grazia Revello ◽

...

Keyword(s):

Endothelial Cells ◽

Clinical Isolate ◽

Human Cytomegalovirus ◽

Ribonucleotide Reductase ◽

Human Fibroblasts ◽

Artificial Chromosome ◽

Cell Types ◽

Factor X ◽

Wild Type ◽

Human Endothelial Cells

ABSTRACT An endothelial cell-tropic and leukotropic human cytomegalovirus (HCMV) clinical isolate was cloned as a fusion-inducing factor X-bacterial artificial chromosome in Escherichia coli, and the ribonucleotide reductase homolog UL45 was deleted. Reconstituted virus RVFIX and RVΔUL45 grew equally well in human fibroblasts and human endothelial cells. Thus, UL45 is dispensable for growth of HCMV in both cell types.

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Human Cytomegalovirus Entry into Epithelial and Endothelial Cells Depends on Genes UL128 to UL150 and Occurs by Endocytosis and Low-pH Fusion

Journal of Virology ◽

10.1128/jvi.80.2.710-722.2006 ◽

2006 ◽

Vol 80 (2) ◽

pp. 710-722 ◽

Cited By ~ 229

Author(s):

Brent J. Ryckman ◽

Michael A Jarvis ◽

Derek D. Drummond ◽

Jay A. Nelson ◽

David C. Johnson

Keyword(s):

Endothelial Cells ◽

Epithelial Cells ◽

Human Cytomegalovirus ◽

Early Stage ◽

Human Fibroblasts ◽

Cell Types ◽

Low Ph ◽

Virus Particles ◽

Peg Treatment ◽

Infection Processes

ABSTRACT Human cytomegalovirus (HCMV) replication in epithelial and endothelial cells appears to be important in virus spread, disease, and persistence. It has been difficult to study infection of these cell types because HCMV laboratory strains (e.g., AD169 and Towne) have lost their ability to infect cultured epithelial and endothelial cells during extensive propagation in fibroblasts. Clinical strains of HCMV (e.g., TR and FIX) possess a cluster of genes (UL128 to UL150) that are largely mutated in laboratory strains, and recent studies have indicated that these genes facilitate replication in epithelial and endothelial cells. The mechanisms by which these genes promote infection of these two cell types are unclear. We derived an HCMV UL128-to-UL150 deletion mutant from strain TR, TRΔ4, and studied early events in HCMV infection of epithelial and endothelial cells, and the role of genes UL128 to UL150. Analysis of wild-type TR indicated that HCMV enters epithelial and endothelial cells by endocytosis followed by low-pH-dependent fusion, which is different from the pH-independent fusion with the plasma membrane observed with human fibroblasts. TRΔ4 displayed a number of defects in early infection processes. Adsorption and entry of TRΔ4 on epithelial cells were poor compared with those of TR, but these defects could be overcome with higher doses of virus and the use of polyethylene glycol (PEG) to promote fusion between virion and cellular membranes. High multiplicity and PEG treatment did not promote infection of endothelial cells by TRΔ4, yet virus particles were internalized. Together, these data indicate that genes UL128 to UL150 are required for HCMV adsorption and penetration of epithelial cells and to promote some early stage of virus replication, subsequent to virus entry, in endothelial cells.

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Differential Requirement of Human Cytomegalovirus UL112-113 Protein Isoforms for Viral Replication

Journal of Virology ◽

10.1128/jvi.00254-17 ◽

2017 ◽

Vol 91 (17) ◽

Cited By ~ 5

Author(s):

Tim Schommartz ◽

Jiajia Tang ◽

Rebekka Brost ◽

Wolfram Brune

Keyword(s):

Endothelial Cells ◽

Dna Replication ◽

Viral Replication ◽

Dna Polymerase ◽

Human Cytomegalovirus ◽

Cell Nucleus ◽

Human Fibroblasts ◽

Gene Products ◽

Viral Dna ◽

Viral Dna Replication

ABSTRACT The UL112-113 gene is one of the few alternatively spliced genes of human cytomegalovirus (HCMV). It codes for four phosphoproteins, p34, p43, p50, and p84, all of which are expressed with early kinetics and accumulate at sites of viral DNA replication within the host cell nucleus. Although these proteins are known to play important, possibly essential, roles in the viral replication cycle, little is known about the contribution of individual UL112-113 protein products. Here we used splice site mutagenesis, intron deletion and substitution, and nonsense mutagenesis to prevent the individual expression of each UL112-113 protein isoform and to investigate the importance of each isoform for viral replication. We show that HCMV mutants lacking p34 or p50 expression replicated to high titers in human fibroblasts and endothelial cells, indicating that these proteins are nonessential for viral replication, while mutant viruses carrying a stop mutation within the p84 coding sequence were severely growth impaired. Viral replication could not be detected upon the inactivation of p43 expression, indicating that this UL112-113 protein is essential for viral replication. We also analyzed the ability of UL112-113 proteins to recruit other viral proteins to intranuclear prereplication compartments. While UL112-113 expression was sufficient to recruit the UL44-encoded viral DNA polymerase processivity factor, it was not sufficient for the recruitment of the viral UL84 and UL117 proteins. Remarkably, both the p43 and p84 isoforms were required for the efficient recruitment of pUL44, which is consistent with their critical role in the viral life cycle. IMPORTANCE Human cytomegalovirus requires gene products from 11 genetic loci for the lytic replication of its genome. One of these loci, UL112-113, encodes four proteins with common N termini by alternative splicing. In this study, we inactivated the expression of each of the four UL112-113 proteins individually and determined their requirement for HCMV replication. We found that two of the UL112-113 gene products were dispensable for viral replication in human fibroblasts and endothelial cells. In contrast, viral replication was severely reduced or absent when one of the other two gene products was inactivated, indicating that they are of crucial importance for the viral replication cycle. We further showed that the latter two gene products are involved in the recruitment of pUL44, an essential cofactor of the viral DNA polymerase, to specific sites within the cell nucleus that are thought to serve as starting points for viral DNA replication.

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A Human Cytomegalovirus gO-Null Mutant Fails To Incorporate gH/gL into the Virion Envelope and Is Unable To Enter Fibroblasts and Epithelial and Endothelial Cells

Journal of Virology ◽

10.1128/jvi.02249-09 ◽

2009 ◽

Vol 84 (5) ◽

pp. 2585-2596 ◽

Cited By ~ 105

Author(s):

Paul T. Wille ◽

Amber J. Knoche ◽

Jay A. Nelson ◽

Michael A. Jarvis ◽

David C. Johnson

Keyword(s):

Endothelial Cells ◽

Human Cytomegalovirus ◽

Cell Types ◽

Null Mutant ◽

Virus Envelope ◽

Virus Particles ◽

Extracellular Virus ◽

Hcmv Strain ◽

Virion Envelope ◽

Er Export

ABSTRACT Human cytomegalovirus (HCMV) depends upon a five-protein complex, gH/gL/UL128-131, to enter epithelial and endothelial cells. A separate HCMV gH/gL-containing complex, gH/gL/gO, has been described. Our prevailing model is that gH/gL/UL128-131 is required for entry into biologically important epithelial and endothelial cells and that gH/gL/gO is required for infection of fibroblasts. Genes encoding UL128-131 are rapidly mutated during laboratory propagation of HCMV on fibroblasts, apparently related to selective pressure for the fibroblast entry pathway. Arguing against this model in the accompanying paper by B. J. Ryckman et al. (J. Virol., 84:2597-2609, 2010), we describe evidence that clinical HCMV strain TR expresses a gO molecule that acts to promote endoplasmic reticulum (ER) export of gH/gL and that gO is not stably incorporated into the virus envelope. This was different from results involving fibroblast-adapted HCMV strain AD169, which incorporates gO into the virion envelope. Here, we constructed a TR gO-null mutant, TRΔgO, that replicated to low titers, spread poorly among fibroblasts, but produced normal quantities of extracellular virus particles. TRΔgO particles released from fibroblasts failed to infect fibroblasts and epithelial and endothelial cells, but the chemical fusogen polyethylene glycol (PEG) could partially overcome defects in infection. Therefore, TRΔgO is defective for entry into all three cell types. Defects in entry were explained by observations showing that TRΔgO incorporated about 5% of the quantities of gH/gL in extracellular virus particles compared with that in wild-type virions. Although TRΔgO particles could not enter cells, cell-to-cell spread involving epithelial and endothelial cells was increased relative to TR, apparently resulting from increased quantities of gH/gL/UL128-131 in virions. Together, our data suggest that TR gO acts as a chaperone to promote ER export and the incorporation of gH/gL complexes into the HCMV envelope. Moreover, these data suggest that it is gH/gL, and not gH/gL/gO, that is present in virions and is required for infection of fibroblasts and epithelial and endothelial cells. Our observations that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial/endothelial cells differ from models for other beta- and gammaherpesviruses that use one of two different gH/gL complexes to enter different cells.

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Human Cytomegalovirus Persistently Infects Aortic Endothelial Cells

Journal of Virology ◽

10.1128/jvi.72.7.5661-5668.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 5661-5668 ◽

Cited By ~ 88

Author(s):

Kenneth N. Fish ◽

Cecilia Soderberg-Naucler ◽

Lisa K. Mills ◽

Stephan Stenglein ◽

Jay A. Nelson

Keyword(s):

Endothelial Cells ◽

Human Cytomegalovirus ◽

Antigen Expression ◽

Cell Types ◽

Aortic Endothelial Cells ◽

Phenotypic Differences ◽

Extracellular Virus ◽

Viral Particles ◽

Infected Cells ◽

Hcmv Replication

ABSTRACT Endothelial cells (EC) have been implicated as constituting an important cell type in the pathogenesis of human cytomegalovirus (HCMV). Microvascular and macrovascular EC exhibit different biochemical and functional properties depending on the organ of origin. Phenotypic differences between microvascular and macrovascular EC may alter the ability of these cells to support HCMV replication. In this study, we compared the replication of HCMV in primary macrovascular aortic EC (AEC) with that in brain microvascular EC (BMVEC). An examination of IE72, pp65, and gB viral antigen expression in BMVEC and AEC by immunoflourescence revealed similar frequencies of infected cells. Intracellular production of virus was 3 log units greater in BMVEC than in AEC, while equal quantities of extracellular virus were produced in both cell types. HCMV infection of BMVEC resulted in rapid cellular lysis, while the virus was nonlytic and continuously released from HCMV-infected AEC for the life span of the culture. An examination of infected cells by electron microscopy revealed the formation of abundant nucleocapsids in both AEC and BMVEC. However, significant amounts of mature viral particles were only detected in the cytoplasm of BMVEC. These observations indicate that levels of HCMV replication in EC obtained from different organs are distinct and suggest that persistently infected AEC may serve as a reservoir of virus.

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Requirement for Both D Domains of the Propolypeptide in von Willebrand Factor Muitimerization and Storage

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649724 ◽

1993 ◽

Vol 70 (06) ◽

pp. 1053-1057 ◽

Cited By ~ 32

Author(s):

Agnès M Journet ◽

Simin Saffaripour ◽

Denisa D Wagner

Keyword(s):

Endothelial Cells ◽

Endoplasmic Reticulum ◽

Von Willebrand Factor ◽

Deletion Mutants ◽

Relative Importance ◽

D2 Domain ◽

Constitutive Secretion ◽

Von Willebrand ◽

And Storage ◽

Willebrand Factor

SummaryBiosynthesis of the adhesive glycoprotein von Willebrand factor (vWf) by endothelial cells results in constitutive secretion of small multimers and storage of the largest multimers in rodshaped granules called Weibel-Palade bodies. This pattern is reproduced by expression of pro-vWf in heterologous cells with a regulated pathway of secretion, that store the recombinant protein in similar elongated granules. In these cells, deletion of the vWf prosequence prevents vWf storage. The prosequence, composed of two homologous domains (D1 and D2), actively participates in vWf multimer formation as well. We expressed deletion mutants lacking either the D1 domain (D2vWf) or the D2 domain (D1vWf) in various cell lines to analyze the relative importance of each domain in vWf muitimerization and storage. Both proteins were secreted efficiently without being retained in the endoplasmic reticulum. Despite this, neither multimerized past the dimer stage and they were not stored. We conclude that several segments of the prosequence are jointly involved in vWf muitimerization and storage.

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In vitro Three Dimensional Scaffold-free Construct of Human Adipose-derived Stem Cells in Coculture with Endothelial Cells and Fibroblasts

Revista de Chimie ◽

10.37358/rc.17.6.5670 ◽

2017 ◽

Vol 68 (6) ◽

pp. 1341-1344

Author(s):

Grigore Berea ◽

Gheorghe Gh. Balan ◽

Vasile Sandru ◽

Paul Dan Sirbu

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Endothelial Cells ◽

Single Cell ◽

Cell Line ◽

Bone Repair ◽

Umbilical Vein ◽

Human Fibroblasts ◽

Three Dimensional ◽

Adipose Derived Stem Cells

Complex interactions between stem cells, vascular cells and fibroblasts represent the substrate of building microenvironment-embedded 3D structures that can be grafted or added to bone substitute scaffolds in tissue engineering or clinical bone repair. Human Adipose-derived Stem Cells (hASCs), human umbilical vein endothelial cells (HUVECs) and normal dermal human fibroblasts (NDHF) can be mixed together in three dimensional scaffold free constructs and their behaviour will emphasize their potential use as seeding points in bone tissue engineering. Various combinations of the aforementioned cell lines were compared to single cell line culture in terms of size, viability and cell proliferation. At 5 weeks, viability dropped for single cell line spheroids while addition of NDHF to hASC maintained the viability at the same level at 5 weeks Fibroblasts addition to the 3D construct of stem cells and endothelial cells improves viability and reduces proliferation as a marker of cell differentiation toward osteogenic line.

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