scholarly journals Genetic Stability of Bacterial Artificial Chromosome-Derived Human Cytomegalovirus during CultureIn Vitro

2016 ◽  
Vol 90 (8) ◽  
pp. 3929-3943 ◽  
Author(s):  
Isa Murrell ◽  
Gavin S. Wilkie ◽  
Andrew J. Davison ◽  
Evelina Statkute ◽  
Ceri A. Fielding ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Cell Types ◽  
Clinical Strain ◽  
Minimal Risk ◽  
Bacterial Artificial Chromosomes ◽  
Wild Type ◽  
Artificial Chromosomes

ABSTRACTClinical human cytomegalovirus (HCMV) strains invariably mutate when propagatedin vitro. Mutations in gene RL13 are selected in all cell types, whereas in fibroblasts mutants in the UL128 locus (UL128L; genes UL128, UL130, and UL131A) are also selected. In addition, sporadic mutations are selected elsewhere in the genome in all cell types. We sought to investigate conditions under which HCMV can be propagated without incurring genetic defects. Bacterial artificial chromosomes (BACs) provide a stable, genetically defined source of viral genome. Viruses were generated from BACs containing the genomes of strains TR, TB40, FIX, and Merlin, as well as from Merlin-BAC recombinants containing variant nucleotides in UL128L from TB40-BAC4 or FIX-BAC. Propagation of viruses derived from TR-BAC, TB40-BAC4, and FIX-BAC in either fibroblast or epithelial cells was associated with the generation of defects around the prokaryotic vector, which is retained in the unique short (US) region of viruses. This was not observed for Merlin-BAC, from which the vector is excised in derived viruses; however, propagation in epithelial cells was consistently associated with mutations in the unique longb′ (UL/b′) region, all impacting on gene UL141. Viruses derived from Merlin-BAC in fibroblasts had mutations in UL128L, but mutations occurred less frequently with recombinants containing UL128L nucleotides from TB40-BAC4 or FIX-BAC. Viruses derived from a Merlin-BAC derivative in which RL13 and UL128L were either mutated or repressed were remarkably stable in fibroblasts. Thus, HCMV containing a wild-type gene complement can be generatedin vitroby deriving virus from a self-excising BAC in fibroblasts and repressing RL13 and UL128L.IMPORTANCEResearchers should aim to study viruses that accurately represent the causative agents of disease. This is problematic for HCMV because clinical strains mutate rapidly when propagatedin vitro, becoming less cell associated, altered in tropism, more susceptible to natural killer cells, and less pathogenic. Following isolation from clinical material, HCMV genomes can be stabilized by cloning into bacterial artificial chromosomes (BACs), and then virus is regenerated by DNA transfection. However, mutations can occur not only during isolation prior to BAC cloning but also when virus is regenerated. We have identified conditions under which BAC-derived viruses containing an intact, wild-type genome can be propagatedin vitrowith minimal risk of mutants being selected, enabling studies of viruses expressing the gene complement of a clinical strain. However, even under these optimized conditions, sporadic mutations can occur, highlighting the advisability of sequencing the HCMV stocks used in experiments.

scholarly journals Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli: a New Approach for Construction of HCMV Mutants

1999 ◽  
Vol 73 (10) ◽  
pp. 8320-8329 ◽  
Author(s):  
Eva-Maria Borst ◽  
Gabriele Hahn ◽  
Ulrich H. Koszinowski ◽  
Martin Messerle
Keyword(s):  
Escherichia Coli ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Artificial Chromosome ◽  
Bacterial Artificial Chromosomes ◽  
Reading Frame ◽  
E Coli ◽  
Artificial Chromosomes ◽  
Internal Repeat ◽  
The Stability

ABSTRACT We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in Escherichia coli (M. Messerle, I. Crnković, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759–14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation inE. coli, during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in E. coli. The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.

scholarly journals 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

2002 ◽  
Vol 76 (18) ◽  
pp. 9551-9555 ◽  
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.

scholarly journals The pentameric complex drives immunologically covert cell–cell transmission of wild-type human cytomegalovirus

2017 ◽  
Vol 114 (23) ◽  
pp. 6104-6109 ◽  
Author(s):  
Isa Murrell ◽  
Carmen Bedford ◽  
Kristin Ladell ◽  
Kelly L. Miners ◽  
David A. Price ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Neutralizing Antibodies ◽  
Artificial Chromosome ◽  
Wild Type ◽  
Free Virus ◽  
Conditional Expression ◽  
Intact Virus ◽  
Cell Transmission ◽  
Cell Cell

Human cytomegalovirus (HCMV) strains that have been passaged in vitro rapidly acquire mutations that impact viral growth. These laboratory-adapted strains of HCMV generally exhibit restricted tropism, produce high levels of cell-free virus, and develop susceptibility to natural killer cells. To permit experimentation with a virus that retained a clinically relevant phenotype, we reconstructed a wild-type (WT) HCMV genome using bacterial artificial chromosome technology. Like clinical virus, this genome proved to be unstable in cell culture; however, propagation of intact virus was achieved by placing the RL13 and UL128 genes under conditional expression. In this study, we show that WT-HCMV produces extremely low titers of cell-free virus but can efficiently infect fibroblasts, epithelial, monocyte-derived dendritic, and Langerhans cells via direct cell–cell transmission. This process of cell–cell transfer required the UL128 locus, but not the RL13 gene, and was significantly less vulnerable to the disruptive effects of IFN, cellular restriction factors, and neutralizing antibodies compared with cell-free entry. Resistance to neutralizing antibodies was dependent on high-level expression of the pentameric gH/gL/gpUL128–131A complex, a feature of WT but not passaged strains of HCMV.

scholarly journals Human Cytomegalovirus US28 Is Important for Latent Infection of Hematopoietic Progenitor Cells

2015 ◽  
Vol 90 (6) ◽  
pp. 2959-2970 ◽  
Author(s):  
Monica S. Humby ◽  
Christine M. O'Connor
Keyword(s):  
Progenitor Cells ◽  
Human Cytomegalovirus ◽  
Latent Infection ◽  
Ex Vivo ◽  
Artificial Chromosome ◽  
Hematopoietic Progenitor Cells ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Recombinant Viruses

ABSTRACTHuman cytomegalovirus (HCMV) resides latently in hematopoietic progenitor cells (HPCs). During latency, only a subset of HCMV genes is transcribed, including one of the four virus-encoded G protein-coupled receptors (GPCRs), US28. Although US28 is a multifunctional lytic protein, its function during latency has remained undefined. We generated a panel of US28 recombinant viruses in the bacterial artificial chromosome (BAC)-derived clinical HCMV strain TB40/E-mCherry. We deleted the entire US28 open reading frame (ORF), deleted all four of the viral GPCR ORFs, or deleted three of the HCMV GPCRs but not the US28 wild-type protein. Using these recombinant viruses, we assessed the requirement for US28 during latency in the Kasumi-3in vitrolatency model system and in primaryex vivo-cultured CD34+HPCs. Our data suggest that US28 is required for latency as infection with viruses lacking the US28 ORF alone or in combination with the remaining HCMV-encoded GPCR results in transcription from the major immediate early promoter, the production of extracellular virions, and the production of infectious virus capable of infecting naive fibroblasts. The other HCMV GPCRs are not required for this phenotype as a virus expressing only US28 but not the remaining virus-encoded GPCRs is phenotypically similar to that of wild-type latent infection. Finally, we found that US28 copurifies with mature virions and is expressed in HPCs upon virus entry although its expression at the time of infection does not complement the US28 deletion latency phenotype. This work suggests that US28 protein functions to promote a latent state within hematopoietic progenitor cells.IMPORTANCEHuman cytomegalovirus (HCMV) is a widespread pathogen that, once acquired, remains with its host for life. HCMV remains latent, or quiescent, in cells of the hematopoietic compartment and upon immune challenge can reactivate to cause disease. HCMV-encoded US28 is one of several genes expressed during latency although its biological function during this phase of infection has remained undefined. Here, we show that US28 aids in promoting experimental latency in tissue culture.

scholarly journals Urine-Derived Epithelial Cells as Models for Genetic Kidney Diseases

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1413
Author(s):  
Tjessa Bondue ◽  
Fanny O. Arcolino ◽  
Koenraad R. P. Veys ◽  
Oyindamola C. Adebayo ◽  
Elena Levtchenko ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Kidney Diseases ◽  
Cell Types ◽  
Cell Models ◽  
Tubular Epithelial Cells ◽  
Disease Mechanisms ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular ◽  
Disease Cell

Epithelial cells exfoliated in human urine can include cells anywhere from the urinary tract and kidneys; however, podocytes and proximal tubular epithelial cells (PTECs) are by far the most relevant cell types for the study of genetic kidney diseases. When maintained in vitro, they have been proven extremely valuable for discovering disease mechanisms and for the development of new therapies. Furthermore, cultured patient cells can individually represent their human sources and their specific variants for personalized medicine studies, which are recently gaining much interest. In this review, we summarize the methodology for establishing human podocyte and PTEC cell lines from urine and highlight their importance as kidney disease cell models. We explore the well-established and recent techniques of cell isolation, quantification, immortalization and characterization, and we describe their current and future applications.

Cultured epithelial cells derived from human foetal pancreas as a model for the study of cystic fibrosis: further analyses on the origins and nature of the cell types

1988 ◽  
Vol 90 (1) ◽  
pp. 73-77
Author(s):  
A. Harris ◽  
L. Coleman
Keyword(s):  
Cystic Fibrosis ◽  
Tissue Culture ◽  
Epithelial Cells ◽  
Culture System ◽  
Cultured Cells ◽  
Cell Types ◽  
Cultured Epithelial Cells ◽  
Different Cell Types ◽  
Foetal Pancreas

The establishment of a tissue-culture system for epithelial cells derived from human foetal pancreas has recently been reported. Further analyses have now been made on these cells in vitro, together with parallel investigation of the distribution of different cell types within the intact foetal pancreas. Results support the view that the cultured cells are ductal in origin and nature. Pancreatic epithelial cell cultures have also been established from foetuses with cystic fibrosis.

scholarly journals Toll-Like Receptor 2 Controls the Gamma Interferon Response to Francisella tularensis by Mouse Liver Lymphocytes

2007 ◽  
Vol 75 (11) ◽  
pp. 5338-5345 ◽  
Author(s):  
Kee-Jong Hong ◽  
Jason R. Wickstrum ◽  
Hung-Wen Yeh ◽  
Michael J. Parmely
Keyword(s):  
Francisella Tularensis ◽  
Cell Types ◽  
Gamma Interferon ◽  
Interferon Response ◽  
Interleukin 12 ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Ifn Γ ◽  
Deficient Mice

ABSTRACT The production of gamma interferon (IFN-γ) is a key step in the protective innate immune response to Francisella tularensis. Natural killer cells and T cells in the liver are important sources of this cytokine during primary F. tularensis infections, and interleukin-12 (IL-12) appears to be an essential coactivating cytokine for hepatic IFN-γ expression. The present study was undertaken to determine whether or not macrophages (Mφ) or dendritic cells (DC) provide coactivating signals for the liver IFN-γ response in vitro, whether IL-12 mediates these effects, and whether Toll-like receptor (TLR) signaling is essential to induce this costimulatory activity. Both bone marrow-derived Mφ and DC significantly augmented the IFN-γ response of F. tularensis-challenged liver lymphocytes in vitro. While both cell types produced IL-12p40 in response to F. tularensis challenge, only DC secreted large quantities of IL-12p70. DC from both IL-12p35-deficient and TLR2-deficient mice failed to produce IL-12p70 and did not costimulate liver lymphocytes for IFN-γ production in response to viable F. tularensis organisms. Conversely, liver lymphocytes from TLR2-deficient mice cocultured with wild-type accessory cells produced IFN-γ at levels comparable to those for wild-type hepatic lymphocytes. These findings indicate that TLR2 controls hepatic lymphocyte IFN-γ responses to F. tularensis by regulating DC IL-12 production. While Mφ also coinduced hepatic IFN-γ production in response to F. tularensis, they did so in a fashion less dependent on TLR2.

scholarly journals Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization

2020 ◽  
Author(s):  
Om Srivast ◽  
Kiran Srivast ◽  
Roy Joseph ◽  
Landon Wilson
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Immunogold Labeling ◽  
Human Lens ◽  
Membrane Association ◽  
Related Protein ◽  
Wild Type ◽  
Age Related ◽  
Ionic Imbalance

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods: Lenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.Results: Compared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice. Conclusions: The results show that an increased lens membrane association of αAN101D--relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

scholarly journals Absence of the Macrophage Mannose Receptor in Mice Does Not Increase Susceptibility to Pneumocystis carinii Infection In Vivo

2003 ◽  
Vol 71 (11) ◽  
pp. 6213-6221 ◽  
Author(s):  
Steve D. Swain ◽  
Sena J. Lee ◽  
Michel C. Nussenzweig ◽  
Allen G. Harmsen
Keyword(s):  
Pneumocystis Carinii ◽  
Hydrolytic Enzymes ◽  
Mannose Receptor ◽  
Opportunistic Pathogen ◽  
Cell Types ◽  
Surface Expression ◽  
Wild Type ◽  
Macrophage Mannose Receptor

ABSTRACT Host defense against the opportunistic pathogen Pneumocystis carinii requires functional interactions of many cell types. Alveolar macrophages are presumed to be a vital host cell in the clearance of P. carinii, and the mechanisms of this interaction have come under scrutiny. The macrophage mannose receptor is believed to play an important role as a receptor involved in the binding and phagocytosis of P. carinii. Although there is in vitro evidence for this interaction, the in vivo role of this receptor in P. carinii clearance in unclear. Using a mouse model in which the mannose receptor has been deleted, we found that the absence of this receptor is not sufficient to allow infection by P. carinii in otherwise immunocompetent mice. Furthermore, when mice were rendered susceptible to P. carinii by CD4+ depletion, mannose receptor knockout mice (MR-KO) had pathogen loads equal to those of wild-type mice. However, the MR-KO mice exhibited a greater influx of phagocytes into the alveoli during infection. This was accompanied by increased pulmonary pathology in the MR-KO mice, as well as greater accumulation of glycoproteins in the alveoli (glycoproteins, including harmful hydrolytic enzymes, are normally cleared by the mannose receptor). We also found that the surface expression of the mannose receptor is not downregulated during P. carinii infection in wild-type mice. Our findings suggest that while the macrophage mannose receptor may be important in the recognition of P. carinii, in vivo, this mechanism may be redundant, and the absence of this receptor may be compensated for.

scholarly journals Human Cytomegalovirus Cell Tropism and Host Cell Receptors

Vaccines ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 70 ◽  
Author(s):  
Gerna ◽  
Kabanova ◽  
Lilleri
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Current Data ◽  
Immunosuppressed Patient ◽  
Cell Tropism ◽  
Virus Envelope

In the 1970s–1980s, a striking increase in the number of disseminated human cytomegalovirus (HCMV) infections occurred in immunosuppressed patient populations. Autopsy findings documented the in vivo disseminated infection (besides fibroblasts) of epithelial cells, endothelial cells, and polymorphonuclear leukocytes. As a result, multiple diagnostic assays, such as quantification of HCMV antigenemia (pp65), viremia (infectious virus), and DNAemia (HCMV DNA) in patient blood, were developed. In vitro experiments showed that only low passage or endothelial cell-passaged clinical isolates, and not laboratory-adapted strains, could reproduce both HCMV leuko- and endothelial cell-tropism, which were found through genetic analysis to require the three viral genes UL128, UL130, and UL131 of the HCMV UL128 locus (UL128L). Products of this locus, together with gH/gL, were shown to form the gH/gL/pUL128L pentamer complex (PC) required for infection of epithelial cells/endothelial cells, whereas gH/gL and gO form the gH/gL/gO trimer complex (TC) required for infection of all cell types. In 2016, following previous work, a receptor for the TC that mediates entry into fibroblasts was identified as PDGFRα, while in 2018, a receptor for the PC that mediates entry into endothelial/epithelial cells was identified as neuropilin2 (Nrp2). Furthermore, the olfactory receptor family member OR14I1 was recently identified as a possible additional receptor for the PC in epithelial cells. Thus, current data support two models of viral entry: (i) in fibroblasts, following interaction of PDGFRα with TC, the latter activates gB to fuse the virus envelope with the cell membrane, whereas (ii) in epithelial cells/endothelial cells, interaction of Nrp2 (and OR14I1) with PC promotes endocytosis of virus particles, followed by gB activation by gH/gL/gO (or gH/gL) and final low-pH entry into the cell.

Sign in / Sign up

Export Citation Format

Share Document