Heparan Sulfate Mediates Infection of High-Neurovirulence Theiler's Viruses

ABSTRACT The mechanisms by which Theiler's murine encephalomyelitis virus (TMEV) binds and enters host cells and the molecules involved are not completely understood. In this study, we demonstrate that the high-neurovirulence TMEV GDVII virus uses the glycosaminoglycan heparan sulfate (HS) as an attachment factor that is required for efficient infection. Studies based on soluble HS-mediated inhibition of attachment and infection, removal of HS with specific enzymes, and blocking with anti-HS antibodies establish that HS mediates GDVII virus entry into mammalian cells. Data from defined proteoglycan-deficient Chinese hamster ovary mutant cells further support the role of HS in GDVII infection and indicate that the extent of sulfation is critical for infection. Neuraminidase treatment of proteoglycan-deficient cells restores permissiveness to GDVII virus, indicating that sialic acid hinders direct access of virus to the protein entry receptor. A model of the potential steps in GDVII virus entry into mammalian cells involving HS is proposed.

Download Full-text

Neurotrophin Receptor TrkC Is an Entry Receptor for Trypanosoma cruzi in Neural, Glial, and Epithelial Cells

Infection and Immunity ◽

10.1128/iai.05403-11 ◽

2011 ◽

Vol 79 (10) ◽

pp. 4081-4087 ◽

Cited By ~ 16

Author(s):

Craig Weinkauf ◽

Ryan Salvador ◽

Mercio PereiraPerrin

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Mammalian Cells ◽

Chinese Hamster ◽

Neuronal Cell ◽

Host Cells ◽

Neurotrophin Receptor ◽

Content Type

ABSTRACTTrypanosoma cruzi, the agent of Chagas' disease, infects a variety of mammalian cells in a process that includes multiple cycles of intracellular division and differentiation starting with host receptor recognition by a parasite ligand(s). Earlier work in our laboratory showed that the neurotrophin-3 (NT-3) receptor TrkC is activated byT. cruzisurfacetrans-sialidase, also known as parasite-derived neurotrophic factor (PDNF). However, it has remained unclear whether TrkC is used byT. cruzito enter host cells. Here, we show that a neuronal cell line (PC12-NNR5) relatively resistant toT. cruzibecame highly susceptible to infection when overexpressing human TrkC but not human TrkB. Furthermore,trkCtransfection conferred an ∼3.0-fold intracellular growth advantage. Sialylation-deficient Chinese hamster ovarian (CHO) epithelial cell lines Lec1 and Lec2 also became much more permissive toT. cruziafter transfection with thetrkCgene. Additionally, NT-3 specifically blockedT. cruziinfection of the TrkC-NNR5 transfectants and of naturally permissive TrkC-bearing Schwann cells and astrocytes, as did recombinant PDNF. Two specific inhibitors of Trk autophosphorylation (K252a and AG879) and inhibitors of Trk-induced MAPK/Erk (U0126) and Akt kinase (LY294002) signaling, but not an inhibitor of insulin-like growth factor 1 receptor, abrogated TrkC-mediated cell invasion. Antibody to TrkC blockedT. cruziinfection of the TrkC-NNR5 transfectants and of cells that naturally express TrkC. The TrkC antibody also significantly and specifically reduced cutaneous infection in a mouse model of acute Chagas' disease. TrkC is ubiquitously expressed in the peripheral and central nervous systems, and in nonneural cells infected byT. cruzi, including cardiac and gastrointestinal muscle cells. Thus, TrkC is implicated as a functional PDNF receptor in cell entry, independently of sialic acid recognition, mediating broadT. cruziinfection bothin vitroandin vivo.

Download Full-text

The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells

PLoS Pathogens ◽

10.1371/journal.ppat.1009803 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009803

Author(s):

Dipanwita Mitra ◽

Mohammad H. Hasan ◽

John T. Bates ◽

Michael A. Bierdeman ◽

Dallas R. Ederer ◽

...

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Virus Entry ◽

Degree Of Polymerization ◽

Host Cells ◽

Virus Infectivity ◽

Wild Type ◽

Enveloped Viruses ◽

Host Membrane ◽

Mutant Cells

Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity.

Download Full-text

The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus infectivity

10.1101/590463 ◽

2019 ◽

Cited By ~ 1

Author(s):

Mohammad H. Hasan ◽

Rinkuben Parmar ◽

Quntao Liang ◽

Hong Qiu ◽

Vaibhav Tiwari ◽

...

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Virus Entry ◽

Degree Of Polymerization ◽

Convincing Evidence ◽

Host Cells ◽

Virus Infectivity ◽

Cmv Infection ◽

Virus Binding ◽

Heparan Sulfates

AbstractHerpesviruses attach to host cells by interacting with cell surface heparan sulfate (HS) proteoglycans prior to specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions results in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide convincing evidence that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) infection and binding by following line of evidences. First, purified CMV extracellular virions preferentially bound to the sulfated longer chain of HS on a glycoarray compared to unsulfated glycosaminoglycans and shorter chain unsulfated HS. Second, the fraction of glycosaminoglycans (GAG) displaying higherdpand sulfation had a major impact on CMV infectivity and titers. Finally, cell lines knocked out for specific sulfotransferases Glucosaminyl 3-O-sulfotransferase (3-O-ST-1 and −4 and double −1/4) produced significantly reduced CMV titers compared to wild-type cells. Similarly, a peptide generated against sulfated-HS significantly reduced virus titers compared to the control peptide. Taken together, the above results highlight the significance of the chain length and sulfation patterns of HS in CMV binding and infectivity.ImportanceThe cell surface heparan sulfates (HS) are exploited by multiple viruses as they provide docking sites during cell entry and therefore are a promising target for the development of novel antivirals. In addition, the molecular diversity in HS chains generates unique binding sites for specific ligands and hence offers preferential binding for one virus over other. In the current study several HS mimics were analyzed for their ability to inhibit cytomegalovirus (CMV) infection. The results were corroborated by parallel studies in mutant mouse cells and virus binding to glycoarrays. Combined together, the data suggests that virus particles preferentially attach to specifically modified HS and thus the process is amenable to targeting by specifically designed HS mimics.

Download Full-text

Engineering mammalian cells to produce plant-specific N-glycosylation on proteins

Glycobiology ◽

10.1093/glycob/cwaa009 ◽

2020 ◽

Vol 30 (8) ◽

pp. 528-538 ◽

Cited By ~ 1

Author(s):

Joachim Steen Larsen ◽

Richard Torbjörn Gustav Karlsson ◽

Weihua Tian ◽

Morten Alder Schulz ◽

Annemarie Matthes ◽

...

Keyword(s):

Cho Cells ◽

Mammalian Cells ◽

Chinese Hamster ◽

Structural Features ◽

Host Cells ◽

Igg Antibody ◽

Endogenous Genes ◽

Species Specific ◽

Full Conversion ◽

Core Fucosylation

Abstract Protein N-glycosylation is an essential and highly conserved posttranslational modification found in all eukaryotic cells. Yeast, plants and mammalian cells, however, produce N-glycans with distinct structural features. These species-specific features not only pose challenges in selecting host cells for production of recombinant therapeutics for human medical use but also provide opportunities to explore and utilize species-specific glycosylation in design of vaccines. Here, we used reverse cross-species engineering to stably introduce plant core α3fucose (α3Fuc) and β2xylose (β2Xyl) N-glycosylation epitopes in the mammalian Chinese hamster ovary (CHO) cell line. We used directed knockin of plant core fucosylation and xylosylation genes (AtFucTA/AtFucTB and AtXylT) and targeted knockout of endogenous genes for core fucosylation (fut8) and elongation (B4galt1), for establishing CHO cells with plant N-glycosylation capacities. The engineering was evaluated through coexpression of two human therapeutic N-glycoproteins, erythropoietin (EPO) and an immunoglobulin G (IgG) antibody. Full conversion to the plant-type α3Fuc/β2Xyl bi-antennary agalactosylated N-glycosylation (G0FX) was demonstrated for the IgG1 produced in CHO cells. These results demonstrate that N-glycosylation in mammalian cells is amenable for extensive cross-kingdom engineering and that engineered CHO cells may be used to produce glycoproteins with plant glycosylation.

Download Full-text

Key Challenges in Designing CHO Chassis Platforms

Processes ◽

10.3390/pr8060643 ◽

2020 ◽

Vol 8 (6) ◽

pp. 643 ◽

Cited By ~ 2

Author(s):

Anis Hamdi ◽

Diana Széliová ◽

David E. Ruckerbauer ◽

Isabel Rocha ◽

Nicole Borth ◽

...

Keyword(s):

Metabolic Engineering ◽

Cell Lines ◽

Mammalian Cells ◽

Large Scale ◽

Modular Design ◽

Chinese Hamster ◽

Production Performance ◽

Host Cells ◽

Cho Cell ◽

Support Design

Following the success of and the high demand for recombinant protein-based therapeutics during the last 25 years, the pharmaceutical industry has invested significantly in the development of novel treatments based on biologics. Mammalian cells are the major production systems for these complex biopharmaceuticals, with Chinese hamster ovary (CHO) cell lines as the most important players. Over the years, various engineering strategies and modeling approaches have been used to improve microbial production platforms, such as bacteria and yeasts, as well as to create pre-optimized chassis host strains. However, the complexity of mammalian cells curtailed the optimization of these host cells by metabolic engineering. Most of the improvements of titer and productivity were achieved by media optimization and large-scale screening of producer clones. The advances made in recent years now open the door to again consider the potential application of systems biology approaches and metabolic engineering also to CHO. The availability of a reference genome sequence, genome-scale metabolic models and the growing number of various “omics” datasets can help overcome the complexity of CHO cells and support design strategies to boost their production performance. Modular design approaches applied to engineer industrially relevant cell lines have evolved to reduce the time and effort needed for the generation of new producer cells and to allow the achievement of desired product titers and quality. Nevertheless, important steps to enable the design of a chassis platform similar to those in use in the microbial world are still missing. In this review, we highlight the importance of mammalian cellular platforms for the production of biopharmaceuticals and compare them to microbial platforms, with an emphasis on describing novel approaches and discussing still open questions that need to be resolved to reach the objective of designing enhanced modular chassis CHO cell lines.

Download Full-text

PERKEMBANGAN TEKNOLOGI SEL MAMALIA CHINESE HAMSTER OVARY (CHO) UNTUK PRODUKSI OBAT BERBASIS PROTEIN

BERITA BIOLOGI ◽

10.14203/beritabiologi.v18i2.3705 ◽

2019 ◽

Vol 18 (2) ◽

pp. 125-133

Author(s):

Adi Santoso

Keyword(s):

Cho Cells ◽

Mammalian Cells ◽

Chinese Hamster ◽

Basic Research ◽

Host Cells ◽

Basic Knowledge ◽

Research Progress ◽

Market Demand ◽

Technological Advances ◽

Development Technology

Chinese hamsters ovary (CHO) and its derivative such as CHO-DXB11 cells, CHO-K1, CHO-DG44 and CHO-S are mammalian cells that are often used for production of therapeutic protein drugs. The CHO cells often used for protein production have several advantages including 1) host cells that are safe to use in drug production, 2) the level of production of proteins produced can be increased by amplifying genes using methotrexate (MTX), 3) having the capacity to make post-translation modificationsand 4) CHO cells can be adapted to grow in suspension. The high need for protein-based drugs triggers the development of basic knowledge and innovation in production of recombinant proteins. The impressive technological advances in CHO cell technology have made these cells can be used to produce proteins around 10 g/liter in order to meet the market demand. The first protein successfully produced using CHO mammalian cells was the therapeutic Tissue Plasminogen Activator (r-tPA, Activase) protein used for stroke patients. The presence of this drug is quickly followed by several other types of drugs. In this review, history of development of CHO cells, the contribution of CHO cells to basic research, progress of effective line cell screening and development technology are discussed.

Download Full-text

Structural and functional characterization of SARS-CoV-2 RBD domains produced in mammalian cells

10.1101/2021.02.23.432424 ◽

2021 ◽

Author(s):

Christoph Gstöttner ◽

Tao Zhang ◽

Anja Resemann ◽

Sophia Ruben ◽

Stuart Pengelley ◽

...

Keyword(s):

Posttranslational Modifications ◽

Mammalian Cells ◽

Vaccine Development ◽

Functional Characterization ◽

Chinese Hamster ◽

Structural Features ◽

Host Cells ◽

Hek293 Cells ◽

Functional Features

AbstractAs the SARS-CoV-2 pandemic is still ongoing and dramatically influences our life, the need for recombinant proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor binding domain (RBD), mediates the interaction with the ACE2 receptor on host cells and may be modulated by its structural features. Therefore, well characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional posttranslational modifications) a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, higher level of sialylation and a combination of core 1 and core 2 type O-glycans. Additionally, from both putative O-glycosylation sites, we could confirm that O-glycosylation was exclusively present at T323, which was previously unknown. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides a workflow for characterization of new RBDs and batch-to-batch comparison.

Download Full-text

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100095017 ◽

1972 ◽

Vol 30 ◽

pp. 350-351

Author(s):

K. Shankar Narayan ◽

Kailash C. Gupta ◽

Tohru Okigaki

Keyword(s):

Ultraviolet Light ◽

Mammalian Cells ◽

Biological Effects ◽

Survival Rates ◽

Chinese Hamster ◽

Cell Types ◽

Differential Sensitivity ◽

Ultrastructural Changes ◽

Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

Download Full-text