PrM- and Cell-Binding Domains of the Dengue Virus E Protein

ABSTRACT The E-prM proteins of flaviviruses are unusual complexes which play important roles in virus assembly and fusion modulation and in potential immunity-inducing vaccines. Despite their importance, little is known about the biogenesis and structural organization of E-prM complexes. Pulse-chase radiolabeling of dengue virus-infected Vero cells demonstrated a rapid interassociation of E and prM proteins, and sucrose gradient sedimentation analysis suggested that E-prM complexes progressed from simple heteromers to more densely sedimenting structures indicating increased multimerization. E-prM heteromers of even higher complexity were observed in virus particles, suggesting an intracellular assembly process which results in the networking of E-prM subunits into a lattice-like structure found in virus particles. Trypsin cleavage of E-prM-containing virus particles resulted in the release of a soluble 45-kDa fragment of the E protein which retained cell-binding activity. The results suggest that E-prM interactions in dengue virus particles are largely mediated by domains in the carboxy-terminal anchoring domain of E, while cell-binding activity is retained in a trypsin-releasable ectodomain of the E protein.

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Discovery of the Liver Hyaluronan Receptor for Endocytosis (HARE) and Its Progressive Emergence as the Multi-Ligand Scavenger Receptor Stabilin-2

Biomolecules ◽

10.3390/biom9090454 ◽

2019 ◽

Vol 9 (9) ◽

pp. 454 ◽

Cited By ~ 4

Author(s):

Weigel

Keyword(s):

Single Molecule ◽

Scavenger Receptor ◽

Binding Activity ◽

Full Length ◽

Cell Binding ◽

Anionic Polymers ◽

Binding Domains ◽

Functional Differences ◽

Clearance Receptor ◽

Hyaluronan Receptor

Since the discovery of a novel liver hyaluronan (HA) clearance receptor in 1981 by Laurent, Fraser and coworkers, 22 different ligands cleared by the renamed receptor (the Hyaluronan Receptor for Endocytosis (HARE); Stabilin-2 (Stab2)) were discovered over 37 years. Ligands fall into three groups: (1) 11 anionic polymers, (2) seven cleaved or modified proteins and (3) four types of cells. Seven synthetic ligands, not found normally in serum or tissues, likely mimic natural molecules cleared by the receptor. In 2002 we purified and cloned HARE, based on HA-binding activity, and two other groups cloned full-length receptor; FEEL-2 and Stab2. Macrophages likely require full-length Stab2 for efficient binding and phagocytosis of bacteria or apoptotic cells, since cell-binding domains are throughout the receptor. In contrast, all 16 known single-molecule binding sites are only within the C-terminal half (190HARE). The HARE isoform is generated by proteolysis, not mRNA splicing. The majority of circulating ligands is cleared by HARE, since sinusoidal endothelial cells of liver, spleen and lymph node express twice as many HARE half-receptors as full-length receptors. Based on their significant binding and functional differences, a modified receptor nomenclature is proposed that designates HARE as the C-terminal half-receptor isoform and Stab2 as the full-length receptor isoform.

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Physiological Osmotic Induction of Leptospira interrogans Adhesion: LigA and LigB Bind Extracellular Matrix Proteins and Fibrinogen

Infection and Immunity ◽

10.1128/iai.01635-06 ◽

2007 ◽

Vol 75 (5) ◽

pp. 2441-2450 ◽

Cited By ~ 177

Author(s):

Henry A. Choy ◽

Melissa M. Kelley ◽

Tammy L. Chen ◽

Annette K. Møller ◽

James Matsunaga ◽

...

Keyword(s):

Extracellular Matrix ◽

Binding Activity ◽

Surface Proteins ◽

Microbial Pathogens ◽

Host Proteins ◽

Amino Terminal ◽

Binding Domains ◽

Multiple Ligands ◽

Carboxy Terminal

ABSTRACTTransmission of leptospirosis occurs through contact of mucous membranes and abraded skin with freshwater contaminated by pathogenicLeptospiraspp. Exposure to physiological osmolarity induces leptospires to express high levels of the Lig surface proteins containing imperfect immunoglobulin-like repeats that are shared or differ between LigA and LigB. We report that osmotic induction of Lig is accompanied by 1.6- to 2.5-fold increases in leptospiral adhesion to immobilized extracellular matrix and plasma proteins, including collagens I and IV, laminin, and especially fibronectin and fibrinogen. Recombinant LigA-unique and LigB-unique repeat proteins bind to these same host ligands. We found that the avidity of LigB in binding fibronectin is comparable to that of theStaphylococcus aureusFnBPA D repeats. Both LigA- and LigB-unique repeats interact with the amino-terminal fibrin- and gelatin-binding domains of fibronectin, which are also recognized by fibronectin-binding proteins mediating the adhesion of other microbial pathogens. In contrast, repeats common to both LigA and LigB do not bind these host proteins, and nonrepeat sequences in the carboxy-terminal domain of LigB show only weak interaction with fibronectin and fibrinogen. A functional role for the binding activity of LigA and LigB is suggested by the ability of the recombinants to inhibit leptospiral adhesion to fibronectin by 28% and 21%, respectively. The binding of LigA and LigB to multiple ligands present in different tissues suggests that these adhesins may be involved in the initial colonization and dissemination stages of leptospirosis. The characterization of the Lig adhesin function should aid the design of Lig-based vaccines and serodiagnostic tests.

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Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile

Glycobiology ◽

10.1093/glycob/cwn048 ◽

2008 ◽

Vol 18 (9) ◽

pp. 698-706 ◽

Cited By ~ 54

Author(s):

T. Dingle ◽

S. Wee ◽

G. L Mulvey ◽

A. Greco ◽

E. N Kitova ◽

...

Keyword(s):

Clostridium Difficile ◽

Host Cell ◽

Functional Properties ◽

Cell Binding ◽

Binding Domains ◽

Carboxy Terminal

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RNA binding domains of heterologous viral proteins substituted for basic residues in the RSV Gag NC domain restore specific packaging of genomic RNA

10.1101/2020.03.12.989541 ◽

2020 ◽

Author(s):

Breanna L. Rice ◽

Timothy L. Lochmann ◽

Leslie J. Parent

Keyword(s):

Electrostatic Interactions ◽

Rna Binding ◽

Specific Binding ◽

Binding Activity ◽

Genomic Rna ◽

Basic Residue ◽

Gag Proteins ◽

Virus Particles ◽

Binding Domains ◽

Rous Sarcoma

AbstractThe Rous sarcoma virus Gag polyprotein transiently traffics through the nucleus, which is required for efficient incorporation of the viral genomic RNA (gRNA) into virus particles. Packaging of gRNA is mediated by two zinc knuckles and basic residues located in the nucleocapsid (NC) domain in Gag. To further examine the role of basic residues located downstream of the zinc knuckles in gRNA encapsidation, we used a gain-of-function approach. We replaced a basic residue cluster essential for gRNA packaging with heterologous basic residue motif (BR) with RNA binding activity from either the HIV-1 Rev protein (Rev BR) or the HSV ICP27 protein (ICP27 BR). Compared to wild type Gag, the mutant ICP27 BR and Rev BR Gag proteins were much more strongly localized to the nucleus and released significantly lower levels of virus particles. Surprisingly, both the ICP27 BR and Rev BR mutants packaged normal levels of gRNA per virus particle when examined in the context of a proviral vector, yet both mutants were noninfectious. These results support the hypothesis that basic residues located in the C-terminal region of NC are required for selective gRNA packaging, potentially via initial electrostatic interactions that facilitate specific binding.

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SARS-CoV-2 cytopathogenesis in cultured cells and in COVID-19 autoptic lung, evidences of lipid involvement.

10.21203/rs.3.rs-39274/v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Roberta Nardacci ◽

Francesca Colavita ◽

Concetta Castilletti ◽

Daniele Lapa ◽

Giulia Matusali ◽

...

Keyword(s):

Lipid Droplets ◽

Cellular Localization ◽

Cultured Cells ◽

Virus Assembly ◽

Vero Cells ◽

Ultrastructural Changes ◽

Type Ii ◽

Virus Particles ◽

Cytopathic Effects ◽

Type Ii Pneumocytes

Abstract Background: The pathogenesis of SARS-CoV-2 remains to be defined. Elucidating SARS-CoV-2 cellular localization within cells and its cytopathic effects requires definition. We performed a comparative ultrastructural study of SARS-CoV-2 infection of Vero-6 cells and lung from COVID-19 patients. Main findings: SARS-CoV-2 induces rapid ultrastructural changes and death in Vero cells. Ultrastructural changes in SARS-CoV-2 infection differ from those in SARS-CoV-1. Type II pneumocytes in lung tissue showed prominent altered morphological features with numerous vacuoles and swollen mitochondria with presence of abundant lipid droplets. The accumulation of lipid droplets was the most striking finding we observed in cultured cells and in infected pneumocytes. Virus particles were also found associated with lipo-lysosomes suggesting that they can play an important step in virus assembly.Interpretation: The cytopathology of SARS-CoV-2 appears to be different to that caused by SARS-CoV-1. Our findings highlight important open topics which may represent future targets to contrast the pathogenicity of SARS-CoV-2.

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Antiviral activity of silymarin and baicalein against dengue virus

Scientific Reports ◽

10.1038/s41598-021-98949-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Zhao Xuan Low ◽

Brian Ming OuYong ◽

Pouya Hassandarvish ◽

Chit Laa Poh ◽

Babu Ramanathan

Keyword(s):

Antiviral Activity ◽

Dengue Virus ◽

Viral Entry ◽

Dengue Infection ◽

Antiviral Agents ◽

Antiviral Drug ◽

Vero Cells ◽

Viral Envelope ◽

E Protein

AbstractDengue is an arthropod-borne viral disease that has become endemic and a global threat in many countries with no effective antiviral drug available currently. This study showed that flavonoids: silymarin and baicalein could inhibit the dengue virus in vitro and were well tolerated in Vero cells with a half-maximum cytotoxic concentration (CC50) of 749.70 µg/mL and 271.03 µg/mL, respectively. Silymarin and baicalein exerted virucidal effects against DENV-3, with a selective index (SI) of 10.87 and 21.34, respectively. Baicalein showed a better inhibition of intracellular DENV-3 progeny with a SI of 7.82 compared to silymarin. Baicalein effectively blocked DENV-3 attachment (95.59%) to the Vero cells, while silymarin prevented the viral entry (72.46%) into the cells, thus reducing viral infectivity. Both flavonoids showed promising antiviral activity against all four dengue serotypes. The in silico molecular docking showed that silymarin could bind to the viral envelope (E) protein with a binding affinity of − 8.5 kcal/mol and form hydrogen bonds with the amino acids GLN120, TRP229, ASN89, and THR223 of the E protein. Overall, this study showed that silymarin and baicalein exhibited potential anti-DENV activity and could serve as promising antiviral agents for further development against dengue infection.

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RNA-Binding Domains of Heterologous Viral Proteins Substituted for Basic Residues in the RSV Gag NC Domain Restore Specific Packaging of Genomic RNA

Viruses ◽

10.3390/v12040370 ◽

2020 ◽

Vol 12 (4) ◽

pp. 370 ◽

Cited By ~ 1

Author(s):

Breanna L. Rice ◽

Timothy L. Lochmann ◽

Leslie J. Parent

Keyword(s):

Electrostatic Interactions ◽

Rna Binding ◽

Binding Activity ◽

Genomic Rna ◽

Basic Residue ◽

Gag Proteins ◽

Virus Particles ◽

Binding Domains ◽

Rous Sarcoma ◽

Sarcoma Virus

The Rous sarcoma virus Gag polyprotein transiently traffics through the nucleus, which is required for efficient incorporation of the viral genomic RNA (gRNA) into virus particles. Packaging of gRNA is mediated by two zinc knuckles and basic residues located in the nucleocapsid (NC) domain in Gag. To further examine the role of basic residues located downstream of the zinc knuckles in gRNA encapsidation, we used a gain-of-function approach. We replaced a basic residue cluster essential for gRNA packaging with heterologous basic residue motif (BR) with RNA-binding activity from either the HIV-1 Rev protein (Rev BR) or the HSV ICP27 protein (ICP27 BR). Compared to wild-type Gag, the mutant ICP27 BR and Rev BR Gag proteins were much more strongly localized to the nucleus and released significantly lower levels of virus particles. Surprisingly, both the ICP27 BR and Rev BR mutants packaged normal levels of gRNA per virus particle when examined in the context of a proviral vector, yet both mutants were noninfectious. These results support the hypothesis that basic residues located in the C-terminal region of NC are required for selective gRNA packaging, potentially by binding non-specifically to RNA via electrostatic interactions.

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Involvement of GER in Friend leukemia virus assembly in high-pressure frozen cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160509 ◽

1990 ◽

Vol 48 (3) ◽

pp. 590-591

Author(s):

W. Djaczenko ◽

M. Müller ◽

A. Benedetto ◽

G. Carbone

Keyword(s):

High Pressure ◽

Virus Assembly ◽

Leukemia Virus ◽

Leukemia Cells ◽

Serial Sections ◽

Myeloma Cells ◽

Virus Particles ◽

Friend Leukemia ◽

Carbon Coated ◽

Friend Leukemia Virus

A thickening of ER membranes in murine myeloma cells was attributed by de Harven to the assembly of intracisternal virus particles. We observed similar thickening of GER membranes in Friend leukemia cells (FLC) apparently associated with Friend leukemia virus (FLV) assembly. We reinvestigated the problem of GER involvement in FLV assembly using high pressure cryofixed FLC.FLC (745A clone growing in suspension and FF clone growing in monolayer) were immersed in Hexadecene (Fluka, Switzerland) and rapidly frozen in Balzers HPM 010 freezing machine working at 2200 bar. All cells were freeze substituted at -90°C in 2% OsO4 in absolute acetone. Serial sections cut to avoid misinterpretations due to the geometry of sections, were collected on carbon coated 100 mesh grids.

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Genomic analysis of C-type lectins

Biochemical Society Symposium ◽

10.1042/bss0690059 ◽

2002 ◽

Vol 69 ◽

pp. 59-72 ◽

Cited By ~ 85

Author(s):

Kurt Drickamer ◽

Andrew J. Fadden

Keyword(s):

Biological Effects ◽

Cell Signalling ◽

Genomic Analysis ◽

Binding Activity ◽

Immunoglobulin Superfamily ◽

Carbohydrate Binding ◽

Carbohydrate Recognition ◽

Calcium Dependent ◽

Binding Domains ◽

Carbohydrate Recognition Domains

Many biological effects of complex carbohydrates are mediated by lectins that contain discrete carbohydrate-recognition domains. At least seven structurally distinct families of carbohydrate-recognition domains are found in lectins that are involved in intracellular trafficking, cell adhesion, cell–cell signalling, glycoprotein turnover and innate immunity. Genome-wide analysis of potential carbohydrate-binding domains is now possible. Two classes of intracellular lectins involved in glycoprotein trafficking are present in yeast, model invertebrates and vertebrates, and two other classes are present in vertebrates only. At the cell surface, calcium-dependent (C-type) lectins and galectins are found in model invertebrates and vertebrates, but not in yeast; immunoglobulin superfamily (I-type) lectins are only found in vertebrates. The evolutionary appearance of different classes of sugar-binding protein modules parallels a development towards more complex oligosaccharides that provide increased opportunities for specific recognition phenomena. An overall picture of the lectins present in humans can now be proposed. Based on our knowledge of the structures of several of the C-type carbohydrate-recognition domains, it is possible to suggest ligand-binding activity that may be associated with novel C-type lectin-like domains identified in a systematic screen of the human genome. Further analysis of the sequences of proteins containing these domains can be used as a basis for proposing potential biological functions.

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Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption

International Journal of Molecular Sciences ◽

10.3390/ijms22147362 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7362

Author(s):

Amina Ben Abla ◽

Guilhem Boeuf ◽

Ahmed Elmarjou ◽

Cyrine Dridi ◽

Florence Poirier ◽

...

Keyword(s):

Specific Binding ◽

Cellular Adhesion ◽

High Yield ◽

Adhesion Assay ◽

Cell Binding ◽

Affinity Tag ◽

Binding Domains ◽

Rational Development ◽

Fibronectin Type Iii ◽

Arginine Glycine Aspartic Acid

Engineering of biomimetic motives have emerged as promising approaches to improving cells’ binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface’s receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5β1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.

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