| 1. |
- Abidine, Yara, et al.
(författare)
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Cellular Chondroitin Sulfate and the Mucin-like Domain of Viral Glycoprotein C Promote Diffusion of Herpes Simplex Virus 1 While Heparan Sulfate Restricts Mobility
- 2022
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Ingår i: Viruses. - : MDPI AG. - 1999-4915. ; 14:8
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Tidskriftsartikel (refereegranskat)abstract
- The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus-GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses.
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| 2. |
- Alqabandi, Maryam, et al.
(författare)
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The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization
- 2021
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Ingår i: BMC Biology. - : BioMed Central. - 1741-7007. ; 19:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, ESCRT-III CHMP2 exists as two isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet.Results: Here, we use a combination of techniques on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Thus, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect.Conclusions: We therefore conclude that CHMP2B and CHMP2A exhibit different mechanical properties and might thus contribute differently to the diverse ESCRT-III-catalyzed membrane remodeling processes.
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| 3. |
- Bally, Marta, et al.
(författare)
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Lipid-based bioanalytical sensors
- 2021
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Ingår i: Handbook of lipid membranes. - Boca Raton : CRC Press. - 9781466555723 - 9781032014418 - 9780429194078 ; , s. 241-269
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Bokkapitel (refereegranskat)abstract
- Lipid assemblies have attracted considerable interest as components in bioanalytical sensors. They provide a native-like environment for the immobilization of membrane proteins and for the study of membrane-related processes. Liposomes are also excellent bioanalytical assay components since selected functionalities can be added to the membrane while their aqueous interior can encapsulate a variety of molecules. This chapter highlights the potential of lipid assemblies in surface-based affinity sensors. It first describes how such sensors are created, providing an overview of lipid immobilization strategies together with a summary of the major transduction techniques used to probe binding at and transport through membrane interfaces. It then reviews the implementation of lipid-based sensors in the study of membrane proteins and membrane-mediated interactions, followed by a discussion of the potential of liposomes as nanoscale labels and as nanoreactors. Finally, it illustrates how external forces can be used to manipulate membrane component for biosensing applications.
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| 4. |
- Bally, Marta, 1981, et al.
(författare)
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Lipid-Based Bioanalytical Sensors
- 2021
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Ingår i: Handbook of Lipid Membranes Molecular, Functional, and Materials Aspects. - Boca Raton : CRC Press. - 9781466555730 ; , s. 241-270, s. 241-269
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Lipid assemblies have attracted considerable interest as components in bioanalytical sensors. They provide a native-like environment for the immobilization of membrane proteins and for the study of membrane-related processes. Liposomes are also excellent bioanalytical assay components since selected functionalities can be added to the membrane while their aqueous interior can encapsulate a variety of molecules. This chapter highlights the potential of lipid assemblies in surface-based affinity sensors. It first describes how such sensors are created, providing an overview of lipid immobilization strategies together with a summary of the major transduction techniques used to probe binding at and transport through membrane interfaces. It then reviews the implementation of lipid-based sensors in the study of membrane proteins and membrane-mediated interactions, followed by a discussion of the potential of liposomes as nanoscale labels and as nanoreactors. Finally, it illustrates how external forces can be used to manipulate membrane component for biosensing applications.
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| 5. |
- Bally, Marta, 1981, et al.
(författare)
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Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context
- 2021
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 413, s. 7157-7178
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Tidskriftsartikel (refereegranskat)abstract
- The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.
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| 6. |
- Becker, Miriam, et al.
(författare)
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Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells
- 2023
-
Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 97:10
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Tidskriftsartikel (refereegranskat)abstract
- Enteric adenovirus types F40 and 41 (EAdVs) are a leading cause of diarrhea and diarrhea-associated death in young children and have recently been proposed to cause acute hepatitis in children. EAdVs have a unique capsid architecture and exhibit — unlike other human adenoviruses — a relatively strict tropism for gastrointestinal tissues with, to date, understudied infection mechanism and unknown target cells. In this study, we turn to potentially limiting host factors by comparing EAdV entry in cell lines with respiratory and intestinal origin by cellular perturbation, virus particle tracking, and transmission electron microscopy. Our analyses highlight kinetic advantages for EAdVs in duodenal HuTu80 cell infection and reveal a larger fraction of mobile particles, faster virus uptake, and infectious particle entry in intestinal cells. Moreover, EAdVs display a dependence on clathrin- and dynamin-dependent pathways in intestinal cells. Detailed knowledge of virus entry routes and host factor requirements is essential to understanding pathogenesis and developing new countermeasures. Hence, this study provides novel insights into the entry mechanisms of a medically important virus with emerging tropism in a cell line originating from a relevant tissue. IMPORTANCE Enteric adenoviruses have historically been difficult to grow in cell culture, which has resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum-derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.
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| 7. |
- Bernasconi, Valentina, 1989, et al.
(författare)
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A vaccine combination of lipid nanoparticles and a cholera toxin adjuvant derivative greatly improves lung protection against influenza virus infection
- 2021
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Ingår i: Mucosal Immunology. - : Elsevier BV. - 1933-0219 .- 1935-3456. ; 14:2, s. 523-536
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Tidskriftsartikel (refereegranskat)abstract
- This is a proof-of-principle study demonstrating that the combination of a cholera toxin derived adjuvant, CTA1-DD, and lipid nanoparticles (LNP) can significantly improve the immunogenicity and protective capacity of an intranasal vaccine. We explored the self-adjuvanted universal influenza vaccine candidate, CTA1-3M2e-DD (FPM2e), linked to LNPs. We found that the combined vector greatly enhanced survival against a highly virulent PR8 strain of influenza virus as compared to when mice were immunized with FPM2e alone. The combined vaccine vector enhanced early endosomal processing and peptide presentation in dendritic cells and upregulated co-stimulation. The augmenting effect was CTA1-enzyme dependent. Whereas systemic anti-M2e antibody and CD4(+)T-cell responses were comparable to those of the soluble protein, the local respiratory tract IgA and the specific Th1 and Th17 responses were strongly enhanced. Surprisingly, the lung tissue did not exhibit gross pathology upon recovery from infection and M2e-specific lung resident CD4(+)T cells were threefold higher than in FPM2e-immunized mice. This study conveys optimism as to the protective ability of a combination vaccine based on LNPs and various forms of the CTA1-DD adjuvant platform, in general, and, more specifically, an important way forward to develop a universal vaccine against influenza.
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| 8. |
- Conca, Dario Valter, et al.
(författare)
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The role of membrane complexity in the early entry stages of SARS-CoV-2 variants
- 2023
-
Ingår i: European Biophysics Journal. - 1432-1017 .- 0175-7571. ; 52:SUPPL 1, s. S176-S176
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The highest density of mutations in SARS-CoV-2 variants is located on the spike glycoprotein (S), which is responsible for receptor ACE2 engagement. This suggests that SARS-CoV-2 is evolving to optimize viral entry. Several molecular studies report differences in the affinity between isolated S and ACE2 among variants. However, overall ACE2 affinity poorly correlates with the increased infectivity of recent variants. We address this discrepancy by considering the virus interaction with the whole plasma membrane and study the role of avidity and membrane complexity in modulating virus-host binding. To this end, we employ an in-vitro model system combining single-particle tracking and native supported lipid bilayers (nSLBs) made from lung epithelial cells. As virion mimics, we developed S-decorated liposomes that allow for direct comparison between variants and BSL-1 handling. Sliposome interaction with nSLBs showed a significant increase in avidity for Omicron compared to Delta and Wuhan strains. Further, using single-molecule force spectroscopy, we reveal a higher affinity for Omicron and Delta S to sensor immobilise heparan sulfate (HS). Our results indicate a shift in the variants’ attachment strategy towards more efficient use of coreceptors and the role of HS as an initial docking site that facilitates virus accumulation at the membrane and ACE2 engagement.
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| 9. |
- Jõemetsa, Silver, 1990, et al.
(författare)
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Independent Size and Fluorescence Emission Determination of Individual Biological Nanoparticles Reveals that Lipophilic Dye Incorporation Does Not Scale with Particle Size
- 2020
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 36:33, s. 9693-9700
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Tidskriftsartikel (refereegranskat)abstract
- Advancements in nanoparticle characterization techniques are critical for improving the understanding of how biological nanoparticles (BNPs) contribute to different cellular processes, such as cellular communication, viral infection, as well as various drug-delivery applications. Since BNPs are intrinsically heterogeneous, there is a need for characterization methods that are capable of providing information about multiple parameters simultaneously, preferably at the single-nanoparticle level. In this work, fluorescence microscopy was combined with surface-based two-dimensional flow nanometry, allowing for simultaneous and independent determination of size and fluorescence emission of individual BNPs. In this way, the dependence of the fluorescence emission of the commonly used self-inserting lipophilic dye 3,3′-dioctadecyl-5,5′-di(4-sulfophenyl)oxacarbocyanine (SP-DiO) could successfully be correlated with nanoparticle size for different types of BNPs, including synthetic lipid vesicles, lipid vesicles derived from cellular membrane extracts, and extracellular vesicles derived from human SH-SY5Y cell cultures; all vesicles had a radius, r, of ∼50 nm and similar size distributions. The results demonstrate that the dependence of fluorescence emission of SP-DiO on nanoparticle size varies significantly between the different types of BNPs, with the expected dependence on membrane area, r2, being observed for synthetic lipid vesicles, while a significant weaker dependence on size was observed for BNPs with more complex composition. The latter observation is attributed to a size-dependent difference in membrane composition, which may influence either the optical properties of the dye and/or the insertion efficiency, indicating that the fluorescence emission of this type of self-inserting dye may not be reliable for determining size or size distribution of BNPs with complex lipid compositions.
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| 10. |
- Kirui, Jared, et al.
(författare)
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The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry
- 2021
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Ingår i: Cells. - : MDPI. - 2073-4409. ; 10:7
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Tidskriftsartikel (refereegranskat)abstract
- Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced cell binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.
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| 11. |
- Liu, Kang-Cheng, et al.
(författare)
-
Membrane insertion mechanism of the caveola coat protein Cavin1
- 2022
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 119:25
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Tidskriftsartikel (refereegranskat)abstract
- Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.
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| 12. |
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| 13. |
- Lubart, Quentin, 1989, et al.
(författare)
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Lipid vesicle composition influences the incorporation and fluorescence properties of the lipophilic sulphonated carbocyanine dye SP-DiO
- 2020
-
Ingår i: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9084 .- 1463-9076. ; 22:16, s. 8781-8790
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Tidskriftsartikel (refereegranskat)abstract
- Lipophilic carbocyanine dyes are widely used as fluorescent cell membrane probes in studies ranging from biophysics to cell biology. While they are extremely useful for qualitative observation of lipid structures, a major problem impairing quantitative studies is that the chemical environment of the lipid bilayer affects both the dye's insertion efficiency and photophysical properties. We present a systematic investigation of the sulphonated carbocyanine dye 3,3′-dioctadecyl-5,5′-di(4-sulfophenyl) (SP-DiO) and demonstrate how its insertion efficiency into pre-formed lipid bilayers and its photophysical properties therein determine its apparent fluorescence intensity in different lipid environments. For this purpose, we use large unilamellar vesicles (LUVs) made of lipids with distinct chain unsaturation, acyl chain length, head group charge, and with variation in membrane cholesterol content as models. Using a combination of absorbance, fluorescence emission, and fluorescence lifetime measurements we reveal that SP-DiO incorporates more efficiently into liquid disordered phases compared to gel phases. Moreover, incorporation into the latter phase is most efficient when the mismatch between the length of the lipid and dye hydrocarbon chains is small. Furthermore, SP-DiO incorporation is less efficient in LUVs composed of negatively charged lipids. Lastly, when cholesterol was included in the LUV membranes, we observed significant spectral shifts, consistent with dye aggregation. Taken together, our study highlights the complex interplay between membrane composition and labeling efficiency with lipophilic dyes and advocates for careful assessment of fluorescence data when attempting a quantitative analysis of fluorescence data with such molecules.
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| 14. |
- Nadeem, Aftab, et al.
(författare)
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A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion
- 2021
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 118:47
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Tidskriftsartikel (refereegranskat)abstract
- Vibrio cholerae, responsible for outbreaks of cholera disease, is a highly motile organism by virtue of a single flagellum. We describe how the flagellum facilitates the secretion of three V. cholerae proteins encoded by a hitherto-unrecognized genomic island. The proteins MakA/B/E can form a tripartite toxin that lyses erythrocytes and is cytotoxic to cultured human cells. A structural basis for the cytolytic activity of the Mak proteins was obtained by X-ray crystallography. Flagellum-facilitated secretion ensuring spatially coordinated delivery of Mak proteins revealed a role for the V. cholerae flagellum considered of particular significance for the bacterial environmental persistence. Our findings will pave the way for the development of diagnostics and therapeutic strategies against pathogenic Vibrionaceae.
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| 15. |
- Nadeem, Aftab, et al.
(författare)
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Phosphatidic acid-mediated binding and mammalian cell internalization of the Vibrio cholerae cytotoxin MakA
- 2021
-
Ingår i: PLoS Pathogens. - : Public Library of Science. - 1553-7366 .- 1553-7374. ; 17:3
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Tidskriftsartikel (refereegranskat)abstract
- Vibrio cholerae is a noninvasive intestinal pathogen extensively studied as the causative agent of the human disease cholera. Our recent work identified MakA as a potent virulence factor of V. cholerae in both Caenorhabditis elegans and zebrafish, prompting us to investigate the potential contribution of MakA to pathogenesis also in mammalian hosts. In this study, we demonstrate that the MakA protein could induce autophagy and cytotoxicity of target cells. In addition, we observed that phosphatidic acid (PA)-mediated MakA-binding to the host cell plasma membranes promoted macropinocytosis resulting in the formation of an endomembrane-rich aggregate and vacuolation in intoxicated cells that lead to induction of autophagy and dysfunction of intracellular organelles. Moreover, we functionally characterized the molecular basis of the MakA interaction with PA and identified that the N-terminal domain of MakA is required for its binding to PA and thereby for cell toxicity. Furthermore, we observed that the ΔmakA mutant outcompeted the wild-type V. cholerae strain A1552 in the adult mouse infection model. Based on the findings revealing mechanistic insights into the dynamic process of MakA-induced autophagy and cytotoxicity we discuss the potential role played by the MakA protein during late stages of cholera infection as an anti-colonization factor.
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| 16. |
- Nadeem, Aftab, et al.
(författare)
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Protein-lipid interaction at low pH induces oligomerization of the MakA cytotoxin from Vibrio cholerae
- 2022
-
Ingår i: eLIFE. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 11
-
Tidskriftsartikel (refereegranskat)abstract
- The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.
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| 17. |
- Norling, Karin, 1988, et al.
(författare)
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Dissimilar Deformation of Fluid- and Gel-Phase Liposomes upon Multivalent Interaction with Cell Membrane Mimics Revealed Using Dual-Wavelength Surface Plasmon Resonance
- 2022
-
Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 38:8, s. 2550-2560
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Tidskriftsartikel (refereegranskat)abstract
- The mechanical properties of biological nanoparticles play a crucial role in their interaction with the cellular membrane, in particular for cellular uptake. This has significant implications for the design of pharmaceutical carrier particles. In this context, liposomes have become increasingly popular, among other reasons due to their customizability and easily varied physicochemical properties. With currently available methods, it is, however, not trivial to characterize the mechanical properties of nanoscopic liposomes especially with respect to the level of deformation induced upon their ligand-receptor-mediated interaction with laterally fluid cellular membranes. Here, we utilize the sensitivity of dual-wavelength surface plasmon resonance to probe the size and shape of bound liposomes (∼100 nm in diameter) as a means to quantify receptor-induced deformation during their interaction with a supported cell membrane mimic. By comparing biotinylated liposomes in gel and fluid phases, we demonstrate that fluid-phase liposomes are more prone to deformation than their gel-phase counterparts upon binding to the cell membrane mimic and that, as expected, the degree of deformation depends on the number of ligand-receptor pairs that are engaged in the multivalent binding.
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| 18. |
- Olofsson, Sigvard, 1948, et al.
(författare)
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Structure and Role of O-Linked Glycans in Viral Envelope Proteins
- 2023
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Ingår i: Annual Review of Virology. - : Annual Reviews. - 2327-056X. ; 10:1, s. 283-304
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Tidskriftsartikel (refereegranskat)abstract
- N- and O-glycans are both important constituents of viral envelope glycoproteins. O-linked glycosylation can be initiated by any of 20 different human polypeptide O-acetylgalactosaminyl transferases, resulting in an important functional O-glycan heterogeneity. O-glycans are organized as solitary glycans or in clusters of multiple glycans forming mucin-like domains. They are functional both in the viral life cycle and in viral colonization of their host. Negatively charged O-glycans are crucial for the interactions between glycosaminoglycan-binding viruses and their host. A novel mechanism, based on controlled electrostatic repulsion, explains how such viruses solve the conflict between optimized viral attachment to target cells and efficient egress of progeny virus. Conserved solitary O-glycans appear important for viral uptake in target cells by contributing to viral envelope fusion. Dual roles of viral O-glycans in the host B cell immune response, either epitope blocking or epitope promoting, may be exploitable for vaccine development. Finally, specific virus-induced O-glycans may be involved in viremic spread.
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| 19. |
- Pulkkinen, Lauri Ilmari Aurelius, et al.
(författare)
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Simultaneous membrane and RNA binding by tick-borne encephalitis virus capsid protein
- 2023
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Ingår i: PLoS Pathogens. - : Public Library of Science. - 1553-7366 .- 1553-7374. ; 19:2
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Tidskriftsartikel (refereegranskat)abstract
- Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate the interactions of the wild-type and truncated capsid proteins with membranes with biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids, which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.
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| 20. |
- Rimkute, Inga, et al.
(författare)
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Histo-blood group antigens of glycosphingolipids predict susceptibility of human intestinal enteroids to norovirus infection
- 2020
-
Ingår i: Journal of Biological Chemistry. - : Elsevier. - 0021-9258 .- 1083-351X. ; 295:47, s. 15974-15987
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Tidskriftsartikel (refereegranskat)abstract
- The molecular mechanisms behind infection and propagation of human restricted pathogens such as human norovirus (HuNoV) have defied interrogation because they were previously unculturable. However, human intestinal enteroids (HIEs) have emerged to offer unique ex vivo models for targeted studies of intestinal biology, including inflammatory and infectious diseases. Carbohydrate-dependent histo-blood group antigens (HBGAs) are known to be critical for clinical infection. To explore whether HBGAs of glycosphingolipids contribute to HuNoV infection, we obtained HIE cultures established from stem cells isolated from jejunal biopsies of six individuals with different ABO, Lewis, and secretor genotypes. We analyzed their glycerolipid and sphingolipid compositions and quantified interaction kinetics and the affinity of HuNoV virus-like particles (VLPs) to lipid vesicles produced from the individual HIE-lipid extracts. All HIEs had a similar lipid and glycerolipid composition. Sphingolipids included HBGA-related type 1 chain glycosphingolipids (GSLs), with HBGA epitopes corresponding to the geno- and phenotypes of the different HIEs. As revealed by single-particle interaction studies of Sydney GII.4 VLPs with glycosphingolipid-containing HIE membranes, both binding kinetics and affinities explain the patterns of susceptibility toward GII.4 infection for individual HIEs. This is the first time norovirus VLPs have been shown to interact specifically with secretor gene-dependent GSLs embedded in lipid membranes of HIEs that propagate GII.4 HuNoV ex vivo, highlighting the potential of HIEs for advanced future studies of intestinal glycobiology and host-pathogen interactions.
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| 21. |
- Thorsteinsson, Konrad, 1991-, et al.
(författare)
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FRET-Based Assay for the Quantification of Extracellular Vesicles and Other Vesicles of Complex Composition
- 2020
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 92:23, s. 15336-15343
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Tidskriftsartikel (refereegranskat)abstract
- Research in the field of extracellular vesicles is rapidly expanding and finding footholds in many areas of medical science. However, the availability of methodologies to quantify the concentration of membrane material present in a sample remains limited. Herein, we present a novel approach for the quantification of vesicle material, specifically the quantification of the total lipid membrane surface area, found in a sample using Förster resonance energy transfer (FRET). In this assay, sonication is used to drive the fusion between vesicles in the sample to be quantified and liposomes containing a pair of FRET fluorophores. The change in emission spectrum upon vesicle fusion is directly related to the total membrane surface area of the sample added, and a calibration curve allows for the quantification of a variety of vesicle species, including enveloped viruses, bacterial outer membrane vesicles, and mammalian extracellular vesicles. Without extensive optimization of experimental parameters, we were able to quantify down to ∼109 vesicles/mL, using as little as 60 μL of the sample. The assay precision was comparable to that of a commercial nanoparticle tracking analysis system. While its limit of detection was slightly higher, the FRET assay is superior for the detection of small vesicles, as its performance is vesicle-size-independent. Taken together, the FRET assay is a simple, robust, and versatile method for the quantification of a variety of purified vesicle samples.
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| 22. |
- Thorsteinsson, Konrad, 1991-
(författare)
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Probing and elucidating the dynamics of virus-membrane interaction via plasma membrane mimics
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Virus infection is initiated by the attachment of a virion to a susceptible cell’s plasma membrane, in a highly dynamic and well-orchestrated process that encompasses various steps and engages numerous viral and cellular factors. These dynamic steps may include initial non-specific binding to ubiquitous cell-membrane ligands, diffusion across the membrane to a suitable entry site and virus engagement with various receptors and co-receptors on the cell surface. Molecules and processes involved may vary across virus species, but it is likely that in all cases the dynamics of virus-membrane interactions need to be carefully fine-tuned to optimize the entry process. Nevertheless, the investigation and characterization of the involved biomolecular interactions are often oversimplified to isolated virus-receptor pairs engagement, ignoring the complexity of the membrane and the dynamic behaviors of the interaction. This doctoral thesis aims to shed light on this critical sphere of virus-membrane interactions, focusing on how viruses dynamically engage with plasma membrane molecules to successfully infect the cell. To facilitate this research, we utilized an innovative approach of using plasma membrane mimics to explore how viruses dynamically interact with the plasma membrane across several chosen contexts.Central to this project was the development and optimization of cell membrane mimics consisting of supported lipid bilayers (SLBs) reflecting the compositional complexity of the membrane. In the first paper, we developed a method to quantify the membrane material in a lipid vesicle sample in terms of total membrane surface area. This method is vital for the full utilization of our membrane mimics, which in many cases require the mixing of different vesicles in specific ratios.Subsequently, cell membrane mimics of complex composition were used to investigate the molecular mechanisms modulating virus-membrane interactions in several chosen contexts. These investigations relied primarily on total internal reflection fluorescent microscopy to characterize the attachment and detachment behavior of individual virus particles from the membrane. Firstly, studies focused on norovirus, a pathogen that infects cells in the gastrointestinal tract. The virus is known to bind to histo-blood group antigens (HGBA), specific glycans found on intestinal epithelial cells membranes. However, susceptibility to norovirus infection varies between individuals, and the difference is correlated to the specific glycan expression of the intestinal cells. This suggests that the differences in susceptibility might be related to differences in virus-membrane interaction dynamics. Using membranes constructed from lipids extracted from human intestinal enteroids (HIE) derived from susceptible and non-susceptible individuals, it was determined that norovirus associates similarly to both susceptible and non-susceptible membranes but dissociates slower from susceptible membranes. Using native supported lipid bilayers (nSLBs), bilayers derived from plasma membrane extracts of the HIEs, we then investigated the contribution of different carbohydrate moieties to interaction kinetics. This investigation revealed that virus binding to fucose residues on HBGAs is only part of the interaction, and that the virus also binds to sialic acid to a similar degree. It was also found that binding occurs primarily to membrane glycoproteins, and not membrane glycolipids.nSLBs were further found to be highly useful complements to virological investigations in a number of contexts. First, we studied the effect of isoform 4 of apolipoprotein E (ApoE4) on herpes simplex virus 1 (HSV-1) binding kinetics to plasma membrane SLBs. ApoE4 is a lipid binding protein that has been found, in conjunction with HSV-1, to be a risk factor for Alzheimer's disease. We showed that membrane-bound ApoE4 does not affect HSV-1 binding kinetics, but that viruses coated with ApoE4 demonstrate faster dissociation from susceptible membranes than non-coated viruses, indicating that the protein facilitates the release of new virions from the infected cell. This provides a mechanistic understanding of the overall pro-viral effect of ApoE, observed in infection experiments.Second, nSLBs from respiratory epithelial cells were used to quantify binding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the plasma membrane. SARS-CoV-2 has caused one of the largest pandemic in modern history and the virus has shown the ability to rapidly mutate, causing periodic surges of new cases, with newer strains spreading more easily. These mutations have often been linked to the viral spike glycoprotein, responsible for viral attachment and entry. Investigations using spike-decorated liposomes as virus-mimetics, revealed that virus-membrane interaction dynamics vary for different variants of concerns. Specifically, the late Omicron variant, a highly transmissible variant, shows significantly increased affinity to susceptible membranes. This increased affinity was primarily due to increased association to the membrane. Experiments also showed that membrane-bound heparan sulfate has an inhibiting effect on virus binding to ACE2, for earlier variants.In summary, we have successfully implemented membrane mimics with different levels of complexity to investigate virus-membrane interactions. This thesis demonstrates their potential in virology research in several contexts, including measuring the avidity of viruses to membranes, evaluating the relative contributions of different attachment factors to kinetics, and the influence of viral and cellular factors on binding.
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| 23. |
- Trybala, Edward, 1955, et al.
(författare)
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Herpes Simplex Virus Type 2 Mucin-Like Glycoprotein mgG Promotes Virus Release from the Surface of Infected Cells
- 2021
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Ingår i: Viruses. - : MDPI AG. - 1999-4915. ; 13:5
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Tidskriftsartikel (refereegranskat)abstract
- The contribution of virus components to liberation of herpes simplex virus type 2 (HSV-2) progeny virions from the surface of infected cells is poorly understood. We report that the HSV-2 mutant deficient in the expression of a mucin-like membrane-associated glycoprotein G (mgG) exhibited defect in the release of progeny virions from infected cells manifested by similar to 2 orders of magnitude decreased amount of infectious virus in a culture medium as compared to native HSV-2. Electron microscopy revealed that the mgG deficient virions were produced in infected cells and present at the cell surface. These virions could be forcibly liberated to a nearly native HSV-2 level by the treatment of cells with glycosaminoglycan (GAG)-mimicking oligosaccharides. Comparative assessment of the interaction of mutant and native virions with surface-immobilized chondroitin sulfate GAG chains revealed that while the mutant virions associated with GAGs similar to fourfold more extensively, the lateral mobility of bound virions was much poorer than that of native virions. These data indicate that the mgG of HSV-2 balances the virus interaction with GAG chains, a feature critical to prevent trapping of the progeny virions at the surface of infected cells.
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