Deciphering cell-herpesvirus interactions using force-distance curve-based atomic force microscopy

Viruses are the most numerous species on earth. Some of them can be the cause of mild to severe diseases with potential deadly outcomes. Viral infection is initiated by the attachment of virus particles to the surface of target host cells. Understanding the molecular interactions taking place between virus particles and cell surface molecules is of fundamental interest for the development of new therapeutics interfering with virus attachment processes. Most of the insights gained on virus-cell interactions are currently obtained via ensemble molecular studies providing average responses of populations of virions. To unveil molecular details arising from biological variability, studies performed at the single virus particle-level could provide new valuable insights into virus attachment mechanisms. The aim of this PhD thesis is therefore to implement new tools to decipher the putative roles of viral surface glycoproteins upon attachment to cell surfaces at the single-virus particle level. This work involves the use and development of atomic force microscopy-based single-virus force spectroscopy (SVFS) to decipher the molecular mechanisms of herpesvirus attachment to cell surface glycosaminoglycans (GAGs). We investigated the attachment properties of single virions of Murid Herpesvirus-4 (MuHV-4) and Herpes Simplex Virus-1 (HSV-1) towards both purified GAGs grafted onto inert surfaces and GAGs on living cell surfaces. We observed that both glycoprotein gp150 (MuHV-4) and the mucin-like region of glycoprotein gC (HSV-1) play regulatory roles upon attachment of virions to GAGs, by regulating the multivalency of other viral glycoprotein-GAG interactions. We further deciphered the putative roles of two apparently redundant glycoproteins of MuHV-4: gH/gL and gp70. We showed that gH/gL provides a larger contribution to virus-GAG interactions than gp70 and that the gH/gL-GAG interactions have a higher inherent stability. The molecular insights gained by SVFS on the complex mechanisms developed by herpesviruses to attach target cells could contribute to the development of new antiviral therapeutics potentially targeting specific viral surface glycoproteins.(AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 202

Initial Step of Virus Entry: Virion Binding to Cell-Surface Glycans.

peer reviewedVirus infection is an intricate process that requires the concerted action of both viral and host cell components. Entry of viruses into cells is initiated by interactions between viral proteins and cell-surface receptors. Various cell-surface glycans function as initial, usually low-affinity attachment factors, providing a first anchor of the virus to the cell surface, and further facilitate high-affinity binding to virus-specific cell-surface receptors, while other glycans function as specific entry receptors themselves. It is now possible to rapidly identify specific glycan receptors using different techniques, define atomic-level structures of virus-glycan complexes, and study these interactions at the single-virion level. This review provides a detailed overview of the role of glycans in viral infection and highlights experimental approaches to study virus-glycan binding along with specific examples. In particular, we highlight the development of the atomic force microscope to investigate interactions with glycans at the single-virion level directly on living mammalian cells, which offers new perspectives to better understand virus-glycan interactions in physiologically relevant conditions. Expected final online publication date for the Annual Review of Virology, Volume 7 is September 29, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates

Initial step of virus entry: virion binding to cell surface glycans

Virus infection is an intricate process that requires the concerted action of both viral and host cell components. Entry of viruses into cells is initiated by interactions between viral proteins and cell surface receptors. Various cell surface glycans function as initial, usually low-affinity attachment factors, providing a first anchor of the viruses to the cell surface and further facilitate high-affinity binding to virus-specific cell surface receptors, while other glycans function as specific entry receptors themselves. It is now possible to rapidly identify specific glycan receptors using different techniques, to define atomic-level structures of virus-glycan complexes and to study these interactions at the single-virion level. This review gives a detailed overview of the role of glycans in viral infection and highlights experimental approaches to study virus-glycan binding along with specific examples. In particular, we highlight the development of the atomic force microscope to investigate interactions with glycans at the single-virion level directly on living mammalian cells offering new perspectives to better understand virus-glycan interactions in physiologically relevant conditions

Heparin-Induced Allosteric Changes in SARS-CoV-2 Spike Protein Facilitate ACE2 Binding and Viral Entry.

Understanding the entry of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) into host cells is crucial in the battle against COVID-19. Using atomic force microscopy (AFM), we probed the interaction between the virus's spike protein and heparan sulfate (HS) as a potential attachment factor. Our AFM studies revealed a moderate-affinity interaction between the spike protein and HS on both model surfaces and living cells, highlighting HS's role in early viral attachment. Remarkably, we observed an interplay between HS and the host cell receptor angiotensin-converting enzyme 2 (ACE2), with HS engagement resulting in enhanced ACE2 binding and subsequent viral entry. Our research furthers our understanding of SARS-CoV-2 infection mechanisms and reveals potential interventions targeting viral entry. These insights are valuable as we navigate the evolving landscape of viral threats and seek effective strategies to combat emerging infectious diseases

Multivalent binding of herpesvirus to living cells is tightly regulated during infection.

Viral infection, initiated by the landing of a virion on a cellular surface, is largely defined by the preliminary interactions established between viral particles and their receptors at the cell surface. While multiple parallel interactions would allow strong virus attachment, a low number of bonds could be preferred to allow lateral diffusion toward specific receptors and to promote efficient release of progeny virions from the cell surface. However, so far, the molecular mechanisms underlying the regulation of the multivalency in virus attachment to receptors are poorly understood. We introduce a new method to force-probe multivalent attachment directly on living cells, and we show, for the first time, direct evidence of a new mechanism by which a herpesvirus surface glycoprotein acts as a key negative regulator in the first step of herpesvirus binding. Using atomic force microscopy, we probe at the single-virion level the number and the strength of the bonds established with heparan sulfate both on model surfaces and on living cells. Our biophysical results, correlated with other techniques, show that the major envelope glycoprotein functions as a regulator of binding valency during both attachment and release steps, determining the binding, diffusion, and release potential of virions at the cellular surface

Single-Virus Force Spectroscopy Discriminates the Intrinsic Role of Two Viral Glycoproteins upon Cell Surface Attachment

Viruses are one of the most efficient pathogenic entities on earth, resulting from millions of years of evolution. Each virus particle carries the minimum number of genes and proteins to ensure their reproduction within host cells, hijacking some host replication machinery. However, the role of some viral proteins is not yet unraveled, with some appearing even redundant. For example, murid herpesvirus 4, the current model for human gammaherpesvirus infection, can bind to cell surface glycosaminoglycans using both glycoproteins gp70 and gH/gL. Here, using atomic force microscopy, we discriminate their relative contribution during virus binding to cell surface glycosaminoglycans. Single-virus force spectroscopy experiments demonstrate that gH/gL is the main actor in glycosaminoglycan binding, engaging more numerous and more stable interactions. We also demonstrated that Fab antibody fragments targeting gH/gL or gp70 appear to be a promising treatment to prevent the attachment of virions to cell surf

Third metacarpal bone mineral density assessment in the standing horse by dual X-day absorptiometry suitability, precision and accuracy

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Third metacarpal bone mineral density assessment in the standing horse by dual X-ray absorptiometry - Suitability, precision and accuracy

Bone mineral density (BMD) is correlated to mechanical properties of bone. In the horse, dual energy X-ray absorptiometry (DXA) has yet only been performed ex-vivo, but a new portable DXA device would be ideal for in-vivo BMD measurement. We explored field suitability, precision and accuracy of this device for in-vivo third metacarpal density assessment. Precision was analysed by calculating measurement variation under repeated measurement tests with (reproducibility) and without (repeatability) limb repositioning. Repeatability and reproducibility were tested ex-vivo, at the some time that intra- and inter-operator reproducibility were assessed in-vivo. In order to test accuracy, bone mineral content (BMC) of several bone samples determined by DXA and ashing were compared. Repeatability was 1.47% and reproducibility 1.69% ex-vivo. In-vivo reproducibility varied between 2.91 and 4.06% for intraoperator test and between 3.13 and 5.53% for interoperator test. BMC measured by DXA and ash weight were highly correlated (R-2 > 0.99). In conclusion, under described conditions this DXA device is usable, accurate and precise. Its sensitiveness reaches 8.23% in an individual longitudinal monitoring. Using the third metacarpal bone as an example, we have shown that this device is suitable for experimental or clinical monitoring

Combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces

ISSN:1750-2799ISSN:1745-2481ISSN:1754-218

Regulatory Mechanisms of the Mucin-Like Region on Herpes Simplex Virus during Cellular Attachment

Mucin-like regions, characterized by a local high density of O-linked glycosylation, are found on the viral envelope glycoproteins of many viruses. Herpes simplex virus type 1 (HSV-1), for example, exhibits a mucin-like region on its glycoprotein gC, a viral protein involved in initial recruitment of the virus to the cell surface via interaction with sulfated glycosaminoglycans. So far, this mucin-like region has been proposed to play a key role in modulating the interactions with cellular glycosaminoglycans, and in particular to promote release of HSV-1 virions from infected cells. However, the molecular mechanisms and the role as a pathogenicity factor remains unclear. Using single virus particle tracking, we show that the mobility of chondroitin sulfate-bound HSV-1 virions is decreased in absence of the mucin-like region. This decrease in mobility correlates with an increase in HSV-1-chondroitin sulfate binding forces as observed using atomic force microscopy-based force spectroscopy. Our data suggest that the mucin-like region modulates virus-glycosaminoglycan interactions by regulating the affinity, type, and number of glycoproteins involved in the virus-glycosaminoglycan interaction. This study therefore presents new evidence for a role of the mucin-like region in balancing the interaction of HSV-1 with glycosaminoglycans and provides further insights into the molecular mechanisms used by the virus to ensure both successful cell entry and release from the infected cell