Fluorescently Labeled Human Papillomavirus Pseudovirions for Use in Virus Entry Experiments

Ventayol, Pilar Samperio; Schelhaas, Mario

doi:10.1002/9780471729259.mc14b04s37

Cited by 6 publications

(7 citation statements)

References 23 publications

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“…In this assay, virus particles that were covalently labeled with a pH-sensitive fluorophore (pHrodo) were employed. pHrodo emits fluorescence exclusively in acidic environments, such as endosomes (65). pHrodo-HPV16 and pHrodo-FPC-HPV16 particles were added to cells, and the pHrodo signal was imaged at different time points p.i.…”

Section: Fpc-hpv16 Shares Important Structural Characteristics With Hmentioning

confidence: 99%

Extracellular Conformational Changes in the Capsid of Human Papillomaviruses Contribute to Asynchronous Uptake into Host Cells

Becker

Greune

Schmidt

et al. 2018

J Virol

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The human papillomavirus type 16 (HPV16) is the leading cause of cervical cancer. For initial infection, HPV16 utilizes a novel endocytic pathway for host cell entry. Unique amongst viruses, uptake occurs asynchronously over a protracted period of time with half-times between 9-12 h. To trigger endocytic uptake, the virus particles need to undergo a series of structural modifications after initial binding to heparan sulfate proteoglycans (HSPG). These changes involve proteolytic cleavage of the major capsid protein L1 by kallikrein-8 (KLK8), exposure of the N-terminus of the minor capsid protein L2 by cyclophilins, and cleavage of this N-terminus by furin. Overall, the structural changes are thought to facilitate the engagement of an elusive secondary receptor for internalization. Here, we addressed whether structural changes are the rate-limiting steps during infectious internalization of HPV16 by using structurally-primed HPV16 particles. Our findings indicate that the structural modifications mediated by cyclophilins and furin, which lead to exposure and cleavage of the L2 N-terminus, respectively, contribute to the slow and asynchronous internalization kinetics, whereas conformational changes elicited by HSPG binding and KLK8 cleavage did not. However, these structural modifications only accounted for 30-50% of the delay in internalization. Therefore, we propose that limited internalization receptor availability for engagement of HPV16 causes slow and asynchronous internalization in addition to rate-limiting structural changes in the viral capsid. HPVs are the main cause for anogenital cancers. Their unique biology is linked to the differentiation program of skin or mucosa. Here, we analyzed another unique aspect of HPV infections using the prototype HPV16. After initial cell binding, HPVs display an unusually protracted residence time on the plasma membrane prior to asynchronous uptake. As viruses typically do not expose themselves to host immune sensing, we analyzed the underlying reasons for this unusual behavior. This study provides evidence that both extracellular structural modifications and possibly a limited availability of the internalization receptor contribute to the slow internalization process of the virus. These findings indicated that perhaps a unique niche for initial infection exists that could allow for rapid infection. In addition, our results may help to develop novel, preventive antiviral measures.

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Section: Fpc-hpv16 Shares Important Structural Characteristics With Hmentioning

confidence: 99%

Extracellular Conformational Changes in the Capsid of Human Papillomaviruses Contribute to Asynchronous Uptake into Host Cells

Becker

Greune

Schmidt

et al. 2018

J Virol

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“…To verify the impact on HPV16 uptake, we employed a bona fide virus endocytosis assay. For this, HPV16 covalently labelled with the pH-sensitive dye pHrodo was used, which gives rise to fluorescence only upon delivery to acidic endosomal organelles (Figure 3D) (Ventayol and Schelhaas, 2015; Becker et al ., 2018). Depletion of Arp3 reduced virus internalization by about 70% (Figure 3D).…”

Section: Resultsmentioning

confidence: 99%

“…HPV16 PsVs were incubated with Alexa Fluor 488, 568, 594 or 647 succinimidyl ester in virion buffer (635 mM NaCl, 0.9 mM CaCl 2 , 0.5 mM MgCl 2 , 2.1 mM KCl in PBS, pH 7.6) using a 1:8 molar ratio of L1 to the dye for 1 h on an overhead rotator (Schelhaas et al ., 2008; Ventayol and Schelhaas, 2015). Free dye was removed by ultracentrifugation using a 15-25-39% OptiPrep step gradient.…”

Section: Star Methodsmentioning

confidence: 99%

“…The PsV concentration was determined by SDS-PAGE and subsequent Coomassie staining. The labeled virus was characterized by binding to glass coverslips and HeLa ATCC cells (Ventayol and Schelhaas, 2015). Labeling of PsVs with pHrodo was achieved following the same protocol.…”

Section: Star Methodsmentioning

confidence: 99%

“…Labeling of PsVs with pHrodo was achieved following the same protocol. Virus characterization was performed in citric acid buffer (pH 4.4) (Ventayol and Schelhaas, 2015).…”

Section: Labeling Of Hpv16 Psvsmentioning

confidence: 99%

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Clathrin-independent endocytosis of Human Papillomaviruses is facilitated by actin nucleation promoting factor WASH

Brinkert

Krebs

et al. 2021

Preprint

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Endocytosis of extracellular or plasma membrane material is a fundamental process. A variety of endocytic pathways exist, several of which are barely understood in terms of mechanistic execution and biological function. Importantly, some mechanisms have been identified and characterized by following virus internalization into cells. This includes a novel endocytic pathway exploited by human papillomavirus type 16 (HPV16). However, its cellular role and mechanism of endocytic vacuole formation remain unclear. Here, HPV16 was used as a tool to examine the mechanistic execution of vesicle formation by combining systematic perturbation of cellular processes with electron and video microscopy. Our results indicate cargo uptake by uncoated, inward-budding pits facilitated by the membrane bending retromer protein SNX2. Actin polymerization-driven vesicle scission is promoted by WASH, an actin regulator typically not found at the plasma membrane. Uncovering a novel role of WASH in endocytosis, we propose to term the new pathway WASH-mediated endocytosis (WASH-ME).

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Meningeal lymphatic endothelial cells fulfill scavenger endothelial cell function and cooperate with microglia in waste removal from the brain

Padberg

Uphoff

Berger

et al. 2021

Glia

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Brain lymphatic endothelial cells (BLECs) constitute a group of loosely connected endothelial cells that reside within the meningeal layer of the zebrafish brain without forming a vascular tubular system. BLECs have been shown to readily endocytose extracellular cargo molecules from the brain parenchyma, however, their functional relevance in relation to microglia remains enigmatic. We here compare their functional uptake efficiency for several macromolecules and bacterial components with microglia in a qualitative and quantitative manner in 5‐day‐old zebrafish embryos. We find BLECs to be significantly more effective in the uptake of proteins, polysaccharides and virus particles as compared to microglia, while larger particles like bacteria are only ingested by microglia but not by BLECs, implying a clear distribution of tasks between the two cell types in the brain area. In addition, we compare BLECs to the recently discovered scavenger endothelial cells (SECs) of the cardinal vein and find them to accept an identical set of substrate molecules. Our data identifies BLECs as the first brain‐associated SEC population in vertebrates, and demonstrates that BLECs cooperate with microglia to remove particle waste from the brain.

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Fluorescently Labeled Human Papillomavirus Pseudovirions for Use in Virus Entry Experiments

Cited by 6 publications

References 23 publications

Extracellular Conformational Changes in the Capsid of Human Papillomaviruses Contribute to Asynchronous Uptake into Host Cells

Extracellular Conformational Changes in the Capsid of Human Papillomaviruses Contribute to Asynchronous Uptake into Host Cells

Clathrin-independent endocytosis of Human Papillomaviruses is facilitated by actin nucleation promoting factor WASH

Meningeal lymphatic endothelial cells fulfill scavenger endothelial cell function and cooperate with microglia in waste removal from the brain

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