Sphingomyelin-Sequestered Cholesterol Domain Recruits Formin-Binding Protein 17 for Constricting Clathrin-Coated Pits in Influenza Virus Entry

Influenza A virus (IAV) is a global health threat. The cellular endocytic machineries harnessed by IAV remain elusive. Here, by tracking single IAV particles and quantifying the internalized IAV, we found that the sphingomyelin (SM)-sequestered cholesterol, but not the accessible cholesterol, is essential for the clathrin-mediated endocytosis (CME) of IAV. The clathrin-independent endocytosis of IAV is cholesterol-independent. Whereas, the CME of transferrin depends on SM-sequestered cholesterol and accessible cholesterol. Furthermore, three-color single-virus tracking and electron microscopy showed that the SM-cholesterol complex nanodomain is recruited to the IAV-containing clathrin-coated structure (CCS) and facilitates neck constriction of the IAV-containing CCS. Meanwhile, formin-binding protein 17 (FBP17), a membrane-bending protein which activates actin nucleation, is recruited to IAV-CCS complex in a manner dependent on the SM-cholesterol complex. We propose that the SM-cholesterol nanodomain at the neck of CCS recruits FBP17 to induce neck constriction by activating actin assembly. These results unequivocally show the physiological importance of the SM-cholesterol complex in IAV entry. Importance: IAV infects the cells by harnessing cellular endocytic machineries. Better understanding of the cellular machineries used for its entry might lead to the development of antiviral strategies, and would also provide important insights into physiological endocytic processes. This work demonstrated that a special pool of cholesterol in plasma membrane, SM-sequestered cholesterol, recruits FBP17 for the constriction of clathrin-coated pits in IAV entry. Meanwhile, the clathrin-independent cell entry of IAV is cholesterol-independent. The internalization of transferrin, the gold-standard cargo endocytosed solely via CME, is much less dependent on the SM-cholesterol complex. These results would provide new insights into IAV infection and pathway/cargo-specific involvement of cholesterol pool(s).

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Coronavirus and Influenza Virus Proteolytic Priming Takes Place in Tetraspanin-Enriched Membrane Microdomains

Journal of Virology ◽

10.1128/jvi.00543-15 ◽

2015 ◽

Vol 89 (11) ◽

pp. 6093-6104 ◽

Cited By ~ 23

Author(s):

James T. Earnest ◽

Michael P. Hantak ◽

Jung-Eun Park ◽

Tom Gallagher

Keyword(s):

Cell Membrane ◽

Membrane Fusion ◽

Target Cell ◽

Influenza A ◽

Virus Entry ◽

Cell Entry ◽

Membrane Microdomains ◽

Enveloped Viruses ◽

Viral Surface ◽

Surface Glycoproteins

ABSTRACTCoronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and virus-priming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudoparticles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin short hairpin RNAs (shRNAs). Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudoparticle transductions but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate coengagement with cell receptors and proteases.IMPORTANCEEnveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus cell entry. Among these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on coronavirus and influenza A virus cell entry and identifies TEMs as sites of viral proteolysis, thereby defining subcellular locations of virus priming with greater precision. Implications of these findings extend to the use of virus entry antagonists, such as protease inhibitors, which might be most effective when localized to these microdomains.

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Clathrin Hub Expression Dissociates the Actin-Binding Protein Hip1R from Coated Pits and Disrupts Their Alignment with the Actin Cytoskeleton

Traffic ◽

10.1034/j.1600-0854.2001.21114.x ◽

2001 ◽

Vol 2 (11) ◽

pp. 851-858 ◽

Cited By ~ 31

Author(s):

Elizabeth M. Bennett ◽

Chih-Ying Chen ◽

Asa E. Y. Engqvist-Goldstein ◽

David G. Drubin ◽

Frances M. Brodsky

Keyword(s):

Actin Cytoskeleton ◽

Binding Protein ◽

Actin Binding ◽

Coated Pits ◽

Actin Binding Protein

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Influenza A Virus Cell Entry, Replication, Virion Assembly and Movement

Frontiers in Immunology ◽

10.3389/fimmu.2018.01581 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 80

Author(s):

Dan Dou ◽

Rebecca Revol ◽

Henrik Östbye ◽

Hao Wang ◽

Robert Daniels

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Cell Entry ◽

Virion Assembly

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LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

Journal of Virology ◽

10.1128/jvi.00562-20 ◽

2020 ◽

Vol 94 (18) ◽

Cited By ~ 7

Author(s):

Xuesen Zhao ◽

Shuangli Zheng ◽

Danying Chen ◽

Mei Zheng ◽

Xinglin Li ◽

...

Keyword(s):

Influenza A ◽

Yellow Fever Virus ◽

Virus Entry ◽

Ectopic Expression ◽

A549 Cells ◽

Cellular Protein ◽

Host Cells ◽

Cellular Proteins ◽

Human Coronavirus ◽

Human Coronaviruses

ABSTRACT C3A is a subclone of the human hepatoblastoma HepG2 cell line with strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2), gamma-interferon-inducible lysosome/endosome-localized thiolreductase (GILT), and lymphocyte antigen 6 family member E (LY6E), the three cellular proteins identified to function in interference with virus entry, were expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-O43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFN-inducible transmembrane 3 (IFITM3) restriction of human coronavirus (CoV) entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a mechanism distinct from other factors that modulate CoV entry. IMPORTANCE Virus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control of host innate and adaptive immune responses. In the last decade, several interferon-inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH, and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as a host factor that facilitates the entry of several human-pathogenic viruses, including human immunodeficiency virus, influenza A virus, and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.

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A myosin-7B–dependent endocytosis pathway mediates cellular entry of α-synuclein fibrils and polycation-bearing cargos

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1918617117 ◽

2020 ◽

Vol 117 (20) ◽

pp. 10865-10875 ◽

Cited By ~ 5

Author(s):

Qi Zhang ◽

Yue Xu ◽

Juhyung Lee ◽

Michal Jarnik ◽

Xufeng Wu ◽

...

Keyword(s):

Cell Surface ◽

Actin Filaments ◽

Membrane Dynamics ◽

Cell Entry ◽

Sulfate Proteoglycan ◽

Actin Network ◽

Coated Pits ◽

Endocytosis Pathway ◽

Underlying Mechanisms ◽

Pathological Event

Cell-to-cell transmission of misfolding-prone α-synuclein (α-Syn) has emerged as a key pathological event in Parkinson’s disease. This process is initiated when α-Syn–bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulators for α-Syn preformed fibrils (PFFs). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting α-Syn PFFs to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFFs. By contrast, MYO7B regulates α-Syn PFF cell entry by maintaining a plasma membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing “scars” on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is restricted to α-Syn PFFs and other polycation-bearing cargos that enter cells via HSPGs. Thus, our study not only defines regulatory factors for α-Syn PFF endocytosis, but also reveals a previously unknown endocytosis mechanism for HSPG-binding cargos in general, which requires forces generated by MYO7B and actin filaments.

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Bayesian estimation to test accuracy for influenza A infection via respiratory clinical signs in the absence of a gold standard

Journal of Veterinary Medicine and Animal Health ◽

10.5897/jvmah2015.0410 ◽

2015 ◽

Vol 7 (10) ◽

pp. 318-327 ◽

Cited By ~ 1

Author(s):

Homwong Nitipong ◽

Marthaler Douglas ◽

Convertino Matteo ◽

Torremorell Montserrat ◽

E Craft Meggan ◽

...

Keyword(s):

Bayesian Estimation ◽

Gold Standard ◽

Influenza A ◽

Clinical Signs ◽

Test Accuracy

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Nonstructural Protein 1 of Influenza A Virus Interacts with Human Guanylate-Binding Protein 1 to Antagonize Antiviral Activity

PLoS ONE ◽

10.1371/journal.pone.0055920 ◽

2013 ◽

Vol 8 (2) ◽

pp. e55920 ◽

Cited By ~ 52

Author(s):

Zixiang Zhu ◽

Zixue Shi ◽

Wenjun Yan ◽

Jianchao Wei ◽

Donghua Shao ◽

...

Keyword(s):

Antiviral Activity ◽

Influenza A Virus ◽

Influenza A ◽

Nonstructural Protein ◽

Binding Protein ◽

Nonstructural Protein 1 ◽

Guanylate Binding Protein

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Influenza A virus uses the aggresome processing machinery for host cell entry

Science ◽

10.1126/science.1257037 ◽

2014 ◽

Vol 346 (6208) ◽

pp. 473-477 ◽

Cited By ~ 125

Author(s):

Indranil Banerjee ◽

Yasuyuki Miyake ◽

Samuel Philip Nobs ◽

Christoph Schneider ◽

Peter Horvath ◽

...

Keyword(s):

Host Cell ◽

Influenza A Virus ◽

Influenza A ◽

Cell Entry ◽

Processing Machinery ◽

Host Cell Entry

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Oncolytic H-1 Parvovirus Enters Cancer Cells through Clathrin-Mediated Endocytosis

Viruses ◽

10.3390/v12101199 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1199

Author(s):

Tiago Ferreira ◽

Amit Kulkarni ◽

Clemens Bretscher ◽

Karsten Richter ◽

Marcelo Ehrlich ◽

...

Keyword(s):

Viral Entry ◽

Small Interfering Rna ◽

Anticancer Agent ◽

Cell Entry ◽

Productive Infection ◽

Acidic Ph ◽

Coated Pits ◽

Late Endosomes ◽

Interfering Rna ◽

Specific Inhibitors

H-1 protoparvovirus (H-1PV) is a self-propagating virus that is non-pathogenic in humans and has oncolytic and oncosuppressive activities. H-1PV is the first member of the Parvoviridae family to undergo clinical testing as an anticancer agent. Results from clinical trials in patients with glioblastoma or pancreatic carcinoma show that virus treatment is safe, well-tolerated and associated with first signs of efficacy. Characterisation of the H-1PV life cycle may help to improve its efficacy and clinical outcome. In this study, we investigated the entry route of H-1PV in cervical carcinoma HeLa and glioma NCH125 cell lines. Using electron and confocal microscopy, we detected H-1PV particles within clathrin-coated pits and vesicles, providing evidence that the virus uses clathrin-mediated endocytosis for cell entry. In agreement with these results, we found that blocking clathrin-mediated endocytosis using specific inhibitors or small interfering RNA-mediated knockdown of its key regulator, AP2M1, markedly reduced H-1PV entry. By contrast, we found no evidence of viral entry through caveolae-mediated endocytosis. We also show that H-1PV entry is dependent on dynamin, while viral trafficking occurs from early to late endosomes, with acidic pH necessary for a productive infection. This is the first study that characterises the cell entry pathways of oncolytic H-1PV.

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