Cryo-Em Imaging of a Pseudotyped Adenovhtus with Ocular Cell Tropism

2000 ◽  
Vol 6 (S2) ◽  
pp. 286-287
Author(s):  
P.L. Stewart ◽  
C.Y. Chiu ◽  
D. Von Seggern ◽  
G.R. Nemerow
Keyword(s):  
Viral Entry ◽  
Human Adenovirus ◽  
Cell Entry ◽  
Soluble Form ◽  
Cell Tropism ◽  
Penton Base ◽  
Single Particle Reconstruction ◽  
Virus Particles ◽  
Entry Pathway ◽  
Reconstruction Methods

Human adenovirus (Ad) provides unique model system for studying the multistage events in viral entry. We are applying cryo-electron microscopy and single particle reconstruction methods to study the cell entry pathway of Ad as well as to examine the structure of reengineered Ad vectors. Ad cell entry is mediated by interactions with two different cellular receptors: the fiber receptor, CAR for most Ad serotypes or a 50 kDa receptor for a subset of serotypes including Ad37, for attachment; and the αvβ3 and αvβ5 integrins for internalization. We have previously reconstructed both Ad2 and Adl2 virus particles complexed with a soluble form of αvβ5 integrin. We learned during our comparative cryo-EM study of the Ad2/αvβ5 and Adl2/αvβ5 complexes that the length of the variable RGD loop in the penton base affects the relative height of the integrin density over the penton base.

scholarly journals Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Qi Zhang ◽  
Catherine Zhengzheng Chen ◽  
Manju Swaroop ◽  
Miao Xu ◽  
Lihui Wang ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Viral Entry ◽  
Drug Repurposing ◽  
Cell Entry ◽  
Entry Pathway ◽  
Viral Particles ◽  
Approved Drugs ◽  
Attachment Factor

Abstract The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-Cov and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, and facilitates the attachment of Spike-bearing viral particles to the cell surface to promote viral entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry and reveals drugs capable of targeting this important step in the viral life cycle.

scholarly journals How antibodies alter the cell entry pathway of dengue virus particles in macrophages

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Nilda V. Ayala-Nunez ◽  
Tabitha E. Hoornweg ◽  
Denise P.I. van de Pol ◽  
Klaas A. Sjollema ◽  
Jacky Flipse ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Cell Entry ◽  
Virus Particles ◽  
Entry Pathway

scholarly journals Cryo-Electron Microscopy Structure of an Adenovirus-Integrin Complex Indicates Conformational Changes in both Penton Base and Integrin

2009 ◽  
Vol 83 (22) ◽  
pp. 11491-11501 ◽  
Author(s):  
Steffen Lindert ◽  
Mariena Silvestry ◽  
Tina-Marie Mullen ◽  
Glen R. Nemerow ◽  
Phoebe L. Stewart
Keyword(s):  
Electron Microscopy ◽  
Conformational Change ◽  
Conformational Changes ◽  
Adenovirus Type ◽  
Cell Entry ◽  
Structural Basis ◽  
Soluble Form ◽  
Penton Base ◽  
Cryo Electron Microscopy ◽  
Moderate Resolution

ABSTRACT A structure of adenovirus type 12 (HAdV12) complexed with a soluble form of integrin αvβ5 was determined by cryo-electron microscopy (cryoEM) image reconstruction. Subnanometer resolution (8 Å) was achieved for the icosahedral capsid with moderate resolution (27 Å) for integrin density above each penton base. Modeling with αvβ3 and αIIbβ3 crystal structures indicates that a maximum of four integrins fit over the pentameric penton base. The close spacing (∼60 Å) of the RGD protrusions on penton base precludes integrin binding in the same orientation to neighboring RGD sites. Flexible penton-base RGD loops and incoherent averaging of bound integrin molecules explain the moderate resolution observed for the integrin density. A model with four integrins bound to a penton base suggests that integrin might extend one RGD-loop in the direction that could induce a conformational change in the penton base involving clockwise untwisting of the pentamer. A global conformational change in penton base could be one step on the way to the release of Ad vertex proteins during cell entry. Comparison of the cryoEM structure with bent and extended models for the integrin ectodomain reveals that integrin adopts an extended conformation when bound to the Ad penton base, a multivalent viral ligand. These findings shed further light on the structural basis of integrin binding to biologically relevant ligands, as well as on the molecular events leading to HAdV cell entry.

scholarly journals Direct Evidence from Single-Cell Analysis that Human α-Defensins Block Adenovirus Uncoating To Neutralize Infection

2010 ◽  
Vol 84 (8) ◽  
pp. 4041-4049 ◽  
Author(s):  
Emily K. Nguyen ◽  
Glen R. Nemerow ◽  
Jason G. Smith
Keyword(s):  
Direct Evidence ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Indirect Evidence ◽  
Human Adenovirus ◽  
Cell Entry ◽  
Adenovirus Infection ◽  
Viral Genomes ◽  
Virus Particles ◽  
Evolutionarily Conserved

ABSTRACT Human α-defensins are evolutionarily conserved effectors of the innate immune response with broadly acting antibacterial activity. Their role in antiviral immunity is less well understood. We previously showed that these antimicrobial peptides are potent inhibitors of human adenovirus infection. Based on biochemical studies and indirect evidence from confocal microscopy, we proposed that defensins bind to and stabilize the virus capsid and neutralize infection by preventing the release of the endosomalytic protein VI. To determine whether defensin action also restricts exposure of the viral genome, we developed a system to evaluate adenovirus uncoating during cell entry by monitoring the exposure of BrdU-labeled viral genomes. This assay allowed us to determine the kinetics of uncoating of virus particles in single cells. Using this assay, we now provide direct evidence that human α-defensins block adenovirus infection by preventing uncoating during cell entry.

scholarly journals Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro

2020 ◽  
Author(s):  
Qi Zhang ◽  
Catherine Z. Chen ◽  
Manju Swaroop ◽  
Miao Xu ◽  
Lihui Wang ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Viral Entry ◽  
Drug Repurposing ◽  
Cell Entry ◽  
Entry Pathway ◽  
Viral Particles ◽  
Approved Drugs ◽  
Attachment Factor

The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-CoV and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, facilitates the attachment of viral particles to the cell surface to promote cell entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry, and reveals drugs capable of targeting this important step in the viral life cycle.

scholarly journals ACE2, TMPRSS2 and L-SIGN expression in placentae from HIV-positive pregnancies exposed to antiretroviral therapy–implications for SARS-CoV-2 placental infection

2021 ◽  
Author(s):  
Smriti Kala ◽  
Ksenia Meteleva ◽  
Lena Serghides
Keyword(s):  
Antiretroviral Therapy ◽  
Pregnant Women ◽  
Viral Entry ◽  
Cell Entry ◽  
Hiv Positive ◽  
Mrna Levels ◽  
Black Race ◽  
Entry Receptor ◽  
Lower Expression ◽  
Human Placentae

Abstract Background SARS-CoV-2 binding receptor ACE2 and the spike protein priming protease TMPRSS2 are co-expressed in human placentae. It is unknown whether their expression is altered in the context of HIV infection and antiretroviral therapy (ART). Methods We compared mRNA levels of SARS-CoV-2 cell-entry mediators ACE2, TMPRSS2 and L-SIGN (an alternative entry receptor) by qPCR in 105 placentae: 45 from pregnant women with HIV (WHIV) exposed to protease inhibitor (PI)-based ART, 17 from WHIV on non-PI-based ART, and 43 from HIV-uninfected women. Results ACE2 levels were lower, while L-SIGN levels were higher in placentae from WHIV on PI-based ART as compared to those on non-PI-based ART and to HIV-uninfected women. TMPRSS2 levels were similar between groups. Black race was significantly associated with lower expression of ACE2 and higher expression of L-SIGN. ACE2 levels were significantly higher in placentae of female fetuses. Discussion We have identified pregnant women of Black race and WHIV who are on PI-based ART to have relatively lower expression of placental ACE2 than those of White race and HIV-uninfected women. This effect may potentially contribute to altered susceptibility to COVID-19 in these women, either favorably; by reduced viral entry, or detrimentally; by loss of ACE2 protection against hyperinflammation.

scholarly journals Characterization of the Early Events in Dengue Virus Cell Entry by Biochemical Assays and Single-Virus Tracking

2007 ◽  
Vol 81 (21) ◽  
pp. 12019-12028 ◽  
Author(s):  
Hilde M. van der Schaar ◽  
Michael J. Rust ◽  
Barry-Lee Waarts ◽  
Heidi van der Ende-Metselaar ◽  
Richard J. Kuhn ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Dengue Virus ◽  
Membrane Fusion ◽  
High Ratio ◽  
Cell Entry ◽  
Viral Membrane ◽  
Virus Particles ◽  
Biochemical Assays ◽  
Particle Ratio ◽  
Infectious Unit

ABSTRACT In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by infectivity assays, leading to an infectious unit-to-particle ratio of approximately 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface density of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was observed that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was observed in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes.

The penton base of human adenovirus type 3 has the RGD motif

Gene ◽  
1994 ◽  
Vol 146 (2) ◽  
pp. 257-259 ◽  
Author(s):  
Alain Cuzange ◽  
Jadwiga Chroboczek ◽  
Bernard Jacrot
Keyword(s):  
Human Adenovirus ◽  
Adenovirus Type ◽  
Penton Base ◽  
Rgd Motif ◽  
Type 3

scholarly journals Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells

2018 ◽  
Vol 92 (19) ◽  
Author(s):  
Shutoku Matsuyama ◽  
Kazuya Shirato ◽  
Miyuki Kawase ◽  
Yutaka Terada ◽  
Kengo Kawachi ◽  
...  
Keyword(s):  
Middle East ◽  
Viral Entry ◽  
Cathepsin L ◽  
Inhibitory Effect ◽  
Middle East Respiratory Syndrome ◽  
Cell Entry ◽  
Target Cells ◽  
Furin Cleavage ◽  
S Protein ◽  
Entry Assay

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin. IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.

Structural and biochemical characterization of a human adenovirus 2/12 penton base chimera

FEBS Journal ◽  
2006 ◽  
Vol 273 (18) ◽  
pp. 4336-4345 ◽  
Author(s):  
Chloe Zubieta ◽  
Laurent Blanchoin ◽  
Stephen Cusack
Keyword(s):  
Biochemical Characterization ◽  
Human Adenovirus ◽  
Penton Base
Sign in / Sign up

Export Citation Format

Share Document