The Structure of the Poliovirus 135S Cell Entry Intermediate at 10-Angstrom Resolution Reveals the Location of an Externalized Polypeptide That Binds to Membranes

ABSTRACT Poliovirus provides a well-characterized system for understanding how nonenveloped viruses enter and infect cells. Upon binding its receptor, poliovirus undergoes an irreversible conformational change to the 135S cell entry intermediate. This transition involves shifts of the capsid protein β barrels, accompanied by the externalization of VP4 and the N terminus of VP1. Both polypeptides associate with membranes and are postulated to facilitate entry by forming a translocation pore for the viral RNA. We have calculated cryo-electron microscopic reconstructions of 135S particles that permit accurate placement of the β barrels, loops, and terminal extensions of the capsid proteins. The reconstructions and resulting models indicate that each N terminus of VP1 exits the capsid though an opening in the interface between VP1 and VP3 at the base of the canyon that surrounds the fivefold axis. Comparison with reconstructions of 135S particles in which the first 31 residues of VP1 were proteolytically removed revealed that the externalized N terminus is located near the tips of propeller-like features surrounding the threefold axes rather than at the fivefold axes, as had been proposed in previous models. These observations have forced a reexamination of current models for the role of the 135S particle in transmembrane pore formation and suggest testable alternatives.

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Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

Journal of Virology ◽

10.1128/jvi.01198-17 ◽

2017 ◽

Vol 91 (20) ◽

Cited By ~ 23

Author(s):

Stefanie Grosse ◽

Magalie Penaud-Budloo ◽

Anne-Kathrin Herrmann ◽

Kathleen Börner ◽

Julia Fakhiri ◽

...

Keyword(s):

Protein Stability ◽

Capsid Protein ◽

Insect Cells ◽

Critical Factor ◽

Capsid Proteins ◽

Wild Type ◽

Particle Assembly ◽

Adeno Associated Virus ◽

Recombinant Vectors

ABSTRACT The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.

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Spectroscopic Studies of Infectious Pancreatic Necrosis Virus, Its Major Capsid Protein, and RNA

Ukrainian Journal of Physics ◽

10.15407/ujpe64.2.120 ◽

2019 ◽

Vol 64 (2) ◽

pp. 120

Author(s):

V. M. Kravchenko ◽

Yu. P. Rud ◽

L. P. Buchatski ◽

Ye. Yu. Stepanenko ◽

D. V. Gryn ◽

...

Keyword(s):

Capsid Protein ◽

Pancreatic Necrosis ◽

Major Capsid Protein ◽

Infectious Pancreatic Necrosis Virus ◽

Viral Rna ◽

Capsid Proteins ◽

Necrosis Virus ◽

Excitation Spectra ◽

Infectious Pancreatic Necrosis ◽

Pancreatic Necrosis Virus

Infectious pancreatic necrosis virus (IPNV) causes the severe disease of salmonid fishes (trout, salmon, etc.) The IPNV virion consists of a double-stranded viral RNA surrounded by a protein capsid. The aim of the work is to determine the role of IPNV virion constituents (capsid proteins and viral RNA) in the formation of spectral properties of the whole IPNV virions. We have measured the UV-Vis absorption, fluorescence, fluorescence excitation, phosphorescence and phosphorescence excitation spectra of IPNV virions, major capsid protein (MCP), and viral RNA dissolved in different buffers. It is shown that the UV absorption of IPNV virions is caused by the absorption of both capsid proteins and viral RNA. The fluorescence of IPNV MCP and virions may be attributed to tyrosine and tyrosine + tryptophan, respectively. The low-temperature phosphorescence of virions can be attributed to that of capsid proteins, rather than viral RNA. The IPNV RNA phosphorescence spectrum exhibits the electronic-vibrational structure and may be due to the emission of adenine links.

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Membrane remodeling by the lytic fragment of sticholysin II: implications for the toroidal pore model

10.1101/620336 ◽

2019 ◽

Author(s):

H. Mesa-Galloso ◽

P.A. Valiente ◽

R.F. Epand ◽

M.E. Lanio ◽

R.M. Epand ◽

...

Keyword(s):

Pore Formation ◽

Membrane Curvature ◽

Membrane Remodeling ◽

Structural Element ◽

Flip Flop ◽

Negatively Curved ◽

N Terminus ◽

Toroidal Pore ◽

Insight Into

AbstractSticholysins are pore-forming toxins of biomedical interest and represent a prototype of proteins acting through the formation of protein-lipid or toroidal pores. Peptides spanning the N-terminus of sticholysins can mimic their permeabilizing activity and together with the full-length toxins have been used as a tool to understand the mechanism of pore formation in membranes. However, the lytic mechanism of these peptides and the lipid shape modulating their activity are not completely clear. In this paper, we combine molecular dynamics (MD) simulations and experimental biophysical tools to dissect different aspects of the pore-forming mechanism of StII1-30, a peptide derived from the N-terminus of sticholysin II. With this combined approach, membrane curvature induction and flip-flop movement of the lipids were identified as two important membrane remodeling steps mediated by StII1-30-pore forming activity. Pore-formation by this peptide was enhanced by the presence of the negatively-curved lipid phosphatidylethanolamine (PE) in membranes. This lipid emerged not only as a facilitator of membrane interactions but also as a structural element of the StII1-30-pore that is recruited to the pore ring upon its assembly. Collectively, these new findings support a toroidal model for the architecture of the pore formed by this peptide and provide new molecular insight into the role of PE as a membrane component that easily accommodates into the ring of toroidal pores aiding in its stabilization. This study contributes to a better understanding of the molecular mechanism underlying the permeabilizing activity of StII1-30 and peptides or proteins acting via a toroidal pore mechanism and offers an informative framework for the optimization of the biomedical application of this and similar molecules.State of significanceWe provide evidence about the ability of StII1-30 to form toroidal pores. Due to pore assembly, StII1-30-pore induces membrane curvature and facilitates flip-flop movement of the lipids. The negatively-curved lipid PE relocates from the membrane into the pore ring, being also a structural element of the pore StII1-30 forms. This peptide emerged as a new tool, together with the full-length toxin, to understand the mechanism of toroidal pore formation in membranes. This study provides new molecular insight into the role of curved lipids as co-factors of toroidal pores, which could aid in its stabilization by easily accommodating into the ring. This framework could underpin strategies for the rational use of peptides or proteins acting via toroidal pores.

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Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase

Journal of Virology ◽

10.1128/jvi.02775-15 ◽

2016 ◽

Vol 90 (7) ◽

pp. 3611-3626 ◽

Cited By ~ 27

Author(s):

Nikolay Kovalev ◽

Isabel Fernández de Castro Martín ◽

Judit Pogany ◽

Daniel Barajas ◽

Kunj Pathak ◽

...

Keyword(s):

Rna Viruses ◽

Viral Rna ◽

Electron Microscopic ◽

Content Type ◽

Endosomal Sorting ◽

Infected Cells ◽

Membrane Bound ◽

Positive Strand Rna

ABSTRACTPlus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs).Tomato bushy stunt virus(TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule)-like structures in the peroxisomal membrane with tight openings toward the cytosol. In this study, using a yeast (Saccharomyces cerevisiae)vps23Δbro1Δ double-deletion mutant, we showed that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell extracts. Dual expression of dominant-negative versions ofArabidopsishomologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression inNicotiana benthamianaleaves. We also demonstrated the critical role of Snf7p (CHMP4), Vps20p, and Vps24p ESCRT-III proteins in tombusvirus replication in yeast andin vitro. Electron microscopic imaging ofvps23Δ yeast revealed the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also showed that the length of the viral RNA affects the sizes of spherules formed inN. benthamianacells. The 4.8-kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ∼600-nucleotide (nt)-long defective interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a “measuring string” during VRC assembly and spherule formation.IMPORTANCEPlant positive-strand RNA viruses, similarly to animal positive-strand RNA viruses, replicate in membrane-bound viral replicase complexes in the cytoplasm of infected cells. Identification of cellular and viral factors affecting the formation of the membrane-bound viral replication complex is a major frontier in current virology research. In this study, we dissected the functions of co-opted cellular ESCRT-I (endosomal sorting complexes required for transport I) and ESCRT-III proteins and the viral RNA in tombusvirus replicase complex formation usingin vitro, yeast-based, and plant-based approaches. Electron microscopic imaging revealed the lack of tombusvirus-induced spherule-like structures in ESCRT-I or ESCRT-III deletion yeasts replicating TBSV RNA, demonstrating the requirement for these co-opted cellular factors in tombusvirus replicase formation. The work could be of broad interest in virology and beyond.

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Mutation of Capsid Protein Phosphorylation Sites Abolishes Cauliflower Mosaic Virus Infectivity

Journal of Virology ◽

10.1128/jvi.76.22.11748-11752.2002 ◽

2002 ◽

Vol 76 (22) ◽

pp. 11748-11752 ◽

Cited By ~ 17

Author(s):

Yvan Chapdelaine ◽

David Kirk ◽

Aletta Karsies ◽

Thomas Hohn ◽

Denis Leclerc

Keyword(s):

Mosaic Virus ◽

Capsid Protein ◽

Plant Cells ◽

Cauliflower Mosaic Virus ◽

Virus Infectivity ◽

Viral Infectivity ◽

Phosphorylation Sites ◽

N Terminus ◽

Derivatives Of

ABSTRACT The cauliflower mosaic virus (CaMV) capsid protein is derived by bidirectional processing of the precapsid protein (CP56). We expressed several derivatives of CP56 in Escherichia coli and used them as substrates for virus-associated kinase and casein kinase II purified from plant cells. Three serine residues located at the N terminus of the mature viral protein CP44 were identified as phosphorylation targets. A mutation of one of them in the viral context had little or no effect on viral infectivity, but a mutation of all three serines abolished infectivity. The mapping of phosphorylation sites in CP44, but not CP39 or CP37, and immunodetection of the Zn finger motif in CP44 and CP39, but not CP37, support the model that CP39 is produced from CP44 by N-terminal processing and CP37 is produced from CP39 by C-terminal processing. We discuss the possible role of phosphorylation in the processing and assembly of CaMV capsid protein.

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Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate

10.1101/2020.01.08.899112 ◽

2020 ◽

Cited By ~ 1

Author(s):

Pranav N.M. Shah ◽

David J. Filman ◽

Krishanthi S. Karunatilaka ◽

Emma L. Hesketh ◽

Elisabetta Groppelli ◽

...

Keyword(s):

Conformational Changes ◽

Cell Entry ◽

Viral Rna ◽

Productive Infection ◽

Human Pathogens ◽

Temperature Dependent ◽

Endosomal Membrane ◽

N Terminus ◽

Clear Identification ◽

Membrane Interactive

ABSTRACTThe virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and opening of holes at the two and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle. However, using established protocols to produce the 135S particle, and single particle cryo-electron microscopy methods, we have identified at least two unique states that we call the early and late 135S particle. Surprisingly, only in the “late” 135S particles have detectable levels of the VP1 N-terminus trapped outside the capsid. Moreover, we observe a distinct density inside the capsid that can be accounted for by VP4 that remains associated with the genome. Taken together our results conclusively demonstrate that the 135S particle is not a unique conformation, but rather a family of conformations that could exist simultaneously.AUTHOR SUMMARYNonenveloped viruses need to provide mechanisms that allow their genomes to be delivered across membrane. This process remains poorly understood. For enterovirus such as poliovirus, genome delivery involves a program of conformational changes that include expansion of the particle and externalization of two normal internal peptides, VP4 and the VP1 N-terminus, which then insert into the cell membrane, triggering endocytosis and the creation of pores that facilitate the transfer of the viral RNA genome across the endosomal membrane. This manuscript describes five high-resolution cryo-EM structures of altered poliovirus particles that represent a number of intermediates along this pathway. The structures reveal several surprising findings, including the discovery of a new intermediate that is expanded but has not yet externalized the membrane interactive peptides, the clear identification of a unique exit site VP1 N-terminus, the demonstration that the externalized VP1 N-terminus partitions between two different sites in a temperature-dependent fashion, direct visualization of an amphipathic helix at the N-terminus of VP1 in an ideal position for interaction with cellular membranes, and the observation that a significant portion of VP4 remains inside the particle and accounts for a feature that had been previously ascribed to part of the viral RNA. These findings represent significant additions to our understanding of the cell entry process of an important class of human pathogens.

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Stannous fluoride treatment of acid-etched caries-like lesions of enamel: A scanning electron microscopic study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100113482 ◽

1984 ◽

Vol 42 ◽

pp. 720-721

Author(s):

M. John Hicks ◽

Leon M. Silverstone ◽

David G. Gantt ◽

Catherine M. Flaitz

Keyword(s):

Acid Etching ◽

Surface Zone ◽

Electron Microscopic ◽

Fluoride Treatment ◽

Stannous Fluoride ◽

Scanning Electron Microscopic Study ◽

Demineralized Enamel ◽

Intact Surface ◽

Topical Fluoride

Although fluoride levels become elevated in sound enamel following a topical fluoride treatment, the caries-preventive effect of fluoride is thought to be due primarily to the role of fluoride in remineralization of clinically undetectable enamel lesions and hypomineralized enamel. During lesion formation, redistribution of fluoride from the enamel surface to the subsurface demineralized enamel occurs. This results in a surface zone with a relatively low fluoride content. In order to maintain an intact surface zone over a carious lesion, it may be necessary to replenish the fluoride levels with an exogenous fluoride source. By acid-etching the lesion surface, a more reactive surface is made available for fluoride interaction. In addition, porosities and etching patterns may be created, allowing for bonding of a caries-resistant resin material to the lesion surface. The purpose of this study was to determine the integrity of the caries-like lesion surface following acid-etching and subsequent stannous fluoride treatment (SnF2).

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Scanning electron microscopic study of collagen fibrillogenesis and extracellular compartmentalization in the chick embryo tendon

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142669 ◽

1986 ◽

Vol 44 ◽

pp. 208-209

Author(s):

Grace C.H. Yang

Keyword(s):

Chick Embryo ◽

Extracellular Space ◽

Fibroblast Cell ◽

Collagen Fibrils ◽

Electron Microscopic ◽

Voltage Electron ◽

Scanning Electron Microscopic Study ◽

Microscopic Studies ◽

And Function

The size and organization of collagen fibrils in the extracellular matrix is an important determinant of tissue structure and function. The synthesis and deposition of collagen involves multiple steps which begin within the cell and continue in the extracellular space. High-voltage electron microscopic studies of the chick embryo cornea and tendon suggested that the extracellular space is compartmentalized by the fibroblasts for the regulation of collagen fibril, bundle, and tissue specific macroaggregate formation. The purpose of this study is to gather direct evidence regarding the association of the fibroblast cell surface with newly formed collagen fibrils, and to define the role of the fibroblast in the control and the precise positioning of collagen fibrils, bundles, and macroaggregates during chick tendon development.

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