scholarly journals In situ Alphavirus Assembly and Budding Mechanism Revealed by Cellular CryoET

2021 ◽  
Author(s):  
David Chmielewski ◽  
Michael Schmid ◽  
Graham Simmons ◽  
Jing Jin ◽  
Wah Chiu
Keyword(s):  
Plasma Membrane ◽  
Electron Tomography ◽  
Virus Budding ◽  
Enveloped Virus ◽  
Infected Cells ◽  
Membrane Bending ◽  
Average Structures

Abstract Chikungunya virus (CHIKV) is a representative alphavirus causing debilitating arthritogenic disease in humans. Alphavirus particles assemble into two icosahedral protein layers: the glycoprotein spike shell embedded in a lipid envelope and the inner nucleocapsid (NC) core. In contrast to matrix-driven assembly of some enveloped viruses, the assembly/budding process of two-layered icosahedral particles remains poorly understood. Here we used cryogenic electron tomography (cryoET) to capture snapshots of the CHIKV assembly process in infected human cells. Subvolume classification of the snapshots revealed 12 intermediate structures, representing different stages of assembly/budding at the plasma membrane. Further subtomogram average structures ranging from subnanometer to nanometer resolutions show that immature, non-icosahedral NCs function as rough scaffolds to trigger icosahedral assembly of the glycoprotein spike lattice, which in turn progressively transforms the underlying NCs into icosahedral cores during budding. Here we resolve a long-standing mechanistic question about the role of spikes and NCs in assembly of two-layered icosahedral shells. Further, data of CHIKV-infected cells treated with budding-inhibiting antibodies shows that spacing spikes apart to prevent their lateral interactions prevents the plasma membrane bending around NC cores, thus blocking virus budding. These findings provide the molecular details of icosahedral enveloped virus formation and antibodies against assembly/budding.

scholarly journals In situ Alphavirus Assembly and Budding Mechanism Revealed by Cellular CryoET

2021 ◽  
Author(s):  
David Chmielewski ◽  
Michael F. Schmid ◽  
Graham Simmons ◽  
Jing Jin ◽  
Wah Chiu
Keyword(s):  
Neutralizing Antibodies ◽  
Molecular Mechanisms ◽  
Electron Tomography ◽  
Lateral Interactions ◽  
Virus Budding ◽  
Structural Mechanism ◽  
Infected Cells ◽  
Assembly Intermediate ◽  
Envelope Layer

Chikungunya virus (CHIKV) is an alphavirus and the etiological agent for debilitating arthritogenic disease in humans. Previous studies with purified virions or budding mutants have not resolved the structural mechanism of alphavirus assembly in situ. Here we used cryogenic electron tomography (cryoET) imaging of CHIKV-infected human cells and subvolume classification to resolve distinct assembly intermediate conformations. These structures revealed that particle formation is driven by the spike envelope layer. Additionally, we showed that asymmetric immature nucleocapsids (NCs) provide scaffolds to trigger assembly of the icosahedral spike lattice, which progressively transforms immature NCs into icosahedral cores during virus budding. Further, cryoET of the infected cells treated with neutralizing antibodies (NAbs) showed that NAb-induced blockage of CHIKV assembly was achieved by preventing spike-spike lateral interactions that are required to bend the plasma membrane around NC cores. These findings provide molecular mechanisms for designing antivirals targeting spike-driven assembly/budding of viruses.

scholarly journals Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus

2016 ◽  
Vol 90 (7) ◽  
pp. 3480-3495 ◽  
Author(s):  
Siddhartha Biswas ◽  
Gary W. Blissard ◽  
David A. Theilmann
Keyword(s):  
Plasma Membrane ◽  
Trichoplusia Ni ◽  
Autographa Californica ◽  
Nucleocapsid Proteins ◽  
Content Type ◽  
Multiple Nucleopolyhedrovirus ◽  
Microtubule Transport ◽  
Infected Cells ◽  
Budded Virus

ABSTRACTThe mechanism by which nucleocapsids ofAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane, leading to the formation of budded virus (BV), is not known. AC141 is a nucleocapsid-associated protein required for BV egress and has previously been shown to be associated with β-tubulin. In addition, AC141 and VP39 were previously shown by fluorescence resonance energy transfer by fluorescence lifetime imaging to interact directly with theDrosophila melanogasterkinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study, we investigated the role of lepidopteran microtubule transport using coimmunoprecipitation, colocalization, yeast two-hybrid, and small interfering RNA (siRNA) analyses. We show that nucleocapsid AC141 associates with the lepidopteranTrichoplusia niKLC and kinesin-1 heavy chain (KHC) by coimmunoprecipitation and colocalization. Kinesin-1, AC141, and microtubules colocalized predominantly at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also coimmunoprecipitated withT. niKLC. Direct analysis of the role ofT. nikinesin-1 by downregulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39 fused with three copies of the mCherry fluorescent protein also colocalized with microtubules. Yeast two-hybrid analysis showed no evidence of a direct interaction between kinesin-1 and AC141 or VP39, suggesting that either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation.IMPORTANCEIn two key processes of the replication cycle of the baculovirusAutographa californicamultiple nucleopolyhedrovirus (AcMNPV), nucleocapsids are transported through the cell. These include (i) entry of budded virus (BV) into the host cell and (ii) egress and budding of nucleocapsids newly produced from the plasma membrane. Prior studies have shown that the entry of nucleocapsids involves the polymerization of actin to propel nucleocapsids to nuclear pores and entry into the nucleus. For the spread of infection, progeny viruses must rapidly exit the infected cells, but the mechanism by which AcMNPV nucleocapsids traverse the cytoplasm is unknown. In this study, we examined whether nucleocapsids interact with lepidopteran kinesin-1 motor molecules and are potentially carried as cargo on microtubules to the plasma membrane in AcMNPV-infected cells. This study indicates that microtubule transport is utilized for the production of budded virus.

scholarly journals Visualizing insulin vesicle neighborhoods in β cells by cryo–electron tomography

2020 ◽  
Vol 6 (50) ◽  
pp. eabc8258
Author(s):  
Xianjun Zhang ◽  
Stephen D. Carter ◽  
Jitin Singla ◽  
Kate L. White ◽  
Peter C. Butler ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Electron Tomography ◽  
Secretory Cells ◽  
Nanometer Scale ◽  
Β Cells ◽  
Cellular Volume ◽  
Cryo Electron Tomography ◽  
Average Size

Subcellular neighborhoods, comprising specific ratios of organelles and proteins, serve a multitude of biological functions and are of particular importance in secretory cells. However, the role of subcellular neighborhoods in insulin vesicle maturation is poorly understood. Here, we present single-cell multiple distinct tomogram acquisitions of β cells for in situ visualization of distinct subcellular neighborhoods that are involved in the insulin vesicle secretory pathway. We propose that these neighborhoods play an essential role in the specific function of cellular material. In the regions where we observed insulin vesicles, a measurable increase in both the fraction of cellular volume occupied by vesicles and the average size (diameter) of the vesicles was apparent as sampling moved from the area near the nucleus toward the plasma membrane. These findings describe the important role of the nanometer-scale organization of subcellular neighborhoods on insulin vesicle maturation.

scholarly journals Nanomachinery Organizing Release at Neuronal and Ribbon Synapses

2019 ◽  
Vol 20 (9) ◽  
pp. 2147 ◽  
Author(s):  
Chakrabarti ◽  
Wichmann
Keyword(s):  
Electron Tomography ◽  
Molecular Architecture ◽  
Inner Hair Cell ◽  
Presynaptic Nerve Terminal ◽  
Ultrastructural Studies ◽  
Ribbon Synapses ◽  
Neuronal Synapses ◽  
Electrophysiological Recordings

A critical aim in neuroscience is to obtain a comprehensive view of how regulated neurotransmission is achieved. Our current understanding of synapses relies mainly on data from electrophysiological recordings, imaging, and molecular biology. Based on these methodologies, proteins involved in a synaptic vesicle (SV) formation, mobility, and fusion at the active zone (AZ) membrane have been identified. In the last decade, electron tomography (ET) combined with a rapid freezing immobilization of neuronal samples opened a window for understanding the structural machinery with the highest spatial resolution in situ. ET provides significant insights into the molecular architecture of the AZ and the organelles within the presynaptic nerve terminal. The specialized sensory ribbon synapses exhibit a distinct architecture from neuronal synapses due to the presence of the electron-dense synaptic ribbon. However, both synapse types share the filamentous structures, also commonly termed as tethers that are proposed to contribute to different steps of SV recruitment and exocytosis. In this review, we discuss the emerging views on the role of filamentous structures in SV exocytosis gained from ultrastructural studies of excitatory, mainly central neuronal compared to ribbon-type synapses with a focus on inner hair cell (IHC) ribbon synapses. Moreover, we will speculate on the molecular entities that may be involved in filament formation and hence play a crucial role in the SV cycle.

scholarly journals Acid Sphingomyelinase Deficiency Increases Susceptibility to Fatal Alphavirus Encephalomyelitis

2006 ◽  
Vol 80 (22) ◽  
pp. 10989-10999 ◽  
Author(s):  
Ching G. Ng ◽  
Diane E. Griffin
Keyword(s):  
Sindbis Virus ◽  
Acid Sphingomyelinase ◽  
Wild Type ◽  
Fatal Disease ◽  
Necrosis Factor Alpha ◽  
Enveloped Virus ◽  
Factor Alpha ◽  
Necrosis Factor

ABSTRACT Sindbis virus (SV), an enveloped virus with a single-stranded, plus-sense RNA genome, is the prototype alphavirus in the Togaviridae family. In mice, SV infects neurons and can cause apoptosis of immature neurons. Sphingomyelin (SM) is the most prevalent cellular sphingolipid, is particularly abundant in the nervous systems of mammals, and is required for alphavirus fusion and entry. The level of SM is tightly regulated by sphingomyelinases. A defect in acid sphingomyelinase (ASMase) results in SM storage and subsequent intracellular accumulation of SM. To better understand the role of the SM pathway in SV pathogenesis, we have characterized SV infection of transgenic mice deficient in the ASMase gene. ASMase knockout (ASM-KO) mice were more susceptible to SV infection than wild-type (WT) or heterozygous (Het) animals. Titers of SV were higher in the brains of ASM-KO mice than in the brains of WT mice. More SV RNA was detected by in situ hybridization, more SV protein was detected by immunohistochemistry, and more terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive cells were present in the cortex and hippocampus of ASM-KO mice than in those of WT or Het mice. Interleukin-6 (IL-6), but not IL-1β or tumor necrosis factor alpha, was elevated in infected ASM-KO mice compared to levels in WT or Het mice, but studies with IL-6-KO mice and recombinant SV expressing IL-6 showed no role for IL-6 in fatal disease. Together these data indicate that the increase in susceptibility of ASM-KO mice to SV infection was the result of more-rapid replication and spread of SV in the nervous system and increased neuronal death.

scholarly journals Endocytic vacuole formation by WASH-mediated endocytosis

2021 ◽  
Author(s):  
Pia Brinkert ◽  
Lena Krebs ◽  
Pilar Samperio Ventayol ◽  
Lilo Greune ◽  
Carina Bannach ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Polymerization ◽  
Endocytic Pathway ◽  
Vacuole Formation ◽  
Cellular Processes ◽  
Human Papillomavirus Type 16 ◽  
Systematic Perturbation ◽  
Membrane Bending ◽  
Endocytic Pathways

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).

scholarly journals SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Steffen Klein ◽  
Mirko Cortese ◽  
Sophie L. Winter ◽  
Moritz Wachsmuth-Melm ◽  
Christopher J. Neufeldt ◽  
...  
Keyword(s):  
Viral Replication ◽  
Electron Tomography ◽  
Membrane Vesicles ◽  
Membrane Curvature ◽  
Virion Assembly ◽  
Ribonucleoprotein Complexes ◽  
Cryo Electron Tomography ◽  
Membrane Bending ◽  
Subtomogram Averaging

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic β-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.

scholarly journals Mechanism of Tetherin Inhibition of Alphavirus Release

2019 ◽  
Vol 93 (7) ◽  
Author(s):  
Judy J. Wan ◽  
Yaw Shin Ooi ◽  
Margaret Kielian
Keyword(s):  
Plasma Membrane ◽  
Virus Particles ◽  
Enveloped Virus ◽  
293 Cells ◽  
Human Tetherin ◽  
Viral Particles ◽  
Antiviral Activities ◽  
Infected Cells ◽  
Cell Type Specific ◽  
Isoform Specificity

ABSTRACTTetherin is an interferon-inducible, antiviral host factor that broadly restricts enveloped virus release by tethering budded viral particles to the plasma membrane. In response, many viruses have evolved tetherin antagonists. The human tetherin gene can express two isoforms, long and short, due to alternative translation initiation sites in the N-terminal cytoplasmic tail. The long isoform (L-tetherin) contains 12 extra amino acids in its N terminus, including a dual tyrosine motif (YDYCRV) that is an internalization signal for clathrin-mediated endocytosis and a determinant of NF-κB activation. Tetherin restricts alphaviruses, which are highly organized enveloped RNA viruses that bud from the plasma membrane. L-tetherin is more efficient than S-tetherin in inhibiting alphavirus release in 293 cells. Here, we demonstrated that alphaviruses do not encode an antagonist for either of the tetherin isoforms. Instead, the isoform specificity reflected a requirement for tetherin endocytosis. The YXY motif in L-tetherin was necessary for alphavirus restriction in 293 cells but was not required for rhabdovirus restriction. L-tetherin’s inhibition of alphavirus release correlated with its internalization but did not involve NF-κB activation. In contrast, in U-2 OS cells, the YXY motif and the L-tetherin N-terminal domain were not required for either robust tetherin internalization or alphavirus inhibition. Tetherin forms that were negative for restriction accumulated at the surface of infected cells, while the levels of tetherin forms that restrict were decreased. Together, our results suggest that tetherin-mediated virus internalization plays an important role in the restriction of alphavirus release and that cell-type-specific cofactors may promote tetherin endocytosis.IMPORTANCEThe mechanisms of tetherin’s antiviral activities and viral tetherin antagonism have been studied in detail for a number of different viruses. Although viral countermeasures against tetherin can differ significantly, overall, tetherin’s antiviral activity correlates with physical tethering of virus particles to prevent their release. While tetherin can mediate virus endocytic uptake and clearance, this has not been observed to be required for restriction. Here we show that efficient tetherin inhibition of alphavirus release requires efficient tetherin endocytosis. Our data suggest that this endocytic uptake can be mediated by tetherin itself or by a tetherin cofactor that promotes uptake of an endocytosis-deficient variant of tetherin.

scholarly journals Multiple Routes of Bluetongue Virus Egress

2020 ◽  
Vol 8 (7) ◽  
pp. 965
Author(s):  
Thomas Labadie ◽  
Edward Sullivan ◽  
Polly Roy
Keyword(s):  
Cell Sorting ◽  
Bluetongue Virus ◽  
Economic Loss ◽  
Structural Protein ◽  
Enveloped Virus ◽  
Main Driver ◽  
Multiple Routes ◽  
Infected Cells ◽  
Virus Egress

Bluetongue virus (BTV) is an arthropod-borne virus infecting livestock. Its frequent emergence in Europe and North America had caused significant agricultural and economic loss. BTV is also of scientific interest as a model to understand the mechanisms underlying non-enveloped virus release from mammalian and insect cells. The BTV particle, which is formed of a complex double-layered capsid, was first considered as a lytic virus that needs to lyse the infected cells for cell to cell transmission. In the last decade, however, a more in-depth focus on the role of the non-structural proteins has led to several examples where BTV particles are also released through different budding mechanisms at the plasma membrane. It is now clear that the non-structural protein NS3 is the main driver of BTV release, via different interactions with both viral and cellular proteins of the cell sorting and exocytosis pathway. In this review, we discuss the most recent advances in the molecular biology of BTV egress and compare the mechanisms that lead to lytic or non-lytic BTV release.

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