scholarly journals Untargeted Lipidomics of Vesicular Stomatitis Virus-Infected Cells and Viral Particles

Viruses ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 3
Author(s):  
Katherine E. Havranek ◽  
Judith Mary Reyes Ballista ◽  
Kelly Marie Hines ◽  
Melinda Ann Brindley
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Viral Entry ◽  
Enrichment Analysis ◽  
Membrane Curvature ◽  
Membrane Properties ◽  
Sphingolipid Metabolism ◽  
Viral Particles ◽  
Lipid Species ◽  
Vesicular Stomatitis ◽  
Viral Lifecycle

The viral lifecycle is critically dependent upon host lipids. Enveloped viral entry requires fusion between viral and cellular membranes. Once an infection has occurred, viruses may rely on host lipids for replication and egress. Upon exit, enveloped viruses derive their lipid bilayer from host membranes during the budding process. Furthermore, host lipid metabolism and signaling are often hijacked to facilitate viral replication. We employed an untargeted HILIC-IM-MS lipidomics approach and identified host lipid species that were significantly altered during vesicular stomatitis virus (VSV) infection. Many glycerophospholipid and sphingolipid species were modified, and ontological enrichment analysis suggested that the alterations to the lipid profile change host membrane properties. Lysophosphatidylcholine (LPC), which can contribute to membrane curvature and serve as a signaling molecule, was depleted during infection, while several ceramide sphingolipids were augmented during infection. Ceramide and sphingomyelin lipids were also enriched in viral particles, indicating that sphingolipid metabolism is important during VSV infection.

scholarly journals Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

2015 ◽  
Vol 89 (23) ◽  
pp. 11750-11760 ◽  
Author(s):  
Timothy K. Soh ◽  
Sean P. J. Whelan
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Proteins ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Viral Particles ◽  
The Matrix ◽  
Vesicular Stomatitis ◽  
Late Domains

ABSTRACTVesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) with the large polymerase protein (L). To study the assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of viruses with insertions in the M gene a replication-competent virus containing a fluorescent M and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintained the same bullet shape appearance as wild-type VSV but had a modest increase in particle length, reflecting the increased genome size. Imaging of the released particles revealed a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the late domains in M, which serve to recruit the endosomal sorting complex required for transport (ESCRT) machinery during budding. Mutations in late domains resulted in the accumulation of virions that failed to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M tagged with enhanced green fluorescent protein and M tagged with mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late-domain mutant into the released virions. In contrast, the intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV.IMPORTANCEAssembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein, to the plasma membrane. The matrix protein contains a motif termed a “late domain” that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate the release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. In the study described here, we developed new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We used those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P.

scholarly journals Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1457
Author(s):  
Maria Fernanda Lay Mendoza ◽  
Marissa Danielle Acciani ◽  
Courtney Nina Levit ◽  
Christopher Santa Maria ◽  
Melinda Ann Brindley
Keyword(s):  
Real Time ◽  
Vesicular Stomatitis Virus ◽  
Viral Entry ◽  
Cell Surface Receptor ◽  
Enveloped Virus ◽  
Glycoprotein G ◽  
Viral Glycoproteins ◽  
Surface Receptor ◽  
Vesicular Stomatitis ◽  
Time Required

Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare the glycoprotein-mediated entry efficiencies of VSV glycoprotein (G), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S), Ebola (EBOV) glycoprotein (GP), Lassa (LASV) GP, and Chikungunya (CHIKV) envelope (E) protein, we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase (NLucP) reporter gene fused to a destabilization domain (PEST), which we used in combination with the live-cell substrate EndurazineTM to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.

scholarly journals Survey of virally mediated permeability changes

1980 ◽  
Vol 190 (3) ◽  
pp. 639-646 ◽  
Author(s):  
K A Foster ◽  
K Gill ◽  
K J Micklem ◽  
C A Pasternak
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Viral Entry ◽  
Sendai Virus ◽  
Ependymal Cells ◽  
Cell Type ◽  
Organ Cultures ◽  
Nasal Turbinate ◽  
Permeability Changes ◽  
Mdbk Cells ◽  
Vesicular Stomatitis

1. Sendai virus causes permeability changes when added to freshly isolated brain cells (cerebellum or ependymal cells) or to a culture of forebrain cells. 2. Sendai virus causes permeability changes when added to organ cultures of ferret lung or nasal turbinate. Influenza virus causes no permeability changes under these conditions. 3. Rabies virus and vesicular-stomatitis virus, in contrast with Sendai virus, do not cause permeability changes in BHK cells or Lettrée cells. 4. Serum from patients suffering from viral hepatitis does not cause permeability changes in human leucocytes; addition to Sendai virus causes permeability changes. 5. It is concluded that permeability changes accompanying viral entry occur only with certain types of paramyxovirus, but that there is little restriction on cell type. 6. MDBK cells infected with Sendai virus show permeability changes during viral release, similar to those that occur during viral entry. Because these changes do not appear to be restricted to paramyxoviruses, they may have considerable clinical significance.

scholarly journals A GP64-Null Baculovirus Pseudotyped with Vesicular Stomatitis Virus G Protein

2001 ◽  
Vol 75 (6) ◽  
pp. 2544-2556 ◽  
Author(s):  
J. T. Mangor ◽  
S. A. Monsma ◽  
M. C. Johnson ◽  
G. W. Blissard
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Viral Entry ◽  
Viral Envelope ◽  
Productive Infection ◽  
Pseudotyped Virus ◽  
Sf9 Cells ◽  
Viral Envelope Protein ◽  
Virion Protein ◽  
Vesicular Stomatitis

ABSTRACT The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. Genetic studies have shown that GP64-null viruses are unable to move from cell to cell and this results from a defect in the assembly and production of budded virions (BV). To further examine requirements for virion budding, we asked whether a GP64-null baculovirus, vAc64−, could be pseudotyped by introducing a heterologous viral envelope protein (vesicular stomatitis virus G protein [VSV-G]) into its membrane and whether the resulting virus was infectious. To address this question, we generated a stably transfected insect Sf9 cell line (Sf9VSV-G) that inducibly expresses the VSV-G protein upon infection with AcMNPV Sf9VSV-G and Sf9 cells were infected with vAc64−, and cells were monitored for infection and for movement of infection from cell to cell. vAc64− formed plaques on Sf9VSV-G cells but not on Sf9 cells, and plaques formed on Sf9VSV-G cells were observed only after prolonged intervals. Passage and amplification of vAc64− on Sf9VSV-G cells resulted in pseudotyped virus particles that contained the VSV-G protein. Cell-to-cell propagation of vAc64− in the G-expressing cells was delayed in comparison to wild-type (wt) AcMNPV, and growth curves showed that pseudotyped vAc64− was generated at titers of approximately 106 to 107 infectious units (IU)/ml, compared with titers of approximately 108 IU/ml for wt AcMNPV. Propagation and amplification of pseudotyped vAc64− virions in Sf9VSV-G cells suggests that the VSV-G protein may either possess the signals necessary for baculovirus BV assembly and budding at the cell surface or may otherwise facilitate production of infectious baculovirus virions. The functional complementation of GP64-null viruses by VSV-G protein was further demonstrated by identification of a vAc64−-derived virus that had acquired the G gene through recombination with Sf9VSV-G cellular DNA. GP64-null viruses expressing the VSV-G gene were capable of productive infection, replication, and propagation in Sf9 cells.

scholarly journals The Membrane-Proximal Stem Region of Vesicular Stomatitis Virus G Protein Confers Efficient Virus Assembly

2000 ◽  
Vol 74 (5) ◽  
pp. 2239-2246 ◽  
Author(s):  
Clinton S. Robison ◽  
Michael A. Whitt
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Virus Assembly ◽  
Membrane Curvature ◽  
Virus Release ◽  
Wild Type ◽  
Virus Budding ◽  
Recombinant Viruses ◽  
Vesicular Stomatitis ◽  
High Level

ABSTRACT In this report, we show that the glycoprotein of vesicular stomatitis virus (VSV G) contains within its extracellular membrane-proximal stem (GS) a domain that is required for efficient VSV budding. To determine a minimal sequence in GS that provides for high-level virus assembly, we have generated a series of recombinant ΔG-VSVs which express chimeric glycoproteins having truncated stem sequences. The recombinant viruses having chimeras with 12 or more membrane-proximal residues of the G stem, and including the G protein transmembrane-cytoplasmic tail domains, produced near-wild-type levels of particles. In contrast, viruses encoding chimeras with shorter or no G-stem sequences produced ∼10- to 20-fold less. This budding domain when present in chimeric glycoproteins also promoted their incorporation into the VSV envelope. We suggest that the G-stem budding domain promotes virus release by inducing membrane curvature at sites where virus budding occurs or by recruiting condensed nucleocapsids to sites on the plasma membrane which are competent for efficient virus budding.

scholarly journals Direct cell-to-cell transmission of vesicular stomatitis virus

1986 ◽  
Vol 85 (1) ◽  
pp. 125-131
Author(s):  
J.D. Vassalli ◽  
T. Lombardi ◽  
A. Wohlwend ◽  
R. Montesano ◽  
L. Orci
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Viral Envelope ◽  
Close Apposition ◽  
Coated Pits ◽  
Neighbouring Cell ◽  
Membrane Interaction ◽  
Viral Particles ◽  
Direct Cell ◽  
Vesicular Stomatitis ◽  
Extracellular Environment

Vesicular stomatitis virus (VSV) infection of kidney-derived, LLC-PK1 epithelial cells resulted in the budding of new viral particles into the basolateral space of the cultures. In lateral regions where cells were in close apposition, the majority of assembling viral particles in the process of budding from the producing cell had their apex already engaged in clathrin-coated pits of the neighbouring cell surface. These observations suggest that the viral envelope-plasma membrane interaction triggers the focal formation of clathrin-coated pits; they also show how VSV infection could spread throughout a tissue with only minimal exposure to a host's extracellular environment.

scholarly journals A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition

2020 ◽  
Author(s):  
M. Eugenia Dieterle ◽  
Denise Haslwanter ◽  
Robert H. Bortz ◽  
Ariel S. Wirchnianski ◽  
Gorka Lasso ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Viral Entry ◽  
Cell Entry ◽  
Immune Protection ◽  
Mechanistic Studies ◽  
Fluorescent Reporter ◽  
Large Panel ◽  
Vesicular Stomatitis ◽  
Highly Correlated ◽  
Humoral Responses

SummaryThere is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, and define correlates of immune protection, and to down-select candidate antivirals. Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein that closely resembles the authentic agent in its entry-related properties. We show that the neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S and that neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and therapeutics and for mechanistic studies of viral entry and its inhibition.

scholarly journals Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins

2020 ◽  
Author(s):  
Maria Fernanda Lay Mendoza ◽  
Marissa Danielle Acciani ◽  
Courtney Nina Levit ◽  
Christopher Santa Maria ◽  
Melinda Ann Brindley
Keyword(s):  
Real Time ◽  
Vesicular Stomatitis Virus ◽  
Viral Entry ◽  
Cell Surface Receptor ◽  
Enveloped Virus ◽  
Viral Glycoproteins ◽  
Surface Receptor ◽  
Entry Requirements ◽  
Vesicular Stomatitis ◽  
Time Required

Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare glycoprotein-mediated entry efficiencies of: VSV G, SARS-CoV-2 S, EBOV GP, LASV GP, and CHIKV E we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase-PEST (NLucP) reporter gene, which we used in combination with the live-cell substrate Endurazine™ to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.

Evaluation of Neutralizing Antibodies against Highly Pathogenic Coronaviruses: A Detailed Protocol for a Rapid Evaluation of Neutralizing Antibodies Using Vesicular Stomatitis Virus (Vsv) Pseudovirus-Based Assay

2020 ◽  
Author(s):  
Sarah A. Almahboub ◽  
Abdullah Algaissi ◽  
Mohamed A. Alfaleh ◽  
M-Zaki ElAssouli ◽  
Anwar M. Hashem
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Highly Pathogenic ◽  
S Protein ◽  
Level 3 ◽  
Vesicular Stomatitis ◽  
Biosafety Level ◽  
Detailed Protocol ◽  
S Proteins

Emerging highly pathogenic human coronaviruses (CoVs) represent a serious ongoing threat to the public health worldwide. The spike (S) proteins of CoVs are surface glycoproteins that facilitate viral entry into host cells via attachment to their respective cellular receptors. The S protein is believed to be a major immunogenic component of CoVs and a target for neutralizing antibodies (nAbs) and most candidate vaccines. Development of a safe and convenient assay is thus urgently needed to determine the prevalence of CoVs nAbs in the population, to study immune response in infected individuals, and to aid in vaccines and viral entry inhibitors evaluation. While live virus-based neutralization assays are used as gold standard serological methods to detect and measure nAbs, handling of highly pathogenic live CoVs requires strict bio-containment conditions in biosafety level-3 laboratories. On the other hand, use of replication-incompetent pseudoviruses bearing CoVs S proteins could represent a safe and useful method to detect nAbs in serum samples under biosafety level-2 conditions. Here, we describe a detailed protocol of a safe and convenient assay to generate vesicular stomatitis virus (VSV)-based pseudoviruses to evaluate and measure nAbs against highly pathogenic CoVs. The protocol covers methods to produce VSV pseudovirus bearing the S protein of the Middle East respiratory syndrome-CoV (MERS-CoV) and the severe acute respiratory syndrome-CoV-2 (SARS-CoV-2), pseudovirus titration, and pseudovirus neutralizing assay. Such assay could be adapted by different laboratories and researchers working on highly pathogenic CoVs without the need to handle live viruses in biosafety level-3 environment.

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