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 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 Many Nonmammalian Cells Exhibit Postentry Blocks to Transduction by Gammaretroviruses Pseudotyped with Various Viral Envelopes, Including Vesicular Stomatitis Virus G Glycoprotein

2001 ◽  
Vol 75 (14) ◽  
pp. 6375-6383 ◽  
Author(s):  
Clarissa Dirks ◽  
A. Dusty Miller
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Reverse Transcription ◽  
Leukemia Virus ◽  
Culture Conditions ◽  
Viral Envelope ◽  
Division Rate ◽  
Retroviral Transduction ◽  
Vesicular Stomatitis ◽  
Viral Envelopes ◽  
Vector Transduction

ABSTRACT Previous studies have suggested that Moloney murine leukemia virus (MoMLV)-based vectors pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) have extensive ability to transduce nonmammalian cells. However, we have identified multiple cell lines from fish (FHM), mosquitoes (Mos-55), moths (Sf9 and High-5), flies (S2), and frogs (XPK2) that are not efficiently transduced by MoMLV-based vectors pseudotyped with many different viral envelope proteins, including VSV-G, while the same vectors are functional in these cells following transfection. A comparison of MoMLV-based vector transduction in mammalian and nonmammalian cells shows that the nonmammalian cells exhibit blocks at either entry, reverse transcription, or integration. Additionally, VSV-G-pseudotyped MoMLV-based vector transduction is attenuated in the zebrafish cell line ZF4 at entry and/or reverse transcription, whereas other transduction processes are unaffected. We show that the variation of transduction by MoMLV-based vectors in mammalian and nonmammalian cells is not due to differences in culture conditions or cell division rate but is likely the result of divergence in cellular factors required for retroviral transduction.

scholarly journals Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells

2009 ◽  
Vol 83 (6) ◽  
pp. 2611-2622 ◽  
Author(s):  
Subash C. Das ◽  
Debasis Panda ◽  
Debasis Nayak ◽  
Asit K. Pattnaik
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Dynamic Imaging ◽  
Viral Envelope ◽  
M Protein ◽  
Recombinant Viruses ◽  
Infected Cells ◽  
Vesicular Stomatitis

ABSTRACT A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

scholarly journals Transfer of carbohydrates on to nascent glycoprotein of vesicular stomatitis virus

1979 ◽  
Vol 181 (2) ◽  
pp. 295-300 ◽  
Author(s):  
J Kruppa
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Viral Envelope ◽  
Pulse Labelling ◽  
Viral Envelope Protein ◽  
Viral Glycoprotein ◽  
Membrane Bound ◽  
Vesicular Stomatitis

I studied the glycosylation in vivo of a viral envelope protein, the glycoprotein of vesicular stomatitis virus (VSV), by pulse labelling of virus-infected HeLa cells with 3H-labelled monosaccharides (mannose, glucosamine). Radioactivity was incorporated into the fraction of membrane-bound polyribosomes, although metabolic conversion of [3H]-mannose into amino acids was negligible. Dissociation of bound polyribosomes revealed that the radioactively co-purified with the peptidyl-tRNA. The nascent peptides were released by alkaline hydrolysis, immunoprecipitated and analysed by polyacrylamide-gel electrophoresis. It is apparent from the size distribution of the [3H]mannose-labelled nascent chains that attachment of carbohydrate starts when approximately half of the amino acid sequence of the viral glycoprotein has been synthesized.

scholarly journals Selection of Novel Vesicular Stomatitis Virus Glycoprotein Variants from a Peptide Insertion Library for Enhanced Purification of Retroviral and Lentiviral Vectors

2006 ◽  
Vol 80 (7) ◽  
pp. 3285-3292 ◽  
Author(s):  
Julie H. Yu ◽  
David V. Schaffer
Keyword(s):  
Protein Engineering ◽  
Vesicular Stomatitis Virus ◽  
Mammalian Cells ◽  
Selection Process ◽  
High Titer ◽  
Lentiviral Vectors ◽  
Viral Envelope ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Virus Glycoprotein ◽  
Vesicular Stomatitis

ABSTRACT The introduction of new features or functions that are not present in an original protein is a significant challenge in protein engineering. For example, modifications to vesicular stomatitis virus glycoprotein (VSV-G), which is commonly used to pseudotype retroviral and lentiviral vectors for gene delivery, have been hindered by a lack of structural knowledge of the protein. We have developed a transposon-based approach that randomly incorporates designed polypeptides throughout a protein to generate saturated insertion libraries and a subsequent high-throughput selection process in mammalian cells that enables the identification of optimal insertion sites for a novel designed functionality. This method was applied to VSV-G in order to construct a comprehensive library of mutants whose combined members have a His6 tag inserted at likely every site in the original protein sequence. Selecting the library via iterative retroviral infections of mammalian cells led to the identification of several VSV-G-His6 variants that were able to package high-titer viral vectors and could be purified by Ni-nitrilotriacetic acid affinity chromatography. Column purification of vectors reduced protein and DNA impurities more than 5,000-fold and 14,000-fold, respectively, from the viral supernatant. This substantially improved purity elicited a weaker immune response in the brain, without altering the infectivity or tropism from wild-type VSV-G-pseudotyped vectors. This work applies a powerful new tool for protein engineering to construct novel viral envelope variants that can greatly improve the safety and use of retroviral and lentiviral vectors for clinical gene therapy. Furthermore, this approach of library generation and selection can readily be extended to other challenges in protein engineering.

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 alpha 2-macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis.

1981 ◽  
Vol 89 (1) ◽  
pp. 29-34 ◽  
Author(s):  
R B Dickson ◽  
M C Willingham ◽  
I Pastan
Keyword(s):  
Electron Microscope ◽  
Cell Surface ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Colloidal Gold ◽  
Microscope Level ◽  
Coated Pits ◽  
Electron Microscope Level ◽  
Receptor Mediated Endocytosis ◽  
Vesicular Stomatitis

alpha 2-Macroglobulin (alpha 2 M) was adsorbed to colloidal gold and used as a new tool in the study of receptor-mediated endocytosis. alpha 2 M-gold is easy to prepare and is clearly visualized at the electron microscope level. When cells were incubated with alpha 2 M-gold at 0 degrees C, gold was visualized both diffusely over the cell surface and concentrated in coated pits. After cells to which alpha 2 M-gold had been bound at 0 degrees C were warmed, the gold was rapidly internalized into uncoated vesicles, previously termed receptosomes. After 30 min of incubation or longer, gold was found in small lysosomes and, later, in large lysosomes and very small vesicles in the region of the Golgi complex. This pattern of localization is similar to that previously described, using peroxidase-labeled anti-alpha 2 M antibodies. By incubating cells with both alpha 2 M-gold and vesicular stomatitis virus (VSV), we studied the internalization of these two markers simultaneously. VSV and alpha 2 M-gold rapidly clustered in the same coated pits and were internalized in the same receptosomes. Proteins and hormones adsorbed to gold may be useful in the study of receptor-mediated endocytosis.

Association of the nucleocapsid protein N of vesicular stomatitis virus with phospholipids vesicles containing the matrix protein M

1984 ◽  
Vol 62 (11) ◽  
pp. 1174-1180 ◽  
Author(s):  
John Capone ◽  
Hara P. Ghosh
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Nucleocapsid Protein ◽  
Matrix Protein ◽  
Lipid Vesicles ◽  
Viral Envelope ◽  
M Protein ◽  
Phospholipid Vesicles ◽  
N Protein ◽  
The Matrix ◽  
Vesicular Stomatitis

The matrix protein M and the nucleocapsid protein N were isolated from vesicular stomatitis virus and reconstituted into artificial phospholipid vesicles. While the M protein could be reconstituted into phospholipid vesicles, the N protein had no affinity for lipid vesicles. The N protein could, however, associate with phospholipid vesicles in the presence of M protein. Identical results were also obtained when an in vitro system synthesizing M and N proteins was used for reconstitution. The results suggest that M protein is involved in virus maturation by interacting with the viral envelope and the N protein of the nucleoprotein core.

scholarly journals Mechanisms of Vesicular Stomatitis Virus Inactivation by Protoporphyrin IX, Zinc-Protoporphyrin IX, and Mesoporphyrin IX

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Christine Cruz-Oliveira ◽  
Andreza F. Almeida ◽  
João M. Freire ◽  
Marjolly B. Caruso ◽  
Maria A. Morando ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Antiviral Drug ◽  
Protoporphyrin Ix ◽  
Lipid Vesicles ◽  
Viral Envelope ◽  
Zinc Protoporphyrin ◽  
Virus Inactivation ◽  
Potential Therapeutic Application ◽  
Antiviral Activities ◽  
Vesicular Stomatitis

ABSTRACT Virus resistance to antiviral therapies is an increasing concern that makes the development of broad-spectrum antiviral drugs urgent. Targeting of the viral envelope, a component shared by a large number of viruses, emerges as a promising strategy to overcome this problem. Natural and synthetic porphyrins are good candidates for antiviral development due to their relative hydrophobicity and pro-oxidant character. In the present work, we characterized the antiviral activities of protoprophyrin IX (PPIX), Zn-protoporphyrin IX (ZnPPIX), and mesoporphyrin IX (MPIX) against vesicular stomatitis virus (VSV) and evaluated the mechanisms involved in this activity. Treatment of VSV with PPIX, ZnPPIX, and MPIX promoted dose-dependent virus inactivation, which was potentiated by porphyrin photoactivation. All three porphyrins inserted into lipid vesicles and disturbed the viral membrane organization. In addition, the porphyrins also affected viral proteins, inducing VSV glycoprotein cross-linking, which was enhanced by porphyrin photoactivation. Virus incubation with sodium azide and α-tocopherol partially protected VSV from inactivation by porphyrins, suggesting that singlet oxygen (1O2) was the main reactive oxygen species produced by photoactivation of these molecules. Furthermore, 1O2 was detected by 9,10-dimethylanthracene oxidation in photoactivated porphyrin samples, reinforcing this hypothesis. These results reveal the potential therapeutic application of PPIX, ZnPPIX, and MPIX as good models for broad antiviral drug design.

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