160. Truncated Vesicular Stomatitis Virus G- Protein Improves Baculovirus Transduction Efficiency In Vitro and In Vivo

ABSTRACT One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing Escherichia coli beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo.

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Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system.

The Journal of Cell Biology ◽

10.1083/jcb.104.3.749 ◽

1987 ◽

Vol 104 (3) ◽

pp. 749-760 ◽

Cited By ~ 47

Author(s):

W E Balch ◽

K R Wagner ◽

D S Keller

Keyword(s):

Endoplasmic Reticulum ◽

G Protein ◽

Vesicular Stomatitis Virus ◽

Golgi Complex ◽

Free System ◽

A Cell ◽

Vesicular Stomatitis ◽

High Mannose

Transport of the vesicular stomatitis virus-encoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (man GlcNAc2) ER form of G protein to the man5GlcNAc5 form by the cis Golgi enzyme alpha-mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the man5GlcNAc2 form without the appearance of the intermediate man GlcNAc2 oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi alpha-mannosidase I inhibitor 1-deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supernatant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro.

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Transient activity of Golgi-like membranes as donors of vesicular stomatitis viral glycoprotein in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.90.3.697 ◽

1981 ◽

Vol 90 (3) ◽

pp. 697-704 ◽

Cited By ~ 37

Author(s):

E Fries ◽

J E Rothman

Keyword(s):

G Protein ◽

Mutant Cell ◽

Chinese Hamster ◽

Sucrose Density Gradient ◽

Viral Glycoprotein ◽

Golgi Membranes ◽

Transient Activity ◽

Vesicular Stomatitis

Previous reports demonstrated that the vesicular stomatitis viral glycoprotein (G protein), initially present in membranes of a Chinese hamster ovary mutant cell line (clone 15B) that is incapable of terminal glycosylation, can be transferred in vitro to exogenous Golgi membranes and there glycosylated (E. Fries and J. E. Rothman, 1980, Proc. Natl. Acad. Sci. U. S. A. 77:3870-3874; and J. E. Rothman and E. Fries, 1981, J. Cell Biol. 89:162-168). Here we present evidence that Golgi-like membranes serve as donors of G protein in this process. Pulse-chase experiments revealed that the donor activity of membranes is greatest at approximately 10 min of chase, a time when G protein has been shown to have arrived in Golgi stacks (J. E. Bergmann, K. T. Tokuyasu, and S. J. Singer, 1981, Proc. Natl. Acad. Sci. U. S. A. 78:1746-1750). Additional evidence that the G protein that is transferred to exogenous Golgi membranes in vitro had already entered the Golgi membranes in vivo was provided by observations that its oligosaccharides had already been trimmed, and that its distribution in a sucrose density gradient was coincident with that of enzymatic markers of Golgi membranes. The capacity of this Golgi-like membrane to serve as donor is transient, declining within 5 min after "trimming" in vivo as the G protein enters a "nontransferable" pool. The rapidity of the process suggests that both the "transferable" and "nontransferable" pools of G protein reside in Golgi-like membranes.

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Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

The Journal of Cell Biology ◽

10.1083/jcb.74.1.43 ◽

1977 ◽

Vol 74 (1) ◽

pp. 43-57 ◽

Cited By ~ 7

Author(s):

MJ Grubman ◽

JA Weinstein ◽

DA Shafritz

Keyword(s):

Protein Synthesis ◽

Vesicular Stomatitis Virus ◽

Cytosol Fraction ◽

Initial Event ◽

Glycoprotein G ◽

Nonspecific Effects ◽

Membrane Bound ◽

Vesicular Stomatitis

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.

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Glycoprotein-Dependent Acidification of Vesicular Stomatitis Virus Enhances Release of Matrix Protein

Journal of Virology ◽

10.1128/jvi.00955-09 ◽

2009 ◽

Vol 83 (23) ◽

pp. 12139-12150 ◽

Cited By ~ 22

Author(s):

Chad E. Mire ◽

Derek Dube ◽

Sue E. Delos ◽

Judith M. White ◽

Michael A. Whitt

Keyword(s):

G Protein ◽

Vesicular Stomatitis Virus ◽

Conformational Changes ◽

Matrix Protein ◽

Low Ph ◽

M Protein ◽

Steady Increase ◽

Protein Dissociation ◽

Vesicular Stomatitis

ABSTRACT To study vesicular stomatitis virus (VSV) entry and uncoating, we generated a recombinant VSV encoding a matrix (M) protein containing a C-terminal tetracysteine Lumio tag (rVSV-ML) that could be fluorescently labeled using biarsenical compounds. Quantitative confocal microscopy showed that there is a transient loss of fluorescence at early times after the initiation of endocytosis of rVSV-ML-Green (rVSV-MLG) virions, which did not occur when cells were treated with bafilomycin A1. The reduction in fluorescence occurred 5 to 10 min postentry, followed by a steady increase in fluorescence intensity from 15 to 60 min postentry. A similar loss of fluorescence was observed in vitro when virions were exposed to acidic pH. The reduction in fluorescence required G protein since “bald” ΔG-MLG particles did not show a similar loss of fluorescence at low pH. Based on the pH-dependent fluorescence properties of Lumio Green, we hypothesize that the loss of fluorescence of rVSV-MLG virions during virus entry is due to a G ectodomain-dependent acidification of the virion interior. Biochemical analysis indicated that low pH also resulted in an enhancement of M protein dissociation from partially permeabilized, but otherwise intact, wild-type virions. From these data we propose that low-pH conformational changes in G protein promote acidification of the virus interior, which facilitates the release of M from ribonucleoprotein particles during uncoating.

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The natural compound Cirsitakaoside enhances antiviral innate responses against vesicular stomatitis virus in vitro and in vivo

International Immunopharmacology ◽

10.1016/j.intimp.2020.106783 ◽

2020 ◽

Vol 86 ◽

pp. 106783

Author(s):

Qianqian Di ◽

Huihui Zhu ◽

Debing Pu ◽

Xibao Zhao ◽

Xiaoli Li ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Natural Compound ◽

Vesicular Stomatitis

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RUBELLA VIRUS CARRIER CULTURES DERIVED FROM CONGENITALLY INFECTED INFANTS

Journal of Experimental Medicine ◽

10.1084/jem.123.5.795 ◽

1966 ◽

Vol 123 (5) ◽

pp. 795-816 ◽

Cited By ~ 66

Author(s):

William E. Rawls ◽

Joseph L. Melnick

Keyword(s):

Vesicular Stomatitis Virus ◽

Viral Persistence ◽

Carrier State ◽

Congenital Rubella ◽

Infected Cells ◽

Eclipse Phase ◽

Vesicular Stomatitis ◽

Simplex Virus

Spontaneous rubella carrier cultures derived from tissues of infants with congenital rubella were studied in an attempt to elucidate a possible mechanism for viral persistence observed in these infants. Chronically infected cells were found to have a reduced growth rate and the cultures appeared to have a shortened life span. The rubella carrier state was not dependent on serum inhibitors or rubella antibodies. Virtually every cell in the carrier population was found to be producing virus. The carrier cultures could not be cured by rubella antibodies. The rubella-infected cells were resistant to superinfection with vesicular stomatitis virus and herpes simplex virus but were susceptible to infection with echovirus 11. The replication of vesicular stomatitis virus was apparently blocked at an intracellular site, for the virus readily adsorbed to the chronically infected cells and entered into an eclipse phase; however no infectious virus developed. No evidence of interferon production by these cells could be obtained. It is postulated that clones of rubella-infected cells in vivo, with properties similar to those in carrier cultures developed in vitro from tissues of in utero infected infants, might explain the observed viral persistence noted in congenital rubella.

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Identification of a Novel Tripartite Complex Involved in Replication of Vesicular Stomatitis Virus Genome RNA

Journal of Virology ◽

10.1128/jvi.77.1.732-738.2003 ◽

2003 ◽

Vol 77 (1) ◽

pp. 732-738 ◽

Cited By ~ 39

Author(s):

Ashim K. Gupta ◽

Daniel Shaji ◽

Amiya K. Banerjee

Keyword(s):

Vesicular Stomatitis Virus ◽

Reverse Genetics ◽

Insect Cells ◽

Recombinant Baculovirus ◽

Wild Type ◽

Bhk Cells ◽

P Protein ◽

Vesicular Stomatitis

ABSTRACT Our laboratory's recent observations that transcriptionally inactive phosphoprotein (P) mutants can efficiently function in replicating vesicular stomatitis virus (VSV) defective interfering particle in a three-plasmid-based (L, P, and N) reverse genetics system in vivo (A. K. Pattnaik, L. Hwang, T. Li, N. Englund, M. Mathur, T. Das, and A. K. Banerjee, J. Virol. 71:8167-8175, 1997) led us to propose that a tripartite complex consisting of L-(N-P) protein may represent the putative replicase for synthesis of the full-length genome RNA. In this communication we demonstrate that such a complex is indeed detectable in VSV-infected BHK cells. Furthermore, coexpression of L, N, and P proteins in Sf21 insect cells by recombinant baculovirus containing the respective genes also resulted in the formation of a tripartite complex, as shown by immunoprecipitation with specific antibodies. A basic amino acid mutant of P protein, P260A, previously shown to be inactive in transcription but active in replication (T. Das, A. K. Pattnaik, A. M. Takacs, T. Li, L. N. Hwang, and A. K. Banerjee, Virology 238:103-114, 1997) was also capable of forming the mutant [L-(N-Pmut)] complex in both insect cells and BHK cells. Sf21 extract containing either the wild-type P protein or the mutant P protein along with the L and N proteins was capable of synthesizing 42S genome-sense RNA in an in vitro replication reconstitution reaction. Addition of N-Pmut or wild-type N-P complex further stimulated the synthesis of the genome-length RNA. These results indicate that the transcriptase and replicase complexes of VSV are possibly two distinct entities involved in carrying out capped mRNAs and uncapped genome and antigenome RNAs, respectively.

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Enhanced Gene Transfer with Fusogenic Liposomes Containing Vesicular Stomatitis Virus G Glycoprotein

Journal of Virology ◽

10.1128/jvi.72.7.6159-6163.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 6159-6163 ◽

Cited By ~ 34

Author(s):

Akihiro Abe ◽

Atsushi Miyanohara ◽

Theodore Friedmann

Keyword(s):

Gene Transfer ◽

Vesicular Stomatitis Virus ◽

Sucrose Density Gradient ◽

Virus Envelope ◽

Lipid Complex ◽

Targeted Gene Delivery ◽

Vesicular Stomatitis ◽

Sucrose Density Gradient Sedimentation

ABSTRACT Exposure of Lipofectin-DNA complexes to the partially purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G) results in loss of serum-mediated inhibition and in enhanced efficiency of gene transfer. Sucrose density gradient sedimentation analysis indicated that the VSV-G associates physically with the DNA-lipid complex to produce a VSV-G liposome. The ability to incorporate surrogate viral or cellular envelope components such as VSV-G into liposomes may allow more-efficient and possibly targeted gene delivery by lipofection, both in vitro and in vivo.

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