scholarly journals Display of Heterologous Proteins on gp64null Baculovirus Virions and Enhanced Budding Mediated by a Vesicular Stomatitis Virus G-Stem Construct

2007 ◽  
Vol 82 (3) ◽  
pp. 1368-1377 ◽  
Author(s):  
Jian Zhou ◽  
Gary W. Blissard
Keyword(s):  
Amino Acids ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Mammalian Cells ◽  
Heterologous Proteins ◽  
C Terminus ◽  
Influenza Virus Hemagglutinin ◽  
Severe Reduction ◽  
Vesicular Stomatitis ◽  
Ha Protein

ABSTRACT The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 envelope glycoprotein is essential for virus entry and plays an important role in virion budding. An AcMNPV construct that contains a deletion of the gp64 gene is unable to propagate infection from cell to cell, and this defect results from both a severe reduction in the production of budded virions and the absence of GP64 on virions. In the current study, we examined GP64 proteins containing N- and C-terminal truncations of the ectodomain and identified a minimal construct capable of targeting the truncated GP64 to budded virions. The minimal budding and targeting construct of GP64 contained 38 amino acids from the mature N terminus of the GP64 ectodomain and 52 amino acids from the C terminus of GP64. Because the vesicular stomatitis virus (VSV) G protein was previously found to rescue infectivity of a gp64null AcMNPV, we also examined a small C-terminal construct of the VSV G protein. We found that a construct containing 91 amino acids from the C terminus of VSV G (termed G-stem) was capable of rescuing AcMNPV gp64null virion budding to wild-type (wt) or nearly wt levels. We also examined the display of chimeric proteins on the gp64null AcMNPV virion. By generating viruses that expressed chimeric influenza virus hemagglutinin (HA) proteins containing the GP64 targeting domain and coinfecting those viruses with a virus expressing the G-stem construct, we demonstrated enhanced display of the HA protein on gp64null AcMNPV budded virions. The combined use of gp64null virions, VSV G-stem-enhanced budding, and GP64 domains for targeting heterologous proteins to virions should be valuable for biotechnological applications ranging from targeted transduction of mammalian cells to vaccine production.

Different properties of two isoforms of annexin XIII in MDCK cells

2000 ◽  
Vol 113 (14) ◽  
pp. 2607-2618 ◽  
Author(s):  
S. Lecat ◽  
P. Verkade ◽  
C. Thiele ◽  
K. Fiedler ◽  
K. Simons ◽  
...  
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Functional Data ◽  
Mdck Cells ◽  
Amino Terminal ◽  
Influenza Virus Hemagglutinin ◽  
Vesicular Stomatitis ◽  
Amino Terminal Domain

Annexins form a family of proteins that are widely expressed and known to bind membranes in the presence of calcium. Two isoforms of the annexin XIII subfamily are expressed in epithelia. We previously reported that annexin XIIIb is apically localized in MDCK cells and that it is involved in raft-mediated delivery of apical proteins. We have now analyzed the properties of annexin XIIIa, which differs from annexin XIIIb by a deletion of 41 amino acids in the amino-terminal domain, and is distributed both apically and basolaterally. Annexin XIIIa binding to membranes is independent of calcium but requires its myristoyl amino-terminal modification, as observed with annexin XIIIb. Our biochemical and functional data show that annexin XIIIa behaves differently in the apical and in the basolateral compartments. Whereas annexin XIIIa apically can associate with rafts independently of calcium, the basolateral pool requires calcium for this. Annexin XIIIa, like annexin XIIIb, stimulates apical transport of influenza virus hemagglutinin but, in contrast, only annexin XIIIa inhibits basolateral transport of vesicular stomatitis virus G protein. Our results suggest that annexin XIIIa and XIIIb have specific roles in epithelial cells, and because of their structural similarities, these isoforms offer interesting tools for unravelling the functions of annexins.

scholarly journals Newly synthesized G protein of vesicular stomatitis virus is not transported to the Golgi complex in mitotic cells.

1985 ◽  
Vol 101 (6) ◽  
pp. 2036-2046 ◽  
Author(s):  
C Featherstone ◽  
G Griffiths ◽  
G Warren
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Independent Method ◽  
Transport Pathway ◽  
Post Translational Modifications ◽  
Vesicular Stomatitis ◽  
G1 Cells ◽  
Mitotic Cells

Newly synthesized G protein of vesicular stomatitis virus is not transported to the surface of cultured mammalian cells during mitosis (Warren et al., 1983, J. Cell Biol. 97:1623-1628). To determine where intracellular transport is inhibited, we have examined the post-translational modifications of G protein, which are indicators of specific compartments on the transport pathway. G protein in mitotic cells had only endo H-sensitive oligosaccharides containing seven or eight mannose residues, but no terminal glucose, and was not fatty acylated. These modifications were indicative of processing only by enzymes of the endoplasmic reticulum (ER). Quantitative immunocytochemistry was used as an independent method to confirm that transport of G protein out of the ER was inhibited. The density of G protein in the ER cisternae was 2.5 times greater than in infected G1 cells treated similarly. Incubation of infected mitotic cells with cycloheximide, which inhibits protein synthesis without affecting transport, did not result in a decrease in the density of G protein in the ER cisternae, demonstrating that G protein cannot be chased out of the ER. These results suggest that intracellular transport stops at or before the first vesicle-mediated step on the pathway.

scholarly journals Attenuated Vesicular Stomatitis Viruses as Vaccine Vectors

1999 ◽  
Vol 73 (5) ◽  
pp. 3723-3732 ◽  
Author(s):  
Anjeanette Roberts ◽  
Linda Buonocore ◽  
Ryan Price ◽  
John Forman ◽  
John K. Rose
Keyword(s):  
Influenza Virus ◽  
Vesicular Stomatitis Virus ◽  
Neutralizing Antibodies ◽  
Intranasal Administration ◽  
Complete Protection ◽  
Lethal Challenge ◽  
Virus Challenge ◽  
Influenza Virus Hemagglutinin ◽  
Vesicular Stomatitis ◽  
Ha Protein

ABSTRACT We showed previously that a single intranasal vaccination of mice with a recombinant vesicular stomatitis virus (VSV) expressing an influenza virus hemagglutinin (HA) protein provided complete protection from lethal challenge with influenza virus (A. Roberts, E. Kretzschmar, A. S. Perkins, J. Forman, R. Price, L. Buonocore, Y. Kawaoka, and J. K. Rose, J. Virol. 72:4704–4711, 1998). Because some pathogenesis was associated with the vector itself, in the present study we generated new VSV vectors expressing HA which are completely attenuated for pathogenesis in the mouse model. The first vector has a truncation of the cytoplasmic domain of the VSV G protein and expresses influenza virus HA (CT1-HA). This nonpathogenic vector provides complete protection from lethal influenza virus challenge after intranasal administration. A second vector with VSV G deleted and expressing HA (ΔG-HA) is also protective and nonpathogenic and has the advantage of not inducing neutralizing antibodies to the vector itself.

scholarly journals Vaccination with a Recombinant Vesicular Stomatitis Virus Expressing an Influenza Virus Hemagglutinin Provides Complete Protection from Influenza Virus Challenge

1998 ◽  
Vol 72 (6) ◽  
pp. 4704-4711 ◽  
Author(s):  
Anjeanette Roberts ◽  
Evelyne Kretzschmar ◽  
Archibald S. Perkins ◽  
John Forman ◽  
Ryan Price ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Vesicular Stomatitis Virus ◽  
Influenza A ◽  
Lethal Dose ◽  
Neutralizing Antibody ◽  
Virus Challenge ◽  
Influenza Virus Hemagglutinin ◽  
Vesicular Stomatitis ◽  
High Level ◽  
Ha Protein

ABSTRACT Since the development of a system for generating vesicular stomatitis virus (VSV) from plasmid DNAs, our laboratory has reported the expression of several different glycoproteins from recombinant VSVs. In one of these studies, high-level expression of an influenza virus hemagglutinin (HA) from a recombinant VSV-HA and efficient incorporation of the HA protein into the virions was reported (E. Kretzschmar, L. Buonocore, M. J. Schnell, and J. K. Rose, J. Virol. 71:5982–5989, 1997). We report here that VSV-HA is an effective intranasal vaccine vector that raises high levels of neutralizing antibody to influenza virus and completely protects mice from bronchial pneumonia caused by challenge with a lethal dose of influenza A virus. Additionally, these recombinant VSVs are less pathogenic than wild-type VSV (serotype Indiana). This vector-associated pathogenicity was subsequently eliminated through introduction of specific attenuating deletions. These live attenuated recombinant VSVs have great potential as vaccine vectors.

scholarly journals Differential Targeting of Vesicular Stomatitis Virus G Protein and Influenza Virus Hemagglutinin Appears During Myogenesis of L6 Muscle Cells

1998 ◽  
Vol 140 (5) ◽  
pp. 1101-1111 ◽  
Author(s):  
P. Rahkila ◽  
V. Luukela ◽  
K. Väänänen ◽  
K. Metsikkö
Keyword(s):  
Influenza Virus ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Glucose Transporter ◽  
Myogenic Differentiation ◽  
Acquired Resistance ◽  
Brefeldin A ◽  
Myoblast Differentiation ◽  
Influenza Virus Hemagglutinin ◽  
Vesicular Stomatitis

Exocytic organelles undergo profound reorganization during myoblast differentiation and fusion. Here, we analyzed whether glycoprotein processing and targeting changed during this process by using vesicular stomatitis virus (VSV) G protein and influenza virus hemagglutinin (HA) as models. After the induction of differentiation, the maturation and transport of the VSV G protein changed dramatically. Thus, only half of the G protein was processed and traveled through the Golgi, whereas the other half remained unprocessed. Experiments with the VSV tsO45 mutant indicated that the unprocessed form folded and trimerized normally and then exited the ER. It did not, however, travel through the Golgi since brefeldin A recalled it back to the ER. Influenza virus HA glycoprotein, on the contrary, acquired resistance to endoglycosidase H and insolubility in Triton X-100, indicating passage through the Golgi. Biochemical and morphological assays indicated that the HA appeared at the myotube surface. A major fraction of the Golgi-processed VSV G protein, however, did not appear at the myotube surface, but was found in intracellular vesicles that partially colocalized with the regulatable glucose transporter. Taken together, the results suggest that, during early myogenic differentiation, the VSV G protein was rerouted into developing, muscle-specific membrane compartments. Influenza virus HA, on the contrary, was targeted to the myotube surface.

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