scholarly journals The M protein of vesicular stomatitis virus associates specifically with the basolateral membranes of polarized epithelial cells independently of the G protein.

1988 ◽  
Vol 107 (5) ◽  
pp. 1707-1715 ◽  
Author(s):  
J E Bergmann ◽  
P J Fusco
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
M Protein ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Polarized Epithelial Cells

Using monoclonal antibodies and indirect immunofluorescence microscopy, we investigated the distribution of the M protein in situ in vesicular stomatitis virus-(VSV) infected MDCK cells. M protein was observed free in the cytoplasm and associated with the plasma membrane. Using the ts045 mutant of VSV to uncouple the synthesis and transport of the VSV G protein we demonstrated that this distribution was not related to the presence of G protein on the cell surface. Sections of epon-embedded infected cells labeled with antibody to the M protein and processed for indirect horseradish peroxidase immunocytochemistry revealed that the M protein was associated specifically with the basolateral plasma membrane. The G and M proteins of VSV have therefore evolved features which bring them independently to the basolateral membrane of polarized epithelial cells and allow virus to bud specifically from that membrane.

scholarly journals Vesicular Stomatitis Virus Glycoprotein Does Not Determine the Site of Virus Release in Polarized Epithelial Cells

2002 ◽  
Vol 76 (8) ◽  
pp. 4103-4107 ◽  
Author(s):  
Gert Zimmer ◽  
Klaus-Peter Zimmer ◽  
Ina Trotz ◽  
Georg Herrler
Keyword(s):  
Epithelial Cells ◽  
Vesicular Stomatitis Virus ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Recombinant Viruses ◽  
Virus Glycoprotein ◽  
Vesicular Stomatitis ◽  
Polarized Epithelial Cells

ABSTRACT In polarized epithelial cells, the vesicular stomatitis virus glycoprotein is segregated to the basolateral plasma membrane, where budding of the virus takes place. We have generated recombinant viruses expressing mutant glycoproteins without the basolateral-membrane-targeting signal in the cytoplasmic domain. Though about 50% of the mutant glycoproteins were found at the apical plasma membranes of infected MDCK cells, the virus was still predominantly released at the basolateral membranes, indicating that factors other than the glycoprotein determine the site of virus budding.

scholarly journals Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells.

1996 ◽  
Vol 133 (3) ◽  
pp. 543-558 ◽  
Author(s):  
A Müsch ◽  
H Xu ◽  
D Shields ◽  
E Rodriguez-Boulan
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Mdck Cells ◽  
Current Model ◽  
Gh3 Cells ◽  
Plasma Membrane Proteins ◽  
Vesicular Release ◽  
Vesicular Stomatitis

Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.

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 Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.

1996 ◽  
Vol 135 (1) ◽  
pp. 153-167 ◽  
Author(s):  
J Peränen ◽  
P Auvinen ◽  
H Virta ◽  
R Wepf ◽  
K Simons
Keyword(s):  
Epithelial Cells ◽  
Membrane Transport ◽  
Vesicular Stomatitis Virus ◽  
Actin Filaments ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Wild Type ◽  
Expression Systems ◽  
Vesicular Stomatitis

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.

scholarly journals Transcytosis of the G protein of vesicular stomatitis virus after implantation into the apical plasma membrane of Madin-Darby canine kidney cells. I. Involvement of endosomes and lysosomes.

1984 ◽  
Vol 99 (3) ◽  
pp. 796-782 ◽  
Author(s):  
M Pesonen ◽  
W Ansorge ◽  
K Simons
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Vesicular Stomatitis ◽  
Darby Canine Kidney ◽  
Canine Kidney

The G protein of vesicular stomatitis virus, implanted into the apical plasma membrane of Madin-Darby canine kidney cells, is rapidly transcytosed to the basolateral membrane. In this and the accompanying paper (Pesonen, M., R. Bravo, and K. Simons, 1984, J. Cell Biol. 99:803-809.) we have studied the intracellular route by which the G protein traverses during transcytosis. Using Percoll density gradient centrifugation and free flow electrophoresis we could demonstrate that the G protein is endocytosed into a nonlysosomal compartment with a density of approximately 1.05 g/cm3, which has many of the characteristics of endosomes. Transcytosis to the basolateral membrane appeared to occur from this compartment. No direct evidence for the involvement of lysosomes in the transcytotic route could be obtained. No G protein was detected in the lysosomes when transcytosis of G protein was occurring. Moreover, at 21 degrees C when passage of G protein to the lysosomes was shown to be arrested, transcytosis of G protein could still be demonstrated.

Visualization of transcytosis in MDCK cells using laser scanning confocal microscopy

1994 ◽  
Vol 52 ◽  
pp. 220-221
Author(s):  
Greg Martin ◽  
Rohit Cariappa ◽  
Ann L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Laser Scanning ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Proteins ◽  
Polarized Epithelial Cells

The plasma membrane of polarized epithelial cells is composed of two structurally and functionally distinct domains -- the apical and basolateral -- that also differ in molecular composition. The routes followed by integral membrane proteins from their site of synthesis to their site of function varies between different kinds of epithelia. Madin-Darby canine kidney (MDCK) cells deliver plasma membrane proteins directly to the correct domain, while polarized hepatocytes deliver all newly synthesized plasma membrane proteins initially to the basolateral membrane, then retrieve and redirect the apical membrane proteins. We are studying the targeting signals and delivery routes of DPPIV, a single transmembrane protein whose destination is the apical domain in polarized epithelial cells.DPPIV transfected into MDCK cells is delivered to the basolateral plasma membrane after long (13hr) treatment with Brefeldin A (BFA). After BFA’s removal these molecules are retrieved from the basolateral membrane and transcytosed to the apical plasma membrane. This protocol provides a useful model for studies of the indirect route of protein sorting in polarized epithelial cells, since DPPIV at the basolateral surface can be labeled with specific antibody and then subsequently followed in living cells.

scholarly journals Plasma Membrane Microdomains Containing Vesicular Stomatitis Virus M Protein Are Separate from Microdomains Containing G Protein and Nucleocapsids

2008 ◽  
Vol 82 (11) ◽  
pp. 5536-5547 ◽  
Author(s):  
B. Dancho Swinteck ◽  
Douglas S. Lyles
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Virus Assembly ◽  
M Protein ◽  
Membrane Microdomains ◽  
Virus Budding ◽  
N Protein ◽  
Vesicular Stomatitis ◽  
Plasma Membrane Microdomains

ABSTRACT Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.

scholarly journals Pseudotypes of Vesicular Stomatitis Virus with CD4 Formed by Clustering of Membrane Microdomains during Budding

2005 ◽  
Vol 79 (11) ◽  
pp. 7077-7086 ◽  
Author(s):  
Erica L. Brown ◽  
Douglas S. Lyles
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Lipid Rafts ◽  
Immunoelectron Microscopy ◽  
Vesicular Stomatitis Virus ◽  
Plasma Membranes ◽  
Membrane Microdomains ◽  
Virus Budding ◽  
Vesicular Stomatitis ◽  
Membrane Components

ABSTRACT Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

scholarly journals Identification of Two Additional Translation Products from the Matrix (M) Gene That Contribute to Vesicular Stomatitis Virus Cytopathology

2002 ◽  
Vol 76 (16) ◽  
pp. 8011-8018 ◽  
Author(s):  
Himangi R. Jayakar ◽  
Michael A. Whitt
Keyword(s):  
Amino Acids ◽  
Vesicular Stomatitis Virus ◽  
Recombinant Virus ◽  
M Protein ◽  
M Gene ◽  
Cell Rounding ◽  
Matrix Gene ◽  
The Matrix ◽  
Infected Cells ◽  
Vesicular Stomatitis

ABSTRACT The matrix (M) protein of vesicular stomatitis virus (VSV) is a multifunctional protein that is responsible for condensation of the ribonucleocapsid core during virus assembly and also plays a critical role in virus budding. The M protein is also responsible for most of the cytopathic effects (CPE) observed in infected cells. VSV CPE include inhibition of host gene expression, disablement of nucleocytoplasmic transport, and disruption of the host cytoskeleton, which results in rounding of infected cells. In this report, we show that the VSV M gene codes for two additional polypeptides, which we have named M2 and M3. These proteins are synthesized from downstream methionines in the same open reading frame as the M protein (which we refer to here as M1) and lack the first 32 (M2) or 50 (M3) amino acids of M1. Infection of cells with a recombinant virus that does not express M2 and M3 (M33,51A) resulted in a delay in cell rounding, but virus yield was not affected. Transient expression of M2 and M3 alone caused cell rounding similar to that with the full-length M1 protein, suggesting that the cell-rounding function of the M protein does not require the N-terminal 50 amino acids. To determine if M2 and M3 were sufficient for VSV-mediated CPE, both M2 and M3 were expressed from a separate cistron in a VSV mutant background that readily establishes persistent infections and that normally lacks CPE. Infection of cells with the recombinant virus that expressed M2 and M3 resulted in cell rounding indistinguishable from that with the wild-type recombinant virus. These results suggest that M2 and M3 are important for cell rounding and may play an important role in viral cytopathogenesis. To our knowledge, this is first report of the multiple coding capacities of a rhabdovirus matrix gene.

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