scholarly journals Sequential coupling between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi transport.

1995 ◽  
Vol 131 (4) ◽  
pp. 875-893 ◽  
Author(s):  
M Aridor ◽  
S I Bannykh ◽  
T Rowe ◽  
W E Balch
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Mammalian Cells ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Vesicle Budding ◽  
Vesicular Traffic ◽  
Golgi Transport ◽  
Sequential Coupling ◽  
Vesicular Stomatitis ◽  
Copi Vesicle

COPI and COPII are vesicle coat complexes whose assembly is regulated by the ARF1 and Sar1 GTPases, respectively. We show that COPI and COPII coat complexes are recruited separately and independently to ER (COPII), pre-Golgi (COPI, COPII), and Golgi (COPI) membranes of mammalian cells. To address their individual roles in ER to Golgi transport, we used stage specific in vitro transport assays to synchronize movement of cargo to and from pre-Golgi intermediates, and GDP- and GTP-restricted forms of Sar1 and ARF1 proteins to control coat recruitment. We find that COPII is solely responsible for export from the ER, is lost rapidly following vesicle budding and mediates a vesicular step required for the build-up of pre-Golgi intermediates composed of clusters of vesicles and small tubular elements. COPI is recruited onto pre-Golgi intermediates where it initiates segregation of the anterograde transported protein vesicular stomatitis virus glycoprotein (VSV-G) from the retrograde transported protein p58, a protein which actively recycles between the ER and pre-Golgi intermediates. We propose that sequential coupling between COPII and COPI coats is essential to coordinate and direct bi-directional vesicular traffic between the ER and pre-Golgi intermediates involved in transport of protein to the Golgi complex.

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 Beta-COP is essential for transport of protein from the endoplasmic reticulum to the Golgi in vitro

1993 ◽  
Vol 122 (6) ◽  
pp. 1155-1167 ◽  
Author(s):  
F Peter ◽  
H Plutner ◽  
H Zhu ◽  
TE Kreis ◽  
WE Balch
Keyword(s):  
Active Form ◽  
Biologically Active ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Vitro Assay ◽  
Vesicle Budding ◽  
Vesicular Carriers ◽  
Golgi Compartment ◽  
Vesicular Stomatitis ◽  
Rat Liver Cytosol

Using a novel in vitro assay which allows us to distinguish vesicle budding from subsequent targeting and fusion steps, we provide the first biological evidence that beta-COP, a component of non-clathrin-coated vesicles believed to mediate intraGolgi transport, is essential for transport of protein from the ER to the cis-Golgi compartment. Incubation in the presence of beta-COP specific antibodies and F(ab) fragments prevents the exit of vesicular stomatitis virus glycoprotein (VSV-G) from the ER. These results demonstrate that beta-COP is required for the assembly of coat complexes mediating vesicle budding. Fractionation of rat liver cytosol revealed that a major biologically active form of beta-COP was found in a high molecular pool (> 1,000 kD) distinct from coatomer and which promoted efficient vesicle budding from the ER. Surprisingly, rab1B could be quantitatively coprecipitated with this beta-COP containing complex and was also essential for function. We suggest that beta-COP functions in an early step during vesicle formation and that rab1B may be recruited as a component of a precoat complex which participates in the export of protein from the ER via vesicular carriers.

scholarly journals Natural killer T cell immunotherapy combined with oncolytic vesicular stomatitis virus or reovirus treatments differentially increases survival in mouse models of ovarian and breast cancer metastasis

2021 ◽  
Vol 9 (3) ◽  
pp. e002096
Author(s):  
Simon Gebremeskel ◽  
Adam Nelson ◽  
Brynn Walker ◽  
Tora Oliphant ◽  
Lynnea Lobert ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Vesicular Stomatitis Virus ◽  
Cell Activation ◽  
Tumor Burden ◽  
Oncolytic Viruses ◽  
Nkt Cell ◽  
Cell Immunotherapy ◽  
Vesicular Stomatitis ◽  
Better Than

BackgroundOncolytic viruses reduce tumor burden in animal models and have generated promising results in clinical trials. However, it is likely that oncolytic viruses will be more effective when used in combination with other therapies. Current therapeutic approaches, including chemotherapeutics, come with dose-limiting toxicities. Another option is to combine oncolytic viruses with immunotherapeutic approaches.MethodsUsing experimental models of metastatic 4T1 breast cancer and ID8 ovarian peritoneal carcinomatosis, we examined natural killer T (NKT) cell-based immunotherapy in combination with recombinant oncolytic vesicular stomatitis virus (VSV) or reovirus. 4T1 mammary carcinoma cells or ID8 ovarian cancer cells were injected into syngeneic mice. Tumor-bearing mice were treated with VSV or reovirus followed by activation of NKT cells via the intravenous administration of autologous dendritic cells loaded with the glycolipid antigen α-galactosylceramide. The effects of VSV and reovirus on immunogenic cell death (ICD), cell viability and immunogenicity were tested in vitro.ResultsVSV or reovirus treatments followed by NKT cell activation mediated greater survival in the ID8 model than individual therapies. The regimen was less effective when the treatment order was reversed, delivering virus treatments after NKT cell activation. In the 4T1 model, VSV combined with NKT cell activation increased overall survival and decreased metastatic burden better than individual treatments. In contrast, reovirus was not effective on its own or in combination with NKT cell activation. In vitro, VSV killed a panel of tumor lines better than reovirus. VSV infection also elicited greater increases in mRNA transcripts for proinflammatory cytokines, chemokines, and antigen presentation machinery compared with reovirus. Oncolytic VSV also induced the key hallmarks of ICD (calreticulin mobilization, plus release of ATP and HMGB1), while reovirus only mobilized calreticulin.ConclusionTaken together, these results demonstrate that oncolytic VSV and NKT cell immunotherapy can be effectively combined to decrease tumor burden in models of metastatic breast and ovarian cancers. Oncolytic VSV and reovirus induced differential responses in our models which may relate to differences in virus activity or tumor susceptibility.

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