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Cellular proteins associated with the interior and exterior of vesicular stomatitis virus virions.

Moerdyk-Schauwecker M, Hwang SI, Grdzelishvili VZ - PLoS ONE (2014)

Bottom Line: Most of these proteins have not been previously shown to be associated with VSV.Functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization.Our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of VSV.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, North Carolina, United States of America; Center for Biomedical Engineering and Science, University of North Carolina at Charlotte, Charlotte, North Carolina, United States of America.

ABSTRACT
Virus particles (virions) often contain not only virus-encoded but also host-encoded proteins. Some of these host proteins are enclosed within the virion structure, while others, in the case of enveloped viruses, are embedded in the host-derived membrane. While many of these host protein incorporations are likely accidental, some may play a role in virus infectivity, replication and/or immunoreactivity in the next host. Host protein incorporations may be especially important in therapeutic applications where large numbers of virus particles are administered. Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus and a candidate vaccine, gene therapy and oncolytic vector. Using mass spectrometry, we previously examined cell type dependent host protein content of VSV virions using intact ("whole") virions purified from three cell lines originating from different species. Here we aimed to determine the localization of host proteins within the VSV virions by analyzing: i) whole VSV virions; and ii) whole VSV virions treated with Proteinase K to remove all proteins outside the viral envelope. A total of 257 proteins were identified, with 181 identified in whole virions and 183 identified in Proteinase K treated virions. Most of these proteins have not been previously shown to be associated with VSV. Functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization. Using western blotting, the presence of several host proteins, including some not previously shown in association with VSV (such as Yes1, Prl1 and Ddx3y), was confirmed and their relative quantities in various virion fractions determined. Our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of VSV.

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Related in: MedlinePlus

1D SDS-PAGE and protein identification by mass spectrometry.(a) Total protein from whole virions or proteinase K (ProK) treated virions was separated by 1D-SDS-PAGE and stained with Coomassie Brilliant Blue R250. Brackets on the right indicate bands cut out and analyzed by mass spectrometry (MS). The position of the molecular mass markers is indicated on the left. The bands containing the viral large polymerase protein (L), glycoprotein (G), nucleocapsid protein (N), phosphoprotein (P), and matrix protein (M) are indicated. Venn diagrams indicate the number of proteins identified by MS, and the degree of overlap in the proteins identified (b) between sample types and (c and d) between technical replicates of the same sample type.
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pone-0104688-g003: 1D SDS-PAGE and protein identification by mass spectrometry.(a) Total protein from whole virions or proteinase K (ProK) treated virions was separated by 1D-SDS-PAGE and stained with Coomassie Brilliant Blue R250. Brackets on the right indicate bands cut out and analyzed by mass spectrometry (MS). The position of the molecular mass markers is indicated on the left. The bands containing the viral large polymerase protein (L), glycoprotein (G), nucleocapsid protein (N), phosphoprotein (P), and matrix protein (M) are indicated. Venn diagrams indicate the number of proteins identified by MS, and the degree of overlap in the proteins identified (b) between sample types and (c and d) between technical replicates of the same sample type.

Mentions: For mass spectrometry (MS) analysis, total protein from 50 µg purified virions or 60 µg ProK treated virions was separated by 1D-SDS-PAGE. These quantities were chosen so that the amount of viral N and P protein was approximately equal in both sample types as determined by Coomassie staining (Fig. 3a) to facilitate comparisons between samples. Importantly, while N, P and L bands were similar in both virion preparations, no full length G protein was visible for ProK treated virions, indicating the ProK treatment was highly successful. After separation by SDS-PAGE, the resolved proteins were cut out in a series of bands as indicated in Figure 3a. These bands were then subjected to in-gel trypsin digest and the resulting peptides were extracted, separated by UPLC and analyzed by tandem MS (MS/MS). In this study, virions were grown on BHK-21 cells as it is the standard cell line for growth of VSV and the high virion yields aid in purification. However, a complete database of Syrian hamster proteins is not available. Instead, peptides were identified by matching them to either a human or a mouse database. The results from both searches were highly similar; therefore only those from the search of the mouse database are presented here.


Cellular proteins associated with the interior and exterior of vesicular stomatitis virus virions.

Moerdyk-Schauwecker M, Hwang SI, Grdzelishvili VZ - PLoS ONE (2014)

1D SDS-PAGE and protein identification by mass spectrometry.(a) Total protein from whole virions or proteinase K (ProK) treated virions was separated by 1D-SDS-PAGE and stained with Coomassie Brilliant Blue R250. Brackets on the right indicate bands cut out and analyzed by mass spectrometry (MS). The position of the molecular mass markers is indicated on the left. The bands containing the viral large polymerase protein (L), glycoprotein (G), nucleocapsid protein (N), phosphoprotein (P), and matrix protein (M) are indicated. Venn diagrams indicate the number of proteins identified by MS, and the degree of overlap in the proteins identified (b) between sample types and (c and d) between technical replicates of the same sample type.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126742&req=5

pone-0104688-g003: 1D SDS-PAGE and protein identification by mass spectrometry.(a) Total protein from whole virions or proteinase K (ProK) treated virions was separated by 1D-SDS-PAGE and stained with Coomassie Brilliant Blue R250. Brackets on the right indicate bands cut out and analyzed by mass spectrometry (MS). The position of the molecular mass markers is indicated on the left. The bands containing the viral large polymerase protein (L), glycoprotein (G), nucleocapsid protein (N), phosphoprotein (P), and matrix protein (M) are indicated. Venn diagrams indicate the number of proteins identified by MS, and the degree of overlap in the proteins identified (b) between sample types and (c and d) between technical replicates of the same sample type.
Mentions: For mass spectrometry (MS) analysis, total protein from 50 µg purified virions or 60 µg ProK treated virions was separated by 1D-SDS-PAGE. These quantities were chosen so that the amount of viral N and P protein was approximately equal in both sample types as determined by Coomassie staining (Fig. 3a) to facilitate comparisons between samples. Importantly, while N, P and L bands were similar in both virion preparations, no full length G protein was visible for ProK treated virions, indicating the ProK treatment was highly successful. After separation by SDS-PAGE, the resolved proteins were cut out in a series of bands as indicated in Figure 3a. These bands were then subjected to in-gel trypsin digest and the resulting peptides were extracted, separated by UPLC and analyzed by tandem MS (MS/MS). In this study, virions were grown on BHK-21 cells as it is the standard cell line for growth of VSV and the high virion yields aid in purification. However, a complete database of Syrian hamster proteins is not available. Instead, peptides were identified by matching them to either a human or a mouse database. The results from both searches were highly similar; therefore only those from the search of the mouse database are presented here.

Bottom Line: Most of these proteins have not been previously shown to be associated with VSV.Functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization.Our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of VSV.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, North Carolina, United States of America; Center for Biomedical Engineering and Science, University of North Carolina at Charlotte, Charlotte, North Carolina, United States of America.

ABSTRACT
Virus particles (virions) often contain not only virus-encoded but also host-encoded proteins. Some of these host proteins are enclosed within the virion structure, while others, in the case of enveloped viruses, are embedded in the host-derived membrane. While many of these host protein incorporations are likely accidental, some may play a role in virus infectivity, replication and/or immunoreactivity in the next host. Host protein incorporations may be especially important in therapeutic applications where large numbers of virus particles are administered. Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus and a candidate vaccine, gene therapy and oncolytic vector. Using mass spectrometry, we previously examined cell type dependent host protein content of VSV virions using intact ("whole") virions purified from three cell lines originating from different species. Here we aimed to determine the localization of host proteins within the VSV virions by analyzing: i) whole VSV virions; and ii) whole VSV virions treated with Proteinase K to remove all proteins outside the viral envelope. A total of 257 proteins were identified, with 181 identified in whole virions and 183 identified in Proteinase K treated virions. Most of these proteins have not been previously shown to be associated with VSV. Functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization. Using western blotting, the presence of several host proteins, including some not previously shown in association with VSV (such as Yes1, Prl1 and Ddx3y), was confirmed and their relative quantities in various virion fractions determined. Our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of VSV.

Show MeSH
Related in: MedlinePlus