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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

Detection of VSV proteins by mass spectrometry.(a) Mean spectral counts per technical replicate for VSV encoded proteins detected in both whole virions and proteinase K (ProK) treated virions. (b) Location of VSV G protein peptides identified in both whole and ProK treated virions as indicated by shaded boxes. Values given below the diagrams indicate the mean spectral counts per technical replicate for the indicated domain(s) with the overall percent sequence coverage given in parentheses.
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pone-0104688-g004: Detection of VSV proteins by mass spectrometry.(a) Mean spectral counts per technical replicate for VSV encoded proteins detected in both whole virions and proteinase K (ProK) treated virions. (b) Location of VSV G protein peptides identified in both whole and ProK treated virions as indicated by shaded boxes. Values given below the diagrams indicate the mean spectral counts per technical replicate for the indicated domain(s) with the overall percent sequence coverage given in parentheses.

Mentions: Although the primary focus of this study was determining host protein incorporation and localization in VSV virions, our search database also included VSV protein sequences allowing for their detection. In untreated samples, a mean of 1231-182 spectra per technical replicate were detected for each of the five VSV encoded proteins (Fig. 4a). The number of spectra went up upon ProK treatment for all VSV proteins except G where there was a sharp reduction. This is consistent with our other data (Fig. 3a) demonstrating that ProK treatment is effectively removing the extracellular domain of the VSV G protein. Removal of the extracellular domain was confirmed by determining the distribution of the spectra and peptides derived from the G protein (Fig. 4b). The mean number of spectra associated with the transmembrane domain and cytoplasmic tail of the G protein were unchanged between whole and ProK treated virions, while ProK treatment decreased the number of spectra associated with the extracellular domain of G. In looking at the distribution of the detected peptides in the G protein, ProK treatment completely eliminated peptides associated with the N-terminal portion of the extracellular domain while a few were still detected in the more C-terminal portion of the domain, suggesting this region has a slight degree of resistance to proteinase cleavage, although the decrease in spectral counts suggests the majority the G proteins were cleaved.


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

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

Detection of VSV proteins by mass spectrometry.(a) Mean spectral counts per technical replicate for VSV encoded proteins detected in both whole virions and proteinase K (ProK) treated virions. (b) Location of VSV G protein peptides identified in both whole and ProK treated virions as indicated by shaded boxes. Values given below the diagrams indicate the mean spectral counts per technical replicate for the indicated domain(s) with the overall percent sequence coverage given in parentheses.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104688-g004: Detection of VSV proteins by mass spectrometry.(a) Mean spectral counts per technical replicate for VSV encoded proteins detected in both whole virions and proteinase K (ProK) treated virions. (b) Location of VSV G protein peptides identified in both whole and ProK treated virions as indicated by shaded boxes. Values given below the diagrams indicate the mean spectral counts per technical replicate for the indicated domain(s) with the overall percent sequence coverage given in parentheses.
Mentions: Although the primary focus of this study was determining host protein incorporation and localization in VSV virions, our search database also included VSV protein sequences allowing for their detection. In untreated samples, a mean of 1231-182 spectra per technical replicate were detected for each of the five VSV encoded proteins (Fig. 4a). The number of spectra went up upon ProK treatment for all VSV proteins except G where there was a sharp reduction. This is consistent with our other data (Fig. 3a) demonstrating that ProK treatment is effectively removing the extracellular domain of the VSV G protein. Removal of the extracellular domain was confirmed by determining the distribution of the spectra and peptides derived from the G protein (Fig. 4b). The mean number of spectra associated with the transmembrane domain and cytoplasmic tail of the G protein were unchanged between whole and ProK treated virions, while ProK treatment decreased the number of spectra associated with the extracellular domain of G. In looking at the distribution of the detected peptides in the G protein, ProK treatment completely eliminated peptides associated with the N-terminal portion of the extracellular domain while a few were still detected in the more C-terminal portion of the domain, suggesting this region has a slight degree of resistance to proteinase cleavage, although the decrease in spectral counts suggests the majority the G proteins were cleaved.

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