Limits...
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.

Show MeSH

Related in: MedlinePlus

Confirmation of host protein incorporation in virion preparations.Protein lysates from uninfected (BHK) and VSV infected (BHK+VSV) BHK-21 cells were separated by SDS-PAGE along with total protein from whole virions, proteinase K (ProK) treated virions, and viral ribonucleoprotein complexes (RNP). Loading of the virion samples was confirmed by Ponceau S staining of the membrane. The position of the viral glycoprotein (G), nucleocapsid protein (N), phosphoprotein (P), and matrix protein (M) is indicated. Western blots using primary antibodies against coiled-coil and C2 domain-containing protein 1A (Cc2d1a), protein tyrosine phosphatase type IVA 2 (Ptp4a2), putative ATP-dependent RNA helicase Pl10 (D1pas1), proto-oncogene tyrosine-protein kinase Yes (Yes1), casein kinase I isoform alpha (Csnk1a1), heat shock cognate 71 kDa protein kDa protein (Hspa8) and E3 ubiquitin-protein ligase Itchy (ITCH), were conducted as indicated to determine the presence of the host proteins. Approximate molecular mass of the proteins is given on the left.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4126742&req=5

pone-0104688-g005: Confirmation of host protein incorporation in virion preparations.Protein lysates from uninfected (BHK) and VSV infected (BHK+VSV) BHK-21 cells were separated by SDS-PAGE along with total protein from whole virions, proteinase K (ProK) treated virions, and viral ribonucleoprotein complexes (RNP). Loading of the virion samples was confirmed by Ponceau S staining of the membrane. The position of the viral glycoprotein (G), nucleocapsid protein (N), phosphoprotein (P), and matrix protein (M) is indicated. Western blots using primary antibodies against coiled-coil and C2 domain-containing protein 1A (Cc2d1a), protein tyrosine phosphatase type IVA 2 (Ptp4a2), putative ATP-dependent RNA helicase Pl10 (D1pas1), proto-oncogene tyrosine-protein kinase Yes (Yes1), casein kinase I isoform alpha (Csnk1a1), heat shock cognate 71 kDa protein kDa protein (Hspa8) and E3 ubiquitin-protein ligase Itchy (ITCH), were conducted as indicated to determine the presence of the host proteins. Approximate molecular mass of the proteins is given on the left.

Mentions: Because protein quantities were chosen so that the amount of N and P protein was approximately equal for all virion samples (Fig. 5), the host protein associated with whole virions that was retained in the treated samples could be estimated. Based on this, proteins were determined to be associated with the viral RNP complex, associated primarily with the interior of the virion but not the RNP or associated primarily with the exterior of the virion. Most proteins were barely detectable in the RNP complexes, suggesting they do not associate with the viral RNP or that the association was disrupted under the conditions used for RNP isolation. Only for E3 ubiquitin-protein ligase Itchy (ITCH) were substantial amounts of protein detected in RNP. The M protein of VSV, as well as other rhabdoviruses, contains a late domain (L-domain) including a proline rich PPxY motif known to bind WW domains found in a number of cellular proteins [62]. VSV M protein has been shown to bind the WW domain of Nedd4 (also detected in this analysis) [63], and can presumably also bind other HECT domain-containing E3 ubiquitin ligases, including ITCH, as has been reported for other viruses with L-domains including PPxY motifs [64]. However, given that ITCH is present in RNP complexes at levels that nearly equal the other two sample types, while M protein is sharply reduced, it seems likely that ITCH may also interact with one of the RNP proteins (N, P or L). Three of the tested proteins, Yes1, casein kinase I isoform alpha (Csnk1a1) and heat shock cognate 71 kDa protein (Hspa8; also known as Hsc70), while barely detectable in RNP, were found at similar levels in whole and ProK treated virions, indicated they are primarily associated with the interior of the virions. In contrast, levels of Ptp4a2 and putative ATP-dependent RNA helicase Pl10 (D1pas1; also known as Ddx3y), dropped sharply in the ProK and RNP samples indicating they are not primarily located in the interior of the virion. One protein, coiled-coil and C2 domain-containing protein 1A (Cc2d1a), identified by MS could not be detected by WB, indicating either protein levels below the threshold of detection, a misidentification of the protein based on homology, or a false-positive identification. The latter seems the least likely as the protein was identified in both sample types, with at least 6 consecutive amino acids matched in manual inspection.


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

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

Confirmation of host protein incorporation in virion preparations.Protein lysates from uninfected (BHK) and VSV infected (BHK+VSV) BHK-21 cells were separated by SDS-PAGE along with total protein from whole virions, proteinase K (ProK) treated virions, and viral ribonucleoprotein complexes (RNP). Loading of the virion samples was confirmed by Ponceau S staining of the membrane. The position of the viral glycoprotein (G), nucleocapsid protein (N), phosphoprotein (P), and matrix protein (M) is indicated. Western blots using primary antibodies against coiled-coil and C2 domain-containing protein 1A (Cc2d1a), protein tyrosine phosphatase type IVA 2 (Ptp4a2), putative ATP-dependent RNA helicase Pl10 (D1pas1), proto-oncogene tyrosine-protein kinase Yes (Yes1), casein kinase I isoform alpha (Csnk1a1), heat shock cognate 71 kDa protein kDa protein (Hspa8) and E3 ubiquitin-protein ligase Itchy (ITCH), were conducted as indicated to determine the presence of the host proteins. Approximate molecular mass of the proteins is given on the left.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104688-g005: Confirmation of host protein incorporation in virion preparations.Protein lysates from uninfected (BHK) and VSV infected (BHK+VSV) BHK-21 cells were separated by SDS-PAGE along with total protein from whole virions, proteinase K (ProK) treated virions, and viral ribonucleoprotein complexes (RNP). Loading of the virion samples was confirmed by Ponceau S staining of the membrane. The position of the viral glycoprotein (G), nucleocapsid protein (N), phosphoprotein (P), and matrix protein (M) is indicated. Western blots using primary antibodies against coiled-coil and C2 domain-containing protein 1A (Cc2d1a), protein tyrosine phosphatase type IVA 2 (Ptp4a2), putative ATP-dependent RNA helicase Pl10 (D1pas1), proto-oncogene tyrosine-protein kinase Yes (Yes1), casein kinase I isoform alpha (Csnk1a1), heat shock cognate 71 kDa protein kDa protein (Hspa8) and E3 ubiquitin-protein ligase Itchy (ITCH), were conducted as indicated to determine the presence of the host proteins. Approximate molecular mass of the proteins is given on the left.
Mentions: Because protein quantities were chosen so that the amount of N and P protein was approximately equal for all virion samples (Fig. 5), the host protein associated with whole virions that was retained in the treated samples could be estimated. Based on this, proteins were determined to be associated with the viral RNP complex, associated primarily with the interior of the virion but not the RNP or associated primarily with the exterior of the virion. Most proteins were barely detectable in the RNP complexes, suggesting they do not associate with the viral RNP or that the association was disrupted under the conditions used for RNP isolation. Only for E3 ubiquitin-protein ligase Itchy (ITCH) were substantial amounts of protein detected in RNP. The M protein of VSV, as well as other rhabdoviruses, contains a late domain (L-domain) including a proline rich PPxY motif known to bind WW domains found in a number of cellular proteins [62]. VSV M protein has been shown to bind the WW domain of Nedd4 (also detected in this analysis) [63], and can presumably also bind other HECT domain-containing E3 ubiquitin ligases, including ITCH, as has been reported for other viruses with L-domains including PPxY motifs [64]. However, given that ITCH is present in RNP complexes at levels that nearly equal the other two sample types, while M protein is sharply reduced, it seems likely that ITCH may also interact with one of the RNP proteins (N, P or L). Three of the tested proteins, Yes1, casein kinase I isoform alpha (Csnk1a1) and heat shock cognate 71 kDa protein (Hspa8; also known as Hsc70), while barely detectable in RNP, were found at similar levels in whole and ProK treated virions, indicated they are primarily associated with the interior of the virions. In contrast, levels of Ptp4a2 and putative ATP-dependent RNA helicase Pl10 (D1pas1; also known as Ddx3y), dropped sharply in the ProK and RNP samples indicating they are not primarily located in the interior of the virion. One protein, coiled-coil and C2 domain-containing protein 1A (Cc2d1a), identified by MS could not be detected by WB, indicating either protein levels below the threshold of detection, a misidentification of the protein based on homology, or a false-positive identification. The latter seems the least likely as the protein was identified in both sample types, with at least 6 consecutive amino acids matched in manual inspection.

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