Data and supplementary information for Kasimatis et al. (2018) G3

Data, primers sequences, and analysis script in support of Kasimatis et al. Auxin-Mediated Sterility Induction System for Longevity and Mating Studies in <i>Caenorhabditis elegans</i>, published in G3: Genes, Genomes, and Genomic

Cellular Proteins Associated with the Interior and Exterior of Vesicular Stomatitis Virus Virions

<div><p>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.</p></div

Overview of sample preparation and experimental approach.

<p>Mass spectrometry based analysis of host protein content was conducted on (a) purified whole virions and (b) purified virions treated with proteinase K to remove surface proteins. G, glycoprotein; M, matrix protein; P, phosphoprotein; L, large polymerase protein; N, nucleocapsid protein; yellow shapes, host proteins.</p

Confirmation of host protein incorporation in virion preparations.

<p>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.</p

1D SDS-PAGE and protein identification by mass spectrometry.

<p>(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.</p

Functional enrichment analysis.

<p>Enrichment analysis and clustering based on gene ontology terms was conducted for (a) all proteins identified and (b) proteins identified in the proteinase K (ProK) treated virions, as described in the materials in methods. The five clusters in each database with the highest enrichment score are depicted here. Numbers above the bars indicate the number of identified proteins associated with each cluster.</p

Additional file 4: of Proteomic and evolutionary analyses of sperm activation identify uncharacterized genes in Caenorhabditis nematodes

Consensus sequence alignments for the Nematode-Specific Peptide family, group D (NSPD). The amino acid sequence is largely conserved, except for the species-specific amino acid repeats in the middle of the gene. (PDF 79 kb

Additional file 2: of Proteomic and evolutionary analyses of sperm activation identify uncharacterized genes in Caenorhabditis nematodes

Proteome data for C. elegans and C. remanei. Un-activated spermatid, membranous organelle, and active sperm proteome data for both species analyzed, including WormBase gene identifiers, protein abundances, and peptide coverage. (XLSX 64 kb

Additional file 6: of Proteomic and evolutionary analyses of sperm activation identify uncharacterized genes in Caenorhabditis nematodes

Sequence alignments for the Nematode-Specific Peptide family, group F (NSPF) orthologous genes. Amino acid sequence is largely conserved across orthologs. (PDF 158 kb

Additional file 1: of Proteomic and evolutionary analyses of sperm activation identify uncharacterized genes in Caenorhabditis nematodes

The un-activated sperm proteome of C. elegans. The majority of the proteome is comprised of the Nematode-Specific Peptide family, group D (NSPD) and the Major Sperm Protein (MSP). Protein abundance is shown as the relative mean normalized spectrum abundance frequency. Proteins found to be unique to either the membranous organelle or activated sperm proteomes are highlighted in teal, while proteins found in both proteomes are shown in gray. Proteins shown in white were not identified in the membranous organelle or activated sperm proteomes, but were found in the previously published un-activated spermatid proteome of Ma et al. [21]. (PDF 863 kb