Analysis of virion associated host proteins in vesicular stomatitis virus using a proteomics approach

<p>Abstract</p> <p>Background</p> <p>Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus and the best studied member of the order <it>Mononegavirales</it>. There is now compelling evidence that enveloped virions released from infected cells carry numerous host (cellular) proteins some of which may play an important role in viral replication. Although several cellular proteins have been previously shown to be incorporated into VSV virions, no systematic study has been done to reveal the host protein composition for virions of VSV or any other member of <it>Mononegavirales</it>.</p> <p>Results</p> <p>Here we used a proteomics approach to identify cellular proteins within purified VSV virions, thereby creating a "snapshot" of one stage of virus/host interaction that can guide future experiments aimed at understanding molecular mechanisms of virus-cell interactions. Highly purified preparations of VSV virions from three different cell lines of human, mouse and hamster origin were analyzed for the presence of cellular proteins using mass spectrometry. We have successfully confirmed the presence of several previously-identified cellular proteins within VSV virions and identified a number of additional proteins likely to also be present within the virions. In total, sixty-four cellular proteins were identified, of which nine were found in multiple preparations. A combination of immunoblotting and proteinase K protection assay was used to verify the presence of several of these proteins (integrin β1, heat shock protein 90 kDa, heat shock cognate 71 kDa protein, annexin 2, elongation factor 1a) within the virions.</p> <p>Conclusion</p> <p>This is, to our knowledge, the first systematic study of the host protein composition for virions of VSV or any other member of the order <it>Mononegavirales</it>. Future experiments are needed to determine which of the identified proteins have an interaction with VSV and whether these interactions are beneficial, neutral or antiviral with respect to VSV replication. Identification of host proteins-virus interactions beneficial for virus would be particularly exciting as they can provide new ways to combat viral infections via control of host components.</p

A role for DNA-dependent activator of interferon regulatory factor in the recognition of herpes simplex virus type 1 by glial cells

<p>Abstract</p> <p>Background</p> <p>The rapid onset of potentially lethal neuroinflammation is a defining feature of viral encephalitis. Microglia and astrocytes are likely to play a significant role in viral encephalitis pathophysiology as they are ideally positioned to respond to invading central nervous system (CNS) pathogens by producing key inflammatory mediators. Recently, DNA-dependent activator of IFN regulatory factor (DAI) has been reported to function as an intracellular sensor for DNA viruses. To date, the expression and functional role of DAI in the inflammatory responses of resident CNS cells to neurotropic DNA viruses has not been reported.</p> <p>Methods</p> <p>Expression of DAI and its downstream effector molecules was determined in C57BL/6-derived microglia and astrocytes, either at rest or following exposure to herpes simplex virus type 1 (HSV-1) and/or murine gammaherpesvirus-68 (MHV-68), by immunoblot analysis. In addition, such expression was studied in ex vivo microglia/macrophages and astrocytes from uninfected animals or mice infected with HSV-1. Inflammatory cytokine production by glial cultures following transfection with a DAI specific ligand (B-DNA), or following HSV-1 challenge in the absence or presence of siRNA directed against DAI, was assessed by specific capture ELISA. The production of soluble neurotoxic mediators by HSV-1 infected glia following DAI knockdown was assessed by analysis of the susceptibility of neuron-like cells to conditioned glial media.</p> <p>Results</p> <p>We show that isolated microglia and astrocytes constitutively express DAI and its effector molecules, and show that such expression is upregulated following DNA virus challenge. We demonstrate that these resident CNS cells express DAI <it>in situ</it>, and show that its expression is similarly elevated in a murine model of HSV-1 encephalitis. Importantly, we show B-DNA transfection can elicit inflammatory cytokine production by isolated glial cells and DAI knockdown can significantly reduce microglial and astrocyte responses to HSV-1. Finally, we demonstrate that HSV-1 challenged microglia and astrocytes release neurotoxic mediators and show that such production is significantly attenuated following DAI knockdown.</p> <p>Conclusions</p> <p>The functional expression of DAI by microglia and astrocytes may represent an important innate immune mechanism underlying the rapid and potentially lethal inflammation associated with neurotropic DNA virus infection.</p

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

Proteomic and evolutionary analyses of sperm activation identify uncharacterized genes in Caenorhabditis nematodes

Abstract Background Nematode sperm have unique and highly diverged morphology and molecular biology. In particular, nematode sperm contain subcellular vesicles known as membranous organelles that are necessary for male fertility, yet play a still unknown role in overall sperm function. Here we take a novel proteomic approach to characterize the functional protein complement of membranous organelles in two Caenorhabditis species: C. elegans and C. remanei. Results We identify distinct protein compositions between membranous organelles and the activated sperm body. Two particularly interesting and undescribed gene families—the Nematode-Specific Peptide family, group D and the here designated Nematode-Specific Peptide family, group F—localize to the membranous organelle. Both multigene families are nematode-specific and exhibit patterns of conserved evolution specific to the Caenorhabditis clade. These data suggest gene family dynamics may be a more prevalent mode of evolution than sequence divergence within sperm. Using a CRISPR-based knock-out of the NSPF gene family, we find no evidence of a male fertility effect of these genes, despite their high protein abundance within the membranous organelles. Conclusions Our study identifies key components of this unique subcellular sperm component and establishes a path toward revealing their underlying role in reproduction

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