Dataset Information

Identification of herpes virus proteines palmitoyled in RPE-1 cells

ABSTRACT: Protein palmitoylation regulates diverse aspects of cellular function and organization and plays a key role in host immune responses to infection. Palmitoylation also modulates the function and localization of virus-encoded proteins. In Serwa et al. (doi:10.1016/j.chembiol.2015.06.024), we employed chemical proteomics tools, bio-orthogonal palmitic acid analogue (heptadec-16-ynoic acid, YnPal), and a multi-functional capture reagent (AzTB) to study palmitoylation events during infection with three strains of herpes simplex virus (HSV-1[17], HSV-1[KOS], HSV-2[186]). We found that a significant fraction of the viral proteome (twelve HSV-encoded proteins) undergoes palmitoylation; we identified a number of virus membrane glycoproteins, structural proteins, and kinases. Furthermore, we identified palmitoylation sites on five HSV-encoded proteins utilising a complementary methodology (Acyl-RAC). This paper also reports selective HSV-induced alterations to palmitoylation and myristoylation of host proteins.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Human Herpesvirus 2 (strain 186) (hhv-2) (human Herpes Simplex Virus 2) Human Herpesvirus 1 (strain 17) (hhv-1) (human Herpes Simplex Virus 1) Human Herpesvirus 1 (strain Kos) (hhv-1) (human Herpes Simplex Virus 1)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Remigiusz Serwa

LAB HEAD: Edward W. Tate

PROVIDER: PXD002708 | Pride | 2022-03-02

REPOSITORIES: Pride

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Publications

Systems Analysis of Protein Fatty Acylation in Herpes Simplex Virus-Infected Cells Using Chemical Proteomics.

Serwa Remigiusz A RA Abaitua Fernando F Krause Eberhard E Tate Edward W EW O'Hare Peter P

Chemistry & biology 20150806 8

Protein fatty acylation regulates diverse aspects of cellular function and organization and plays a key role in host immune responses to infection. Acylation also modulates the function and localization of virus-encoded proteins. Here, we employ chemical proteomics tools, bio-orthogonal probes, and capture reagents to study myristoylation and palmitoylation during infection with herpes simplex virus (HSV). Using in-gel fluorescence imaging and quantitative mass spectrometry, we demonstrate a gen ...[more]

PMID: 26256475

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Project description:Differentiated NT2 cells are a potentially useful model system to study herpes simples virus (HSV) replication in human neurons. These cells can be irreversibly differentiated into NT-neurons in the presence of retinoic acid and non-dividing cultures NT-neurons can be maintained for up to several months without retinoic acid. HSV is capable of infecting and replicating in differentiated NT-neurons and thus differentiated NT-neurons provide a ready source of human post-mitotic cells which can be useful to study certain aspects of the interaction of HSV and the neuron. Microarray analysis was used to examine how HSV-1 infection modulates cellular transcription in human NT-neurons, focusing on changes that take place during the first 24 hours following infection and compared to changes resulting from infection with inactivated virus. In addition, the transcriptional changes resulting from virus infection of neurons were compared to those observed in primary human fibroblasts. At early times after HSV infection a small number of cellular transcripts in NT-Neurons appear to be increased in abundance. Most of these transcripts that are up-regulated after infection are not unique to neuronal cells, and possibly represent a common cellular response to HSV infection. A few transcripts were not detected in infected human fibroblasts and may represent part of a transcriptional profile specific to this type of human neuronal cell. In contrast to the situation at early times after infection, analysis of cellular transcripts in NT-neurons at late times after infection is complicated by the overall profound decrease in cellular transcription. Thus, microarray analysis appeared to be most useful as a screening method for transcription changes following infection at early times after virus infection of NT-neurons. Two-condition experiment, HSV-1 infected versus uninfected NTNeurons, 4 independent biological replicate sets (uninfected/infected) in dual channel array setup (Cy3/Cy5).

Project description:Differentiated NT2 cells are a potentially useful model system to study herpes simples virus (HSV) replication in human neurons. These cells can be irreversibly differentiated into NT-neurons in the presence of retinoic acid and non-dividing cultures NT-neurons can be maintained for up to several months without retinoic acid. HSV is capable of infecting and replicating in differentiated NT-neurons and thus differentiated NT-neurons provide a ready source of human post-mitotic cells which can be useful to study certain aspects of the interaction of HSV and the neuron. Microarray analysis was used to examine how HSV-1 infection modulates cellular transcription in human NT-neurons, focusing on changes that take place during the first 24 hours following infection and compared to changes resulting from infection with inactivated virus. In addition, the transcriptional changes resulting from virus infection of neurons were compared to those observed in primary human fibroblasts. At early times after HSV infection a small number of cellular transcripts in NT-Neurons appear to be increased in abundance. Most of these transcripts that are up-regulated after infection are not unique to neuronal cells, and possibly represent a common cellular response to HSV infection. A few transcripts were not detected in infected human fibroblasts and may represent part of a transcriptional profile specific to this type of human neuronal cell. In contrast to the situation at early times after infection, analysis of cellular transcripts in NT-neurons at late times after infection is complicated by the overall profound decrease in cellular transcription. Thus, microarray analysis appeared to be most useful as a screening method for transcription changes following infection at early times after virus infection of NT-neurons. Two-condition experiment, HSV-1 infected versus uninfected NTNeurons, 3 independent biological replicate sets (uninfected/infected) in dual channel array setup (Cy3/Cy5).

Project description:We performed chromatin immunoprecipitation on microarray (ChIP-chip) transcriptional profiling of the SsrB regulator of Salmonella enterica Typhimurium to better characterize its regulon and to identify the DNA-recognition element coordinating its specific interaction at cis-regulatory sites. SsrB, the response regulator of the two component regulatory system SsrA-SsrB encoded within the Salmonella Pathogenicity Island (SPI-2), directs transcriptional activation of the closely associated type three secretion system (T3SS) also encoded at this locus. Immunoprecipitaiton of SsrB translationally fused to a C-terminal FLAG-tag from formaldehyde cross-linked genomic DNA was performed under SPI-2 activating and non-activating conditions. Downstream analysis and experimental work identified specific interactions within SPI-2 at the previously identified ssrA, ssaB, sseA, ssaG, ssaM promoters and identified an additional cryptic promoter driving expression upstream of ssaR. Additionally, a previously unknown SsrB interaction site within ssaE was shown to be the major contributor driving the expression of the downstream genes and not the previously identified interaction site within the intergenic region of ssaE-sseA. Interactions were also identified outside of SPI-2 upstream of other T3SS-associated genes encoded within other genomic islands, and for other uncharacterized genes. Integration of this data with transcriptional microarray work, previously published DNase I footprinting data, bacteria 1-hybrid investigations and comparative genomics analyses of cis-regulatory regions within the orthologous Sodalis symbiosis region 3 (SSR-3) of Sodalis glossinidius enabled identification of a conserved 18bp palindrome which was experimentally validated as being required for transcriptional activation of SsrB dependant genes. SsrB-FLAG immunoprecipitations were performed under SsrB activating and non-activating conditions from formaldehyde cross-linked bacterial lysates. Nine immunoprecipitation reactions were pooled into three replicate samples for the activating condition and three reactions were pooled into one sample for the non-activating control condition. The immunoprecipitated DNA in addition to the non-immunoprecipitated control DNA for each of the four samples were labeled with Cy3 or Cy5 fluorophores respectively and were concurrently hybridized to four arrays on a single slide.

Dataset Information

Identification of herpes virus proteines palmitoyled in RPE-1 cells

Publications

Systems Analysis of Protein Fatty Acylation in Herpes Simplex Virus-Infected Cells Using Chemical Proteomics.

Similar Datasets

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