Dataset Information

IP-MS analysis

ABSTRACT: It has recently been shown that HSV-1 infection induces widespread defect in host gene transcription termination while viral genes are unaffected. However, The underlying mechanism remain poorly understood. According to our sequencing data, ICP27 as an immediate early protein of HSV-1 was involved in disrupting host transcription termination. To elucidate the underlying mechanism, we performed IP-MS to figure out interaction proteins with ICP27. Other than export proteins, 3’ end processing factors strongly associated with ICP27 based on this IP-MS data. Pre-mRNA 3’ end processing is required for transcription termination. As expected, this MS data as well as our in vitro and in vivo data suggested that ICP27 functions in pre-mRNA 3’ end processing to regulate transcription termination in a sequence dependent manner.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yue Chen

LAB HEAD: Yue Chen

PROVIDER: PXD012837 | Pride | 2020-05-26

REPOSITORIES: Pride

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Publications

Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27.

Wang Xiuye X Hennig Thomas T Whisnant Adam W AW Erhard Florian F Prusty Bhupesh K BK Friedel Caroline C CC Forouzmand Elmira E Hu William W Erber Luke L Chen Yue Y Sandri-Goldin Rozanne M RM Dölken Lars L Shi Yongsheng Y

Nature communications 20200115 1

Infection by viruses, including herpes simplex virus-1 (HSV-1), and cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes. However, the underlying mechanisms remain unclear. Here, we demonstrate that the HSV-1 immediate early protein ICP27 induces DoTT by directly binding to the essential mRNA 3' processing factor CPSF. It thereby induces the assembly of a dead-end 3' processing complex, blocking mRNA 3' cleavage. Remarkably ...[more]

PMID: 31941886

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Project description:The overall goal of the study was to use in vivo data combined with functional genomics to define gene expression signatures representative of a spectrum of HSV CNS infections. Innate immune deficiencies result in a spectrum of severe clinical outcomes following infection. In particular, there is a strong association between loss of the signal transducer and activator of transcription (Stat) pathway, breach of the blood-brain barrier (BBB), and virus-induced neuropathology. The gene signatures that characterize resistance, disease, and mortality in the virus-infected nervous system have not been defined. Herpes simplex virus type 1 (HSV-1) is commonly associated with encephalitis in humans, and humans and mice lacking Stat1 display increased susceptibility to HSV central nervous system (CNS) infections. In this study, two HSV-1 strains were used, KOS (wild type [WT]), and Δvhs, an avirulent recombinant lacking the virion host shutoff (vhs) function. In addition, two mouse strains were used: strain 129 (control) and a Stat1-deficient (Stat1(-/-)) strain. Using combinations of these virus and mouse strains, we established a model of infection resulting in three different outcomes: viral clearance without neurological disease (Δvhs infection of control mice), neurological disease followed by viral clearance (Δvhs infection of Stat1(-/-) mice and WT infection of control mice), or neurological disease followed by death (WT infection of Stat1(-/-) mice). Through the use of functional genomics on the infected brain stem and liver, we determined gene signatures that were representative of the three infection outcomes. Gender matched, 6- to 8- week old immunocompetent, control 129S6 and 129S6 Stat1 knockout mice were infected corneally with 2x10^6 PFU of either wild type HSV-1, a vhs-null HSV virus, or mock-infected. Brain stems and liver of individual mice were isolated at days 1, 3, 5 and 7 post-inoculation for microarray analysis. For microarray analysis, samples were collected from n=2 animals (1 male, 1 female) per mouse strain and virus strain for each time point. Equal masses of tissue were pooled from two mock-infected mice per time point and run on microarray.

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 fibroblasts, 3 independent biological replicate sets (uninfected/infected) in dual channel array setup (Cy3/Cy5).

Dataset Information

IP-MS analysis

Publications

Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27.

Similar Datasets

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