Contribution of IFIX to nuclear and cytoplasmic proteome changes during immune signaling
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive HF
ORGANISM(S): Homo Sapiens (human) Human Alphaherpesvirus 1
TISSUE(S): Primary Cell, Cell Culture, Fibroblast
SUBMITTER: 
Tim Howard
LAB HEAD: Ileana M Cristea
PROVIDER: PXD025101 | Pride | 2021-06-02
REPOSITORIES: Pride
Similar Datasets
Project description:The formation of multimerized protein assemblies has emerged as a core component of immune signal amplification, yet the biochemical basis of this phenomenon remains unclear for many mammalian proteins within host defense pathways. The interferon-inducible protein 16 (IFI16) is a viral DNA sensor that oligomerizes upon binding to nuclear viral DNA and induces downstream antiviral responses. Here, we first generated oligomerization-incompetent IFI16 mutants that exhibit severely reduced ability to induce antiviral cytokine expression, suppress herpes simplex virus 1 (HSV-1) protein levels, and restrict viral progeny production. Using immunoaffinity purification and targeted mass spectrometry, we establish that oligomerization promotes IFI16 interactions with several proteins involved in transcriptional regulation, including PAF1C, UBTF, and ND10
2019-07-10 | PXD014349 | Pride
Project description:Rapid and dynamically shifting protein-protein interactions (PPIs) underlie the capacity of cells to recognize viral infections and ignite the downstream cascades that constitute the innate-immune response. Herpesviruses share an ancient history of coevolution with their hosts and have developed reciprocal methods to suppress or hijack immune response proteins — underscoring the biological complexity of host-pathogen interactions during the immune response. Accordingly, conventional approaches to studying PPIs downstream of innate immune sensing fail to capture both the global scope and the dynamic on-off behavior of protein complexes as they are altered throughout cellular space and time. To overcome this barrier, we have applied Thermal Proteome Profiling Mass Spectrometry (TPP-MS) to systemically characterize PPIs that coordinate the innate-immune response to Herpes Simplex Virus 1 (HSV-1) infection in primary human fibroblasts. Further, by advancing the power of thermal protein co ggregation analysis (TPCA) to infer associations de novo and to globally track these PPIs, we developed a time-resolved portrait of cellular and viral protein associations with IFI16, a nuclear DNA sensor that serves as a central platform for HSV-1 immune responses. Our TPCA analysis, along with high-resolution microscopy and molecular virological techniques, linked IFI16 sensing of viral DNA in the nuclear periphery to the master DNA damage response (DDR) regulatory kinase, DNA-PK — the activation of which we show to be necessary for the antiviral and inflammatory responses to infection. Finally, phospho-peptide enrichment and MS analysis of DNA-PK substrates revealed IFI16 to be targeted by DNA-PK after both DNA damage and viral infection. Functional analysis of this DDR-dependent IFI16 phosphorylation revealed the specific modified residue required for IFI16-driven cytokine responses. Altogether, our study represents the first systems-wide characterization of the global dynamics of PPIs during HSV-1 infection and uncovers a missing link in the immune signaling pathway that places IFI16 and DNA-PK at the center of herpesvirus innate immunity.
2021-05-11 | PXD023167 | Pride
Project description:The control of host processes which influence cell-cell communication is central to determining the outcome of a virus infection in tissue. Despite this, it remains unclear how cells either in close or distant proximity to an infected cell differentially respond to the primary infection. Here, we established a virus infection microenvironment to resolve molecular features and functional consequences of cell spatial address within this localized niche by mass spectrometry. To rule out the possibility that neighboring cells are infected by the viral progeny from the primary infection, cidofovir is added in the system to prevent viral genome replication in order to prevent the spread of infection.
2023-05-11 | PXD040582 | Pride
Project description:The AF4/FMR2 proteins AFF1 and AFF4 act as a scaffold to assemble the Super Elongation Complex (SEC) that strongly activates transcriptional elongation of HIV-1 and cellular genes. Although they can dimerize, it is unclear whether the dimers exist and function within a SEC in vivo. Furthermore, it is unknown whether AFF1 and AFF4 function similarly in mediating SEC-dependent activation of diverse genes. Providing answers to these questions, our current study shows that AFF1 and AFF4 reside in separate SECs that display largely distinct gene target specificities. While the AFF1-SEC is more potent in supporting HIV-1 transactivation by the viral Tat protein, the AFF4-SEC is more important for HSP70 induction upon heat shock. The functional difference between AFF1 and AFF4 in Tat-transactivation has been traced to a single amino acid variation between the two proteins, which causes them to enhance the affinity of Tat for P-TEFb, a key SEC component, with different efficiency. Finally, genome-wide analysis confirms that the genes regulated by AFF1- and AFF4-SEC are largely non-overlapping and perform distinct functions. Thus, the SEC represents a family of related complexes that exist to increase the regulatory diversity and gene control options during transactivation of diverse cellular and viral genes. RNA-seq in HeLa cells of wild-type and after RNAi of AFF1 or AFF4.
2015-05-20 | E-GEOD-69021 | biostudies-arrayexpress
Project description:The host cell cycle is a major target for manipulation by viral pathogens, which often replicate in certain cell cycle stages. The prevalent pathogen human cytomegalovirus (HCMV), for instance, only replicates during G1 and therefore employs diverse mechanisms for regulating cell cycle progression. Here, we demonstrate that the human enzyme sirtuin 2 (SIRT2) is required for HCMV-mediated cell cycle dysregulation. By combining virology assays with mass pectrometry-based proteome, acetylome, and interactome analyses, we have defined a pro-virus function for SIRT2 in regulating the G1 to S phase transition.
2023-10-13 | PXD041171 | Pride
Project description:In mammalian cells, widespread acceleration of cytoplasmic mRNA degradation is linked to impaired RNA polymerase II (Pol II) transcription. This mRNA decay-induced transcriptional repression occurs during infection with gammaherpesviruses including Kaposi’s sarcoma associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68), which encode an mRNA endonuclease that initiates widespread RNA decay. Here, we show that MHV68-induced mRNA decay leads to a genome-wide reduction of Pol II occupancy at mammalian promoters. Viral genes, despite the fact that they require Pol II for transcription, escape this transcriptional repression. Protection is not governed by viral promoter sequences; instead, location on the viral genome is both necessary and sufficient to escape the transcriptional repression effects of mRNA decay. We hypothesize that the ability to escape from transcriptional repression is linked to the localization of viral DNA in replication compartments, providing a means for these viruses to counteract decay-induced viral transcript loss.
2019-12-10 | PXD015786 | Pride
Project description:Human respiratory syncytial virus (RSV) is a globally prevalent negative-stranded RNA virus, which can cause life-threatening respiratory infections in young children, elderly people and immunocompromised patients. Its transcription termination factor M2-1 plays an essential role in viral transcription, but the mechanisms underpinning its function are still unclear. We investigated the cellular interactome of M2-1 using green fluorescent protein (GFP)-trap immunoprecipitation on RSV infected cells coupled with mass spectrometry analysis. We identified 137 potential cellular binding partners of M2-1, among which many proteins associated either with translation or mRNA metabolism, and particularly mRNA maturation and stabilization. Specific immunoprecipitation using the GFP-trap was used to confirm the ability of the polyA-binding protein cytoplasmic 1 (PABPC1), a candidate with a major role in both cellular translation and mRNA stabilization, to interact individually with M2-1. PABPC1 was also shown to colocalize with M2-1 from its accumulation in IBAGs to its liberation in the cytoplasm. These results strongly suggest a continued association of M2-1 with viral mRNA and mRNA metabolism factors from transcription to translation, and imply that M2-1 may have an additional role in the fate of viral mRNA downstream of transcription.
2019-10-11 | PXD013761 | Pride
Project description:Productive infection by human immunodeficiency virus type-1 (HIV-1) requires the import of viral replication complexes into the nuclei of infected cells. Myxovirus resistance 2 (MX2/MxB) blocks this step, halting nuclear accumulation of viral DNA and virus replication. Here, we identify positions in MX2 whose phosphorylation status reduces or enhances antiviral function (hypomorphic and hypermorphic variants, respectively). Importantly, hypermorphic mutant proteins not only increased inhibitory activity against wild-type HIV-1 but can also exhibit antiviral capabilities against HIV-1 capsid mutant viruses that are resistant to wild-type MX2. Furthermore, some of these proteins were also able to inhibit retroviruses that are insensi- tive to MX2. Therefore, we propose that phosphorylation comprises a major element of MX2 regulation and substrate determination.
2022-07-21 | PXD035305 | Pride
Project description:Viral infections are associated with extensive remodeling of the cellular proteome. Viruses encode gene products that manipulate host proteins to redirect cellular processes or subvert antiviral immune responses. One way in which host antiviral proteins antagonize viral infection is by associating with viral genomes and inhibiting essential viral processes. Adenovirus (AdV) encodes gene products from the early E4 region which are necessary for productive infection. Some cellular antiviral proteins are known to be targeted by AdV E4 gene products, resulting in their degradation or mislocalization. However, the full repertoire of host proteins manipulated by viral E4 proteins has not been defined. To identify cellular proteins and processes manipulated by viral products, we developed a global, un-biased proteomics approach to analyze changes to the host proteome during infection with Adenovirus serotype 5 (Ad5) virus. We combined quantification of total protein abundance in the proteome together with an analysis of proteins associated with viral genomes using isolation of Proteins on Nascent DNA (iPOND). By Integrating these proteomics datasets, we identified cellular factors that are degraded in an E4-dependent manner or are associated with the viral genome in the absence of E4 proteins. We further show that some identified proteins exert inhibitory effects on Ad5 infection. Our systems-level analysis reveals cellular processes that are manipulated during Ad5 infection and points to host factors counteracted by early viral proteins as they remodel the host proteome to promote efficient infection. Importance Viral infections stimulate myriad changes to the host cell proteome. As viruses harness cellular processes and counteract host defenses, they impact abundance, modifications, or localization of cellular proteins. Elucidating the dynamic changes to the cellular proteome during viral replication is integral to understanding how virus-host interactions influence the outcome of infection. Adenovirus serotype 5 (Ad5) encodes early gene products from the E4 genomic region that are known to alter host response pathways and promote replication, but the full extent of proteome modifications they mediate is not known. We use an integrated proteomics approach to quantitate protein abundance and protein associations with viral DNA during virus infection. Systems-level analysis identifies cellular proteins and processes impacted in an E4-dependent manner that could overcome potentially inhibitory host defenses. This study provides a global view of Adenovirus-mediated proteome remodeling which can serve as a model to investigate virus-host interactions of DNA viruses.
2021-08-11 | PXD025339 | Pride
Project description:Nearly all biological processes rely on the finely-tuned coordination of protein interactions across cellular space and time. Accordingly, generating protein interactome datasets has become routine in biological studies, yet effectively interpreting these datasets remains computationally challenging. Here, we describe the development of Inter-ViSTA, a computational web platform for protein Interaction Visualization in Space and Time Analysis. Inter-ViSTA enables users to quickly build animated interaction networks by automatically synthesizing information on protein abundances, functional annotations, multiprotein complexes, and subcellular localizations. We then leverage Inter-ViSTA, in conjunction with quantitative mass spectrometry and molecular virology, to define the virus-host protein interactions of the human cytomegalovirus (HCMV) anti-apoptotic protein, pUL37x1 (vMIA). We find that spatially and temporally controlled protein interactions underly pUL37x1 functions during infection, facilitating the pro-viral remodeling of both mitochondria and peroxisomes. Reciprocal isolations, microscopy, and CRISPR-based genetic tools further characterize these associations, revealing new mechanisms at the core of the pUL37x1 manipulation of mitochondrial integrity, such as interactions with the MICOS and SAMM50 complexes. Finally, we show that pUL37x1 activates the peroxisomal protein PEX11ß to regulate peroxisome fission during infection, a key metabolic aspect of virus assembly and spread.
2020-08-03 | PXD017163 | Pride