Dataset Information

Cytonuclear Fractionation TPCA in HSV-1 RF Infection

ABSTRACT: We developed a cytonuclear fractionated TPCA workflow, enabling the probing of global protein-protein interaction (PPI) networks with spatial resolution. We show that fractionation improves downstream PPI prediction, as well as increases the number of PPIs that can be detected. We apply this new workflow to study the spatiotemporal dynamics of host and viral proteins during infection with a mutant strain of HSV-1, HSV-1 RF.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Human Herpesvirus 1 (strain 17) (hhv-1) (human Herpes Simplex Virus 1)

TISSUE(S): Foreskin Fibroblast, Cell Culture

SUBMITTER: Tavis Reed

LAB HEAD: Ileana M. Cristea

PROVIDER: PXD059014 | Pride | 2025-07-07

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20221130_TR_Mock_A_Ratio_Frac_1.raw	Raw
	20221130_TR_Mock_A_Ratio_Frac_2.raw	Raw
	20221130_TR_Mock_A_Ratio_Frac_3.raw	Raw
	20221130_TR_Mock_A_Ratio_Frac_4.raw	Raw
	20221130_TR_Mock_A_Ratio_Frac_5.raw	Raw

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Publications

Sequestration of ribosome biogenesis factors in HSV-1 nuclear aggregates revealed by spatially resolved thermal profiling.

Metzger Peter J PJ Reed Tavis J TJ Lum Krystal K KK Botello Jordy F JF Jiang Lifei L Brangwynne Clifford P CP Troyanskaya Olga G OG Cristea Ileana M IM

Science advances 20250627 26

Viruses exploit host cell reliance on compartmentalization to facilitate their replication. Herpes simplex virus type 1 (HSV-1) modulates the subcellular localization of host proteins to suppress immune activation, license viral gene expression, and achieve translational shutoff. To spatially resolve dynamic protein-protein interaction (PPI) networks during infection with an immunostimulatory HSV-1 strain, we integrated nuclear/cytoplasmic fractionation with thermal proximity coaggregation analy ...[more]

PMID: 40577456

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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.