The human PYHIN proteins, AIM2, IFI16, IFIX, and MNDA, are critical regulators of immune response, transcription, apoptosis, and cell cycle. However, their protein interactions and underlying mechanisms remain largely uncharacterized. Here, we provide the interaction network for all PYHIN proteins and define a function in sensing of viral DNA for the previously uncharacterized IFIX protein. By designing a cell-based inducible system and integrating microscopy, immunoaffinity capture, quantitative mass spectrometry, and bioinformatics, we identify over 300 PYHIN interactions reflective of diverse functions, including DNA damage response, transcription regulation, intracellular signaling, and antiviral response. In view of the IFIX interaction with antiviral factors, including nuclear PML bodies, we further characterize IFIX and demonstrate its function in restricting herpesvirus replication. We discover that IFIX detects viral DNA in both the nucleus and cytoplasm, binding foreign DNA via its HIN domain in a sequence-non-specific manner. Furthermore, IFIX contributes to the induction of interferon response. Our results highlight the value of integrative proteomics in deducing protein function and establish IFIX as an antiviral DNA sensor important for mounting immune responses.

The interferon-inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes expression of antiviral cytokines. How IFIX exerts its host defense functions and whether it is inhibited by the virus remain unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection. We find that IFIX has a dynamic localization during infection that changes from diffuse nuclear and nucleoli distribution in uninfected cells to discrete nuclear puncta early in infection. This is rapidly followed by a reduction in IFIX protein levels. Indeed, using immunoaffinity purification and mass spectrometry, we define IFIX interactions during HSV-1 infection, finding an association with a proteasome subunit and proteins involved in ubiquitin-proteasome processes. Using synchronized HSV-1 infection, microscopy, and proteasome-inhibition experiments, we demonstrate that IFIX co-localizes with nuclear proteasome puncta shortly after 3 h of infection and that its pyrin domain is rapidly degraded in a proteasome-dependent manner. We further demonstrate that, in contrast to several other host defense factors, IFIX degradation is not dependent on the E3 ubiquitin ligase activity of the viral protein ICP0. However, we show IFIX degradation requires immediate-early viral gene expression, suggesting a viral host suppression mechanism. The IFIX interactome also demonstrated its association with transcriptional regulatory proteins, including the 5FMC complex. We validate this interaction using microscopy and reciprocal isolations and determine it is mediated by the IFIX HIN domain. Finally, we show IFIX suppresses immediate-early and early viral gene expression during infection. Altogether, our study demonstrates that IFIX antiviral functions work in part via viral transcriptional suppression and that HSV-1 has acquired mechanisms to block its functions via proteasome-dependent degradation.

In mammalian cells, early defenses against infection by pathogens are mounted through a complex network of signaling pathways shepherded by immune-modulatory pattern-recognition receptors. As obligate parasites, the survival of viruses is dependent upon the evolutionary acquisition of mechanisms that tactfully dismantle and subvert the cellular intrinsic and innate immune responses. Here, we review the diverse mechanisms by which viruses that accommodate DNA genomes are able to circumvent activation of cellular immunity. We start by discussing viral manipulation of host defense protein levels by either transcriptional regulation or protein degradation. We next review viral strategies used to repurpose or inhibit these cellular immune factors by molecular hijacking or by regulating their post-translational modification status. Additionally, we explore the infection-induced temporal modulation of apoptosis to facilitate viral replication and spread. Lastly, the co-evolution of viruses with their hosts is highlighted by the acquisition of elegant mechanisms for suppressing host defenses via viral mimicry of host factors. In closing, we present a perspective on how characterizing these viral evasion tactics both broadens the understanding of virus-host interactions and reveals essential functions of the immune system at the molecular level. This knowledge is critical in understanding the sources of viral pathogenesis, as well as for the design of antiviral therapeutics and autoimmunity treatments.

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Activation of the mitogen-activated protein kinase (MAPK) pathway targets the putative tumor suppressor protein inducible cAMP early repressor (ICER) to ubiquitin-mediated proteasomal degradation [Yehia et al. JBC 2001; 276: 35272-35279]. We demonstrate that ICER proteasomal degradation is implicated in Ras/MAPK-mediated melanoma tumorigenesis. In a system using Tyr/Tet-Ras INK4a-/- transgenic mice and melanoma cells in culture termed R545 cells isolated from Tyr/Tet-Ras INK4a-/- mice [Chin et al. Nature 1999; 400: 468-472], melanoma genesis and melanoma maintenance is strictly dependent upon expression of H-RasV12G. We found that ICER protein was not expressed during melanoma genesis but was strongly expressed in regressing melanomas. Similarly in R545 cells, ICER protein expression was negatively regulated by H-RasV12G. The expression of ICER mRNA was not affected by H-RasV12G expression, suggesting that ICER regulation was post-translational. Indeed, pharmacological inhibition of Ras activity or the proteasome abolished the degradation of ICER caused by H-RasV12G expression indicating that RAS oncogene regulates the expression of ICER protein by targeting ICER to proteasomal degradation. By engineering clones of R545 melanoma cells stably transfected with ICER we were able to determine the prerequisite for Ras-induced tumorigenesis. The reconstitution of physiological levels of ICER showed a significant decrease in cell growth, as well as inhibition of anchorage-independent cell growth and tumorigenicity in nude mice. ICER was found to efficiently repress the expression of cyclin D1 in R545 cells due to the binding of ICER to the CRE in the cyclin D1 promoter. Taken together, we postulate that ICER protein might be targeted to degradation in human tumors where Ras is mutated.