Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation

Abstract

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.

PYHIN1 regulates pro-inflammatory cytokine induction rather than innate immune DNA sensing in airway epithelial cells

“…Because nucleic acid sensing would not be involved in pro-inflammatory cytokine-stimulated mRNA induction, we reasoned that PYHIN1 may directly regulate promoter induction of IL-6 and TNF␣. This would be consistent with the fact that another PYHIN protein, IFI16, has been shown to directly modulate both viral and host gene promoters in certain circumstances (42,43), and with the fact that, like IFI16, PYHIN1 can be detected in the nucleus (31,32). Furthermore, the fact that PYHIN1 was required for both IL-1and TNF␣-mediated IL-6 induction ( Fig.…”

“…This led to an important new insight into PYHIN1 function, namely that it was required for optimal IL-6 and TNF␣ transcriptional induction after cytokine stimulation of cells. Hence, similar to IFI16, PYHIN1 has now been implicated in DNA sensing (31), viral restriction (32), and host immune gene regulation (this paper). IFI16 was previously shown to be required for maximal promoter induction of type I IFNs and IFN-stimulated genes, but not IL-6, in human THP-1 monocytes (42).…”

“…Further, PYHIN1 directly bound dsDNA via its HIN domain, and dsDNA transfection of HEK293 cells ectopically expressing PYHIN1 led to IFN␤ mRNA induction (31). PYHIN1 was also recently shown to suppress HSV-1 viral gene expression in infected cells, and to be targeted by the virus for degradation (32).…”

β-Carboline derivatives as novel antivirals for herpes simplex virus

“…of pre-replicative sites containing the helicase-primase complex, appearing as punctate foci in the nucleus [34]. ICP0, a HSV IE protein, binds to several cellular proteins as well as viral promoters resulting in increased expression of viral E and L genes.…”

The Host-Pathogen Ecosystem Viewed Through the Prism of Proteomics

“…However, the vesicle-containing secretome contained components of the translational machinery, leading the authors to propose a model in which secretion and inhibition of translation plays a role in host defense against bacteria. Also, working on immune response, Crow and Cristea provide insights into the antiviral functions of the interferon-inducible protein IFIX in response to infection of primary human fibroblasts with the DNA virus HSV-1 (15). Using protein interaction studies, MS, microscopy, and functional assays, IFIX is found to have a dual role in defense via both stimulation of innate immune response and inhibition of virus gene expression.…”