Charge-Mediated Pyrin Oligomerization Nucleates Antiviral IFI16 Sensing of Herpesvirus DNA

Abstract

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 identify the pyrin domain (PYD) residues that mediate IFI16 oligomerization in a charge-dependent manner. Based on structure modeling, these residues are predicted to be surface exposed within distinct α-helices. By generating oligomerization-deficient mutants, we demonstrate that IFI16 homotypic clustering is necessary for its assembly onto parental viral genomes at the nuclear periphery upon herpes simplex virus 1 (HSV-1) infection. Preventing oligomerization severely hampered the capacity of IFI16 to induce antiviral cytokine expression, suppress viral protein levels, and restrict viral progeny production. Restoring oligomerization via residue-specific charge mimics partially rescued IFI16 antiviral roles. We show that pyrin domains from PYHIN proteins are functionally interchangeable, facilitating cooperative assembly with the IFI16 HINs, highlighting an inherent role for pyrin domains in antiviral response. Using immunoaffinity purification and targeted mass spectrometry, we establish that oligomerization promotes IFI16 interactions with proteins involved in transcriptional regulation, including PAF1C, UBTF, and ND10 bodies. We further discover PAF1C as an HSV-1 restriction factor. Altogether, our study uncovers intrinsic properties that govern IFI16 oligomerization, which serves as a signal amplification platform to activate innate immune responses and to recruit transcriptional regulatory proteins that suppress HSV-1 replication. IMPORTANCE The ability of mammalian cells to detect the genomes of nuclear-replicating viruses via cellular DNA sensors is fundamental to innate immunity. Recently, mounting evidence is supporting the universal role of polymerization in these host defense factors as a signal amplification strategy. Yet, what has remained unclear are the intrinsic properties that govern their immune signal transmission. Here, we uncover the biochemical basis for oligomerization of the nuclear DNA sensor, IFI16. Upon infection with herpes simplex virus 1 (HSV-1) in human fibroblasts, we characterize the contribution of IFI16 oligomerization to downstream protein interactions and antiviral functions, including cytokine induction and suppression of HSV-1 replication. Until now, the global characterization of oligomerization-dependent protein interactions for an immune receptor has never been explored. Our integrative quantitative proteomics, molecular CRISPR/Cas9-based assays, mutational analyses, and confocal microscopy shed light on the dynamics of immune signaling cascades activated against pathogens.

Early Nuclear Events after Herpesviral Infection

“…However, it was also shown that a fraction of cGAS can locate to the nucleus in primary cells, and that cGAS stabilizes IFI16, and thereby promotes, the sensing of HSV-1 [17]. Moreover, several reports suggest that IFI16 could be directly associated with herpesviral DNA in the nucleus [16,18], most likely mediated through oligomerization via its Pyrin domains [19]. As a consequence of oligomerization, IFI16 can form filamentous structures in viral replication compartments after infection with an ICP0-deficient HSV-1, and it is thought that these structures restrict viral gene expression [20].…”

“…More studies are needed to identify the cellular enzymes that mediate respective histone modifications and whether they are directly recruited by IFI16 to viral genomes. IFI16 is rapidly recruited to incoming viral genomes [18,22], and several studies show IFI16 oligomerization in the proximity of viral genomes at later time points using confocal and live cell microscopy [19,31]. Intriguingly, there was no evidence for the association of viral DNA with IFI16 or PML-NBs after nuclear entry of adenoviruses [32,33].…”

Cytosolic DNA Sensors and CNS Responses to Viral Pathogens

“…Furthermore, additional HSV-1 products have been demonstrated to target down-stream signaling molecules in the cGAS STING pathway. For instance, Upton et al, 2012;Pham et al, 2013;Kuriakose et al, 2016;Lin et al, 2016;Maelfait et al, 2017;Guo et al, 2018;Daniels et al, 2019;Ingram et al, 2019;Rothan et al, 2019;Yang et al, Gao D. et al, 2013;Li et al, 2013;Sun et al, 2013;Herzner et al, 2015;Ma F. et al, 2015;Bode et al, 2016;Vermeire et al, Unterholzner et al, 2010;Conrady et al, 2012;Orzalli et al, 2012;Ansari et al, 2013;Cuchet-Lourenco et al, 2013;Jakobsen et al, 2013;Dell'Oste et al, 2014;Diner et al, 2015;Dutta et al, 2015;Iqbal et al, 2016;Pisano et al, 2017;Lum et al, 2019;Roy et al, 2019;Yang et al, Fernandes-Alnemri et al, 2009;Hornung et al, 2009;Rathinam et al, 2010;Reinholz et al, 2013;Sagulenko et al, 2013;Ekchariyawat et al, 2015;Corrales et al, 2016;Gray et al, 2016;…”

“…While there is conflicting evidence for the role of IFI16 in IFN-β responses to foreign DNA challenge, multiple lines of investigation indicate that this DNA sensor contributes to IFN-β and ISG expression following viral infection (Orzalli et al, 2012 ; Jakobsen et al, 2013 ; Ansari et al, 2015 ; Diner et al, 2015 ; Ma F. et al, 2015 ; Li et al, 2016 ; Zhang D. et al, 2016 ; Jønsson et al, 2017 ; Lum et al, 2019 ; Yang et al, 2020 ). For example, nuclear IRF3 translocation and subsequent IFN production in HSV-1 infected corneal epithelial cells has been shown to be dependent, at least in part, on p204 expression (Conrady et al, 2012 ), while TBK-1 phosphorylation and subsequent IFN-β expression by HSV-1 infected HFFs was found to require IFI16 and STING (Orzalli et al, 2012 ).…”

Function and Regulation of Nuclear DNA Sensors During Viral Infection and Tumorigenesis

“…In the past few years, accumulating evidence has demonstrated that nuclear DNA sensors, such as IFN-γ-inducible protein 16 (IFI16), heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), and nuclear cGAS, also exert critical roles in initiating host antiviral immunity ( 10 ). However, compared to the wealth of knowledge about cytosolic DNA sensors and the other PRRs, studies for the roles and underlying mechanisms of nuclear DNA sensors are only just emerging ( 11 – 13 ). Recent evidence indicates that nuclear DNA sensors are also involved in tumor development beyond pathogenic DNA recognition.…”