Proximity proteomics reveals a role for IFI16 during human coronavirus infection

1. Problem Statement

Human coronaviruses (HCoVs), including SARS-CoV-2, rely heavily on hijacking host cell machinery to replicate and propagate. While it is known that viral non-structural proteins (NSPs) interact with host factors, the specific host proteins that facilitate this interaction across the spectrum of HCoVs remain incompletely characterized. Specifically, the functional role of the host protein IFI16 (Interferon-inducible protein 16) in the context of HCoV replication was unclear. Given IFI16's known function as a cytosolic DNA sensor and regulator of Type I Interferon (IFN) responses, understanding its dual potential role—either as a restriction factor or a proviral factor—during coronavirus infection is critical for elucidating viral pathogenesis and host defense mechanisms.

2. Methodology

The study employed a multi-faceted approach combining proximity-dependent biotinylation, mass spectrometry, and genetic manipulation:

• Proximity Proteomics (Split-TurboID): The researchers utilized the split-TurboID system, a proximity-dependent biotinylation technique, to identify proteins physically close to specific viral targets. They fused TurboID fragments to SARS-CoV-2 NSP8 and NSP10, two non-structural proteins known to form a complex involved in viral replication.
• Mass Spectrometry (MS): Following biotinylation, the biotinylated host proteins were enriched and identified via high-resolution mass spectrometry to map the interactome of the NSP8/NSP10 complex.
• Genetic Perturbation (CRISPR-Cas9 and CRISPRi): To validate the functional significance of identified hits, the team used CRISPR-Cas9 to generate complete knockouts (KO) and CRISPR interference (CRISPRi) to achieve partial knockdown (KD) of the candidate protein, IFI16, in A549 lung epithelial cells.
• Viral Assays: The impact of IFI16 depletion was assessed by measuring:
  • Viral infection rates.
  • Viral RNA replication levels.
  • Viral protein synthesis.
  • Expression levels of Type I Interferons (IFNs).

3. Key Contributions

• Identification of a Novel Host-Viral Interaction: The study successfully identified IFI16 as a proximal partner of the SARS-CoV-2 NSP8 and NSP10 complex, establishing a direct physical or functional link between the viral replication machinery and this specific host sensor.
• Paradigm Shift on IFI16 Function: Contrary to the canonical view of IFI16 solely as an antiviral restriction factor (via Type I IFN induction), this work demonstrates that IFI16 acts as a proviral factor for human coronaviruses.
• Mechanistic Decoupling: The research provides evidence that IFI16's role in promoting viral replication is independent of its traditional function as a regulator of Type I IFN gene expression.

4. Results

• Proteomic Screening: Split-TurboID screening revealed IFI16 as a high-confidence proximal partner of the NSP8/NSP10 complex.
• Impact on Infection: Both CRISPR-mediated knockout and knockdown of IFI16 in A549 cells resulted in a significant decrease in human coronavirus infection efficiency.
• Replication and Synthesis: Cells lacking IFI16 exhibited reduced levels of viral RNA replication and viral protein synthesis, indicating that IFI16 is required for efficient viral production.
• Interferon Response: Surprisingly, the loss of IFI16 led to reduced expression of Type I IFNs. Since Type I IFNs are typically antiviral, one might expect that reducing them would increase viral replication. However, the virus replication decreased despite the lower IFN levels.
• Conclusion on Mechanism: The data suggests that IFI16 promotes viral replication through a mechanism that is distinct from, and overrides, its role in modulating the Type I IFN response. The proviral effect of IFI16 is not a byproduct of IFN suppression but likely stems from its direct interaction with the viral replication complex.

5. Significance

This study fundamentally alters the understanding of the host-virus interplay during coronavirus infection. By identifying IFI16 as a proviral factor, the paper suggests that:

1. Therapeutic Targets: IFI16 could be a potential target for antiviral therapies; inhibiting IFI16 might restrict viral replication without necessarily triggering the complex immune evasion strategies viruses use against IFN pathways.
2. Viral Replication Machinery: It highlights that the SARS-CoV-2 NSP8/NSP10 complex actively recruits or relies on host DNA sensors like IFI16 for its function, expanding the known interactome of viral replication complexes.
3. Complexity of Host Defense: The findings underscore that host factors can have dual or context-dependent roles; a protein known for antiviral defense (IFI16) can be co-opted by the virus to facilitate its own replication, independent of its canonical signaling pathways.