IFIT3 Human

What is human IFIT3 and what distinguishes it structurally from other IFIT proteins?

Human IFIT3 (also known as ISG60) is an interferon-stimulated gene product characterized by unique helix-turn-helix motifs called tetratricopeptide repeats (TPR). These TPR motifs serve as scaffolds that facilitate protein-protein and protein-RNA interactions, which are crucial for IFIT3's biological functions . Unlike other IFIT family members, human IFIT3 contains a distinctive C-terminal domain (CTD) comprising 87 amino acids that is critical for its interaction with IFIT1 . This structural feature is species-specific and absent in mouse Ifit3, highlighting an important evolutionary divergence in this protein family .

To study IFIT3's structure experimentally, researchers typically employ approaches including:

• Protein crystallography to determine three-dimensional structure

• Circular dichroism spectroscopy to confirm proper protein folding

• Truncation experiments to identify functional domains

How does human IFIT3 differ from mouse Ifit3 in structure and function?

The differences between human IFIT3 and mouse Ifit3 are significant and have important implications for translational research:

• Structural variation: Human IFIT3 possesses an 87-amino acid C-terminal domain that is completely absent in mouse Ifit3 .

• Protein-protein interactions: Human IFIT3 directly binds to human IFIT1 through its C-terminal domain, but mouse Ifit3 does not appreciably bind to either mouse Ifit1 or human IFIT1 . Experimental chimeric constructs (Ifit3-IFIT3 CTD) have demonstrated that adding the human C-terminal domain to mouse Ifit3 confers binding to human IFIT1 .

• Regulatory capabilities: Human IFIT3 can modulate IFIT1 protein stability and RNA binding specificity, a function absent in the mouse ortholog due to the missing C-terminal domain .

These species-specific differences underscore why mouse models may not always accurately represent human IFIT3 functions, necessitating careful experimental design when studying IFIT-mediated immunity across species.

Which IFIT family proteins directly interact with IFIT3 and how can these interactions be experimentally verified?

Among IFIT family members, IFIT1 has been definitively shown to directly interact with IFIT3, while IFIT2 and IFIT5 do not demonstrate strong direct binding to IFIT1 in biochemical assays . These interaction patterns can be verified through multiple complementary approaches:

• Co-precipitation assays: Using MBP-tagged IFIT1 with native IFIT3 demonstrates strong binding between these proteins that is not observed with IFIT2 or IFIT5 .

• Reversible tagging experiments: Switching the MBP tag to IFIT3 while leaving IFIT1 untagged confirms the specificity of the interaction .

• Domain mapping: Truncation experiments with IFIT3 fragments (IFIT3 1–403 and IFIT3 403–490) can identify the minimal domains required for interaction .

• Mutational analysis: HyPare computational analysis followed by site-directed mutagenesis of key residues (K426, E439, L445, S451, I453, and F457A) can disrupt the IFIT3-IFIT1 interaction while maintaining proper protein folding .

The IFIT3-IFIT1 interaction appears to occur primarily through "Interface 3" as defined by structural studies, with specific amino acid residues mediating this protein-protein contact .

How does IFIT3 regulate IFIT1 function and stability?

IFIT3 exhibits dual regulatory functions in relation to IFIT1:

• Protein stabilization: IFIT3 binding extends the half-life of IFIT1, effectively increasing its steady-state levels in cells . This stabilization function may explain why IFIT1 levels are often correlated with IFIT3 expression in various experimental contexts.

• Allosteric regulation: IFIT3 binding allosterically regulates the IFIT1 RNA-binding channel, enhancing the specificity of recognition for cap 0 (m⁷GpppN) RNA while not affecting recognition of cap 1 (m⁷GpppNm) or 5′-ppp RNA . This interaction fine-tunes IFIT1's ability to distinguish between host and viral RNA structures.

These regulatory functions are mechanistically important for the restriction of viruses lacking 2′-O methylation in their RNA cap structures, demonstrating how IFIT proteins cooperate to provide specificity in antiviral responses .

What mechanisms underlie IFIT3's antiviral activity against RNA versus DNA viruses?

IFIT3 employs distinct mechanisms to restrict RNA and DNA viruses:

For RNA viruses:

• IFIT3 works cooperatively with IFIT1, which directly binds to viral cap 0 RNA structures lacking 2′-O methylation .

• This recognition of non-self RNA prevents viral translation and replication .

For DNA viruses (specifically adenovirus):

• IFIT3 represses immediate early gene expression (particularly E1A) without affecting viral genome entry into the nucleus .

• IFIT3 expression triggers phosphorylation of TBK1, IRF3, and STAT1, leading to increased expression of IFNβ and other ISGs .

• This antiviral activity against adenovirus requires both IFIT1 and IFIT2 partner proteins .

• The restriction occurs independently of viral pathogen-associated molecular patterns (PAMPs), establishing an antiviral state even in the absence of viral sensing .

These dual mechanisms highlight IFIT3's versatility in countering diverse virus families through both direct and indirect pathways.

How does IFIT3 influence interferon signaling pathways?

IFIT3 serves as a potent activator of interferon signaling through multiple pathways:

• TBK1-IRF3 axis: IFIT3 stimulates IFN production by bridging MAVS and TBK1, leading to IRF3 phosphorylation and IFNβ expression . This interaction facilitates TBK1 activation, which in turn activates IRF3 and NF-κB to induce IFNβ gene expression.

• STING pathway: IFIT3 activates the STING pathway, which synergizes with the MAVS pathway to enhance interferon production . Depletion of STING phenocopies the effect of MAVS or TBK1 depletion in blocking IFIT3-induced IFN signaling .

• Amplification loop: IFIT3 expression leads to STAT1 phosphorylation and increased expression of multiple ISGs including IFIT1, ISG15, OAS3, and MX1 . This creates a positive feedback loop that amplifies the antiviral response.

Experimental evidence shows that IFIT3-expressing cells have elevated levels of TBK1 and IRF3 phosphorylation compared to control cells, even in the absence of viral infection, indicating that IFIT3 can activate these pathways independently of viral PAMPs .

What experimental systems are most suitable for studying IFIT3 functions?

Several experimental systems have proven effective for studying IFIT3 functions:

• Cell culture models:

  • Human diploid fibroblasts immortalized by human telomerase (HDF-TERT cells) provide a reliable system for studying IFIT3's effects on viral replication .

  • A549 cells are also suitable and can both produce and respond to IFNs .

  • When selecting cell lines, researchers should verify their ability to produce and respond to IFNs to ensure relevant IFIT3 functions can be observed.

• Protein interaction studies:

  • In vitro co-precipitation assays using MBP-tagged proteins have successfully demonstrated IFIT3 interactions with other IFIT family members .

  • Structural studies combining crystallography with biochemical experiments can reveal binding interfaces and regulatory mechanisms .

• Viral restriction models:

  • Adenovirus infection at low MOI with quantification of viral DNA replication at 48 hours post-infection provides a reliable readout of IFIT3's antiviral activity .

  • Measurements of viral gene expression (both mRNA and protein levels) offer mechanistic insights into where in the viral life cycle IFIT3 exerts its effects .

• Interferon signaling analysis:

  • Western blotting for phosphorylated signaling proteins (TBK1, IRF3, STAT1) reveals IFIT3's effects on signaling cascades .

  • RT-qPCR for IFNβ and ISGs quantifies the downstream effects of IFIT3 expression .

What genetic manipulation strategies are effective for IFIT3 functional studies?

Several genetic approaches have proven effective for manipulating IFIT3 in experimental settings:

How does IFIT3 coordinate with MAVS and STING pathways for optimal antiviral responses?

IFIT3 operates at the intersection of the MAVS and STING pathways to orchestrate comprehensive antiviral responses:

• MAVS pathway activation:

  • IFIT3 bridges MAVS and TBK1, facilitating TBK1 activation .

  • This interaction occurs on mitochondria, where MAVS is localized .

  • MAVS depletion in IFIT3-expressing cells blocks IFN signaling and reverses adenovirus replication restriction .

• STING pathway involvement:

  • IFIT3 activation of the STING pathway appears to synergize with MAVS-mediated signaling .

  • STING depletion phenocopies the effect of MAVS or TBK1 depletion in blocking IFIT3-induced interferon signaling .

  • This suggests cross-talk between the MAVS and STING pathways coordinated by IFIT3.

• PAMP-independent activation:

  • A particularly noteworthy feature of IFIT3's activity is its ability to activate both pathways independently of viral pathogen-associated molecular patterns (PAMPs) .

  • This constitutes a novel mechanism for establishing an antiviral state even before viral sensing occurs.

The coordinated activation of both MAVS and STING pathways provides a more robust antiviral response than either pathway alone, explaining IFIT3's potent antiviral effects even against DNA viruses like adenovirus .

What is the significance of IFIT3's species-specific variation for translational research?

The significant species-specific differences in IFIT3 structure and function have important implications for translational research:

• Structural divergence:

  • The 87-amino acid C-terminal domain present in human IFIT3 but absent in mouse Ifit3 constitutes a major evolutionary difference .

  • This structural variation leads to fundamental differences in protein-protein interactions and regulatory functions .

• Interaction networks:

  • Human IFIT3 binds directly to human IFIT1, but mouse Ifit3 does not bind appreciably to either mouse Ifit1 or human IFIT1 .

  • These different interaction profiles likely result in distinct regulatory networks in humans versus mice.

• Translational challenges:

  • Mouse models of viral infection may not accurately reflect the role of IFIT3 in human antiviral responses due to these fundamental differences .

  • Researchers must exercise caution when extrapolating findings from mouse studies to human IFIT biology.

• Engineering approaches:

  • Chimeric constructs (e.g., mouse Ifit3 with human IFIT3 CTD) can be used to "humanize" mouse systems for more accurate translational studies .

  • Such approaches may help bridge the gap between murine and human experimental systems.

These species-specific variations highlight the importance of using appropriate experimental systems when studying IFIT3 for potential therapeutic applications or as a biomarker in human disease.