Recombinant Human Interferon-induced transmembrane protein 2 (IFITM2)

What is the primary function of IFITM2 in innate immunity?

IFITM2 is an interferon-induced transmembrane protein that functions as a critical component of the innate immune response against viral infections. Its primary mechanism involves inhibiting viral entry by preventing fusion of viral membranes with endosomal membranes, thus blocking the release of viral contents into the host cell cytoplasm. While IFITM2 permits endocytosis of viral particles, it effectively blocks the subsequent fusion events required for productive infection . Recent studies have revealed that IFITM2 also enhances type I interferon (IFN) signaling by interacting with microbial RNA sensing machinery components, particularly MDA5 (melanoma differentiation-associated protein 5), establishing a positive feed-forward loop that amplifies antiviral responses .

Against which viruses has IFITM2 demonstrated restrictive activity?

IFITM2 exhibits broad-spectrum antiviral activity against multiple clinically significant viruses. Experimental evidence has confirmed its effectiveness against:

• Influenza A virus

• SARS coronaviruses (SARS-CoV and SARS-CoV-2)

• Marburg virus (MARV)

• Ebola virus (EBOV)

• Dengue virus (DNV)

• West Nile virus (WNV)

• Human immunodeficiency virus type 1 (HIV-1)

• Hepatitis C virus (HCV)

• Vesicular stomatitis virus (VSV)

Specifically, IFITM2 can inhibit viral entry mediated by various viral glycoproteins, including influenza virus hemagglutinin protein, MARV and EBOV GP1,2, SARS-CoV and SARS-CoV-2 spike proteins, and VSV G protein .

How does IFITM2 differ functionally from other IFITM family members?

IFITM2 demonstrates distinct functional characteristics compared to other IFITM family members. While IFITM2 and IFITM3 often exhibit similar restriction profiles, IFITM1 frequently shows different patterns of viral inhibition. For example, in studies with Rift Valley Fever Virus, IFITM2 and IFITM3 demonstrated restrictive activity, while IFITM1 did not . Similarly, with SARS-CoV-2, IFITM2 and IFITM3 inhibited spike-pseudotyped vesicular stomatitis virus infection, whereas IFITM1 showed no restrictive effect . In HCV infection, IFITM2 and IFITM3 display coordinated activity by specifically inhibiting late stages of viral entry, trapping virions in the endosomal pathway and targeting them for lysosomal degradation, which may complement the distinct antiviral mechanisms of IFITM1 .

What are the optimal expression systems for studying recombinant human IFITM2?

For recombinant IFITM2 production, Escherichia coli expression systems have been successfully employed to generate human full-length IFITM2 protein with >90% purity suitable for various applications including SDS-PAGE, ELISA, Western blotting, and mass spectrometry . For cellular studies examining IFITM2 function, researchers typically use either:

• Overexpression systems: Transfection of human cell lines (HEK293, Calu-3, etc.) with plasmids encoding Myc- or FLAG-tagged IFITM2 allows for controlled expression and facilitates interaction studies through co-immunoprecipitation experiments .

• RNA interference approaches: siRNA-mediated knockdown of endogenous IFITM2 (using ON-TARGET plus siRNA) allows for loss-of-function studies to determine the necessity of IFITM2 in various antiviral contexts .

When designing experiments, it's important to include appropriate controls, such as empty vector controls for overexpression studies or non-targeting siRNA controls for knockdown experiments, to properly attribute observed effects to IFITM2 specifically.

What methods are effective for studying IFITM2's interactions with other proteins?

To investigate IFITM2's protein interactions, researchers have successfully employed several complementary approaches:

• Co-immunoprecipitation (Co-IP): This technique has effectively demonstrated IFITM2's interaction with MDA5. For optimal results, co-transfect cells with differentially tagged constructs (e.g., Myc-IFITM2 and FLAG-MDA5), then immunoprecipitate with antibodies against one tag and probe for the interaction partner using antibodies against the other tag in Western blots .

• Protein domain mapping: To identify functional domains involved in protein-protein interactions, researchers have utilized truncated variants or site-directed mutagenesis of IFITM2. Studies have shown that the N-terminal domain of IFITM2 plays a critical role in its antiviral activity and ability to activate IFN-β signaling .

• Proximity-based labeling: While not explicitly mentioned in the search results, techniques such as BioID or APEX2 proximity labeling could be valuable for identifying novel interaction partners of IFITM2 within intact cells.

How can researchers effectively measure IFITM2's impact on viral infection?

To quantitatively assess IFITM2's effects on viral infection, researchers employ multiple complementary approaches:

• Viral RNA quantification: RT-qPCR assays can measure viral genomic copies in cells with manipulated IFITM2 expression. This approach was effectively used to demonstrate that IFITM2 restricts EMCV infection in HEK293 cells dependent on IFN-β secretion .

• Infectious virus quantification: Plaque assays or TCID50 (tissue culture infectious dose) assays can determine infectious viral titers in culture supernatants. These methods revealed that IFITM2 knockdown reduced SARS-CoV-2 variant of concern (VOC) production by 4-6 orders of magnitude in Calu-3 cells .

• Viral protein expression: Western blotting or flow cytometry can measure viral protein levels as indicators of infection progression.

• Pseudovirus systems: For highly pathogenic viruses, pseudotyped viruses (e.g., VSV pseudotyped with SARS-CoV-2 spike) provide a safer alternative for studying viral entry mechanisms .

How does IFITM2 mechanistically inhibit viral entry?

IFITM2 employs several mechanisms to restrict viral entry:

• Endosomal membrane modification: IFITM2 alters the fluidity and curvature of endosomal membranes, making them resistant to fusion with viral membranes. This prevents the release of viral genetic material into the cytoplasm despite successful endocytosis of viral particles .

• Viral trafficking disruption: In hepatocytes, IFITM2 works coordinately with other IFITM proteins to trap endocytosed HCV virions in the endosomal pathway and redirect them for lysosomal degradation, effectively preventing productive infection .

• Inhibition of fusion protein function: IFITM2 can specifically inhibit the function of viral fusion proteins, including influenza hemagglutinin, SARS-CoV-2 spike protein, and Ebola GP1,2, preventing the conformational changes necessary for membrane fusion .

This multifaceted approach to viral restriction makes IFITM2 an effective barrier against a broad spectrum of enveloped viruses that require endosomal entry pathways.

What is the significance of IFITM2 in SARS-CoV-2 variants of concern (VOCs) infection?

IFITM2 plays a critical and somewhat paradoxical role in SARS-CoV-2 VOC infections:

• Proviral role: Contrary to its typically antiviral function, IFITM2 appears to be hijacked by SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, and Omicron) as a cofactor for efficient infection. Depletion of endogenous IFITM2 in human lung cells (Calu-3) almost completely prevented productive infection of these variants .

• Magnitude of dependency: The dependency on IFITM2 is remarkably strong, with siRNA-mediated knockdown reducing infectious virus production by 4-6 orders of magnitude for most variants. This reduction brought viral titers from over 10 million infectious particles/mL to near or below detection limits (≤100 infectious particles/mL) .

• Therapeutic potential: An antibody targeting the N-terminus of IFITM2 demonstrated inhibitory effects against SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells, suggesting potential therapeutic applications .

This unusual dependency of SARS-CoV-2 VOCs on IFITM2 represents a significant finding with implications for understanding viral transmission, pathogenicity, and potential therapeutic interventions.

How do IFITM2 expression levels and localization influence its antiviral activity?

The antiviral efficacy of IFITM2 is significantly influenced by its expression levels and subcellular localization:

• IFN-inducible expression: As an interferon-stimulated gene, IFITM2 expression increases substantially following type I IFN stimulation, creating a positive feedback loop that amplifies antiviral responses .

• Subcellular localization: While not explicitly detailed in the search results, IFITM2 predominantly localizes to late endosomes and lysosomes, positioning it strategically to interfere with viruses that utilize these compartments for entry. Alterations in IFITM2 localization, such as through mutations affecting its endosomal targeting signals, can significantly impact its antiviral activity.

• Expression threshold effects: Studies with SARS-CoV-2 indicate that the relationship between IFITM2 expression levels and viral restriction is not always linear. Very high expression levels were found to inhibit viral entry, while moderate expression levels sometimes facilitated infection, suggesting complex threshold effects .

How does IFITM2 enhance type I interferon signaling pathways?

IFITM2 enhances type I interferon signaling through several key mechanisms:

• MDA5 interaction: IFITM2 physically interacts with melanoma differentiation-associated protein 5 (MDA5), a cytosolic pattern recognition receptor that detects viral RNA. This interaction enhances MDA5-triggered IFN-β activation, as demonstrated through co-immunoprecipitation experiments and functional assays .

• Selective enhancement: IFITM2 specifically promotes MDA5 expression at both mRNA and protein levels during viral infection but does not affect downstream signaling effectors such as MAVS, TBK1, and IRF3. This selectivity suggests a targeted enhancement of the initial viral RNA sensing step in the type I IFN pathway .

• Positive feedback loop: IFITM2 not only responds to type I IFN but also enhances its production, creating a positive feed-forward loop that amplifies antiviral responses. This bidirectional relationship positions IFITM2 as both an effector and an inducer of the interferon response .

This enhancement of type I IFN signaling through MDA5 represents a distinct mechanism by which IFITM2 contributes to antiviral immunity beyond its direct inhibition of viral entry.

What is the relationship between IFITM2 and MAVS in antiviral responses?

The relationship between IFITM2 and mitochondrial antiviral signaling protein (MAVS) reveals important insights into IFITM2's role in antiviral signaling:

• MAVS dependency: Experimental evidence demonstrates that IFITM2's ability to restrict EMCV replication is dependent on MAVS expression. When MAVS was knocked down via RNAi, IFITM2 overexpression failed to effectively restrict EMCV, with a significant increase in viral genomic copies observed .

• Indirect interaction: Unlike its direct interaction with MDA5, IFITM2 does not appear to directly affect MAVS expression levels or activation. Instead, IFITM2 enhances signaling upstream of MAVS by promoting MDA5 expression and activation .

• Sequential signaling: The data suggests a sequential model where IFITM2 enhances MDA5 activation, which then signals through MAVS to ultimately induce IFN-β production, completing the signaling pathway necessary for effective antiviral responses .

This MAVS-dependent restriction highlights the importance of intact RIG-I-like receptor signaling pathways for IFITM2's full antiviral functionality.

How do researchers explain the seemingly contradictory pro- and anti-viral activities of IFITM2 in SARS-CoV-2 infection?

The paradoxical roles of IFITM2 in SARS-CoV-2 infection represent a significant area of investigation, with several proposed explanations:

• Expression level-dependent effects: The effect of IFITM2 on SARS-CoV-2 appears to be concentration-dependent. At very high expression levels (as in overexpression systems), IFITM2 may restrict viral entry, while at physiological or moderate levels, it may serve as a cofactor for efficient infection .

• Viral variant-specific interactions: Different SARS-CoV-2 variants may interact distinctly with IFITM2. Studies have shown that variants of concern (VOCs) such as Alpha, Beta, Gamma, Delta, and Omicron have evolved to efficiently hijack IFITM2 for productive infection .

• Cell type-specific effects: The proviral or antiviral effects of IFITM2 may be influenced by the cellular context, including the expression of other cofactors or restriction factors that modulate IFITM2 function.

• Subcellular localization determinants: The conflicting results regarding IFITM2's role in SARS-CoV-2 infection may relate to its distribution within cellular compartments, which could differ between experimental systems or be altered by the virus itself .

These contradictory findings underscore the complex and context-dependent nature of virus-host interactions and highlight the importance of comprehensive experimental approaches when studying restriction factors.

What experimental discrepancies exist in the literature regarding IFITM2's effect on SARS-CoV-2?

Several notable experimental discrepancies have been reported regarding IFITM2's impact on SARS-CoV-2 infection:

• Pseudovirus vs. authentic virus: Studies using SARS-CoV-2 Spike-pseudotyped VSV found that IFITM2 overexpression inhibited infection, while another study reported that IFITM3 (closely related to IFITM2) had no inhibitory effect on Spike-containing pseudovirus infection .

• Cell-to-cell fusion assays: Contradictory results have been reported regarding IFITM2's effect on SARS-CoV-2 Spike-mediated cell fusion. One study observed inhibition of Spike-induced cell-to-cell fusion by IFITMs, while another did not detect inhibition when IFITM2 or IFITM3 was expressed in target cells .

• Knockout vs. knockdown approaches: Different methodological approaches (gene knockout versus siRNA-mediated knockdown) have sometimes yielded different results, potentially due to differences in the completeness of protein depletion or compensatory mechanisms.

These discrepancies highlight the importance of experimental design, model system selection, and validation across multiple methodologies when investigating complex virus-host interactions.

How might structural modifications of IFITM2 affect its antiviral properties?

Advanced research into IFITM2 structure-function relationships reveals several promising avenues for investigation:

• N-terminal domain modifications: The N-terminal domain of IFITM2 plays a critical role in its antiviral activity and ability to activate IFN-β. Targeted modifications of this domain could potentially enhance or alter IFITM2's antiviral spectrum or potency .

• Post-translational modifications: While not explicitly mentioned in the search results, IFITM proteins are known to undergo various post-translational modifications including palmitoylation, ubiquitination, and phosphorylation. Engineering IFITM2 variants with altered modification sites could yield insights into regulatory mechanisms and potentially create versions with enhanced antiviral activity.

• Chimeric IFITM proteins: Creating chimeric proteins combining domains from different IFITM family members (IFITM1, IFITM2, and IFITM3) could help identify the specific structural determinants of virus specificity and restriction potency.

• Antibody-mediated targeting: The finding that an antibody targeting the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication suggests potential for developing therapeutic antibodies or antibody-inspired small molecules that could modulate IFITM2 function in disease contexts .

What are the implications of IFITM2 dependency for developing broad-spectrum antiviral strategies?

The critical role of IFITM2 in viral infections suggests several strategic approaches for antiviral development:

• Dual-targeting approaches: For viruses like SARS-CoV-2 that depend on IFITM2 for efficient replication, developing agents that simultaneously target the virus and modulate IFITM2 interaction could provide synergistic antiviral effects and reduce the potential for resistance development .

• Variant-agnostic interventions: Since diverse SARS-CoV-2 variants (Alpha through Omicron) all show strong dependency on IFITM2, interventions targeting this interaction could potentially remain effective against future emerging variants .

• Tissue-specific considerations: Given IFITM2's importance in lung cells (demonstrated in Calu-3 cells and iPSC-derived alveolar epithelial type II cells), delivery systems that can efficiently target pulmonary tissue would be particularly valuable for respiratory viruses that depend on IFITM2 .

• Combination with IFN-based therapies: Understanding IFITM2's role in both enhancing and being enhanced by type I IFN signaling suggests potential for combination approaches that leverage both direct antiviral activity and immune modulation .

How can researchers reconcile in vitro findings about IFITM2 with in vivo pathogenesis?

Translating in vitro findings about IFITM2 to in vivo contexts presents several methodological challenges and opportunities:

• Animal models with humanized IFITM2: While the search results do not detail animal models for IFITM2 specifically, studies with IFITM3-knockout mice have demonstrated increased susceptibility to influenza virus . Similar approaches with IFITM2-knockout or humanized IFITM2 models could help validate in vitro findings in physiologically relevant systems.

• Human genetic association studies: Identifying and characterizing naturally occurring IFITM2 genetic variants in human populations and their association with viral disease outcomes could provide valuable insights into IFITM2's in vivo significance.

• Ex vivo systems: Using primary human tissues or organoids with manipulated IFITM2 expression could bridge the gap between cell line studies and in vivo relevance.

• Multi-omics approaches: Integrating transcriptomic, proteomic, and metabolomic analyses of IFITM2-modulated systems could help identify in vivo relevant pathways and potential compensatory mechanisms that might not be apparent in simplified in vitro models.

By addressing these translational questions, researchers can develop a more comprehensive understanding of IFITM2's role in viral pathogenesis and its potential as a therapeutic target.