Recombinant Human Interferon alpha-inducible protein 27, mitochondrial (IFI27)

What is IFI27 and where is it encoded in the human genome?

IFI27 (interferon alpha-inducible protein 27) is an interferon-stimulated gene encoded on chromosome 14 at position q32.12 . It functions as a regulatory component in innate immune responses, particularly in response to viral infections. The protein is primarily localized in the cytoplasm of cells, as demonstrated by immunofluorescence analysis in bladder cancer cells . IFI27 contains specific amino acid sequences (residues 60-65, 68, 69, and 82-86) that are predicted to bind RNA according to bioinformatic predictions using RNABindRplus .

Experimental approaches to study IFI27 structure typically involve:

• Protein expression and purification systems

• Immunofluorescence microscopy for localization studies

• Bioinformatic prediction tools for structural analysis

• Western blotting for protein detection and quantification

How is IFI27 expression regulated in different cell types?

IFI27 expression is primarily induced by type I interferons in most, if not all, IFN-responsive cells . Studies have demonstrated that:

• In human A549 cells, IFI27 mRNA levels increase by 4.5-fold after poly(I:C) transfection and 3.7-fold after recombinant IFN-α treatment

• Protein levels correspondingly increase by 5.6-fold and 4.3-fold, respectively

• IFI27 expression is highly impaired in cells deficient for IFNAR1 (a subunit of the type I IFN receptor)

• During viral infections, IFI27 expression is dramatically upregulated:

  • 60-fold at 24 hours post-infection (hpi) and 55-fold at 48 hpi during IAV infection

  • 3-fold at 24 hpi and 40-fold at 48 hpi during SARS-CoV-2 infection

In bladder cancer, IFI27 exhibits low expression levels in both cancer tissues and cell lines compared to normal tissues .

What experimental models are most appropriate for studying IFI27 function?

Based on published research, the following experimental models have proven effective for studying IFI27:

In vitro models:

• Human A549 cells (lung adenocarcinoma cell line): Widely used for viral infection studies with IAV

• A549-hACE-2 cells: Modified to express human ACE-2 receptor, making them susceptible to SARS-CoV-2 infection

• Human 293T cells: Used for transient transfection experiments with IFI27 expression vectors

• Bladder cancer cell lines (T24, UM-UC-3, 5637, J82): Used to study IFI27's role in cancer progression

In vivo models:

• Recombinant IAV expressing human genes: A valid strategy to study ISG responses in vivo

• Footpad-popliteal lymph node mouse model: Used to study the effect of IFI27 on tumor lymphatic metastasis

Genetic manipulation approaches:

• CRISPR-Cas9 knockout: Using sgRNAs (e.g., 5′-GTGCCATGGGCTTCACTGCGG-3′) cloned into pX330 plasmid

• siRNA knockdown: Two different siRNAs specific for IFI27 have been successfully used

• Overexpression systems: pCAGGS plasmid expressing IFI27 fused to an HA tag

What is the dual role of IFI27 in innate immune response regulation?

IFI27 exhibits a complex, context-dependent role in immune regulation:

Negative regulation of antiviral responses:
IFI27 acts as a negative regulator of innate immune responses triggered by cytoplasmic RNA recognition and binding. This is evidenced by:

• In IFI27 knockout (KO) A549 cells, expression of interferon-stimulated genes (IFIT2, IFNL1, CXCL10) is significantly upregulated compared to wild-type cells after poly(I:C) transfection

• Similar results were observed in siRNA IFI27-knocked-down A549 cells

• In 293T cells transiently expressing IFI27, IFNL1 and CXCL10 expression was attenuated by 9-fold and 2,750-fold, respectively, after Sendai virus infection

• Conversely, 293T cells knocked-down for IFI27 showed much higher induction of IFNL1 and CXCL10 (approximately 15-fold and 6-fold, respectively) than control cells

Positive effect on viral replication:
IFI27 appears to promote replication of certain viruses:

• IFI27 KO A549 cells showed a 9-fold decrease in IAV titers at 24 hpi compared to wild-type cells

• Silencing IFI27 using siRNAs resulted in an 8-fold decrease in viral titers at 24 and 48 hpi compared to control cells

This dual role suggests IFI27 might help viruses evade host immune responses by dampening interferon responses.

What molecular mechanisms explain IFI27's RNA binding capabilities?

IFI27 has demonstrated ability to bind RNA, particularly double-stranded RNA (dsRNA):

RNA binding mechanism:

• Bioinformatic predictions using RNABindRplus identified 13 amino acids in IFI27 that likely bind RNA, including residues 60-65, 68, 69, and 82-86

• Experimental validation showed:

  • IFI27 binds to poly(I:C)-conjugated agarose beads but not poly(C)-conjugated beads

  • IFI27 binds to streptavidin-conjugated agarose beads when cells are transfected with biotinylated poly(I:C)

Methodological approach to study RNA binding:

• Transfect cells with expression plasmids (pCAGGS-IFI27-HA)

• Expose cellular lysates to agarose beads conjugated to poly(I:C) or poly(C) as control

• Analyze binding through Western blot detection

• Alternatively, transfect cells with biotinylated poly(I:C), bind extracts to streptavidin-conjugated agarose beads, and detect IFI27 by Western blot

This RNA binding capability likely explains how IFI27 negatively modulates innate immune responses, potentially by sequestering viral RNAs and preventing their recognition by pattern recognition receptors.

How does IFI27 influence cancer progression and immunotherapy response?

IFI27 demonstrates significant effects in cancer contexts, particularly in bladder cancer:

Anti-tumor effects:

• IFI27 is predominantly expressed in the cytoplasm of bladder cancer cells but exhibits low expression levels in bladder cancer tissues and cell lines

• Low IFI27 expression correlates with poor prognosis in bladder cancer patients

• IFI27 overexpression inhibits bladder cancer proliferation, migration, epithelial-mesenchymal transition, and lymph node metastasis

Immunotherapy enhancement:

• PD-1 antibody immunotherapy upregulates IFI27 while downregulating FOXP3 (a key transcription factor for regulatory T cells)

• IFI27 inhibits bladder cancer progression by:

  • Suppressing regulatory T cell infiltration

  • Enhancing anti-tumor immune responses

These findings position IFI27 as a potential molecular marker for improving immunotherapy efficacy in bladder cancer.

Experimental approaches to study IFI27 in cancer:

• Colony formation assays with IFI27-overexpressing or IFI27-knockdown cells

• Footpad-popliteal lymph node model in mice to assess lymphatic metastasis

• Bioluminescence imaging to track tumor progression

• Flow cytometric analysis to evaluate immune cell infiltration

• Combination with PD-1 antibody immunotherapy to assess synergistic effects

What contradictory findings exist in IFI27 research and how can they be reconciled?

Several contradictions or paradoxes exist in the current understanding of IFI27 function:

1. Pro-viral vs. Anti-viral activity:

• IFI27 is an interferon-stimulated gene, typically associated with antiviral activity

• Yet, IFI27 knockdown decreases viral titers for IAV and SARS-CoV-2, suggesting it actually promotes viral replication

• This paradox may be explained by IFI27's role in attenuating excessive interferon responses, creating a more favorable environment for viral replication

2. ISG induction vs. ISG suppression:

3. Cancer context variability:

Reconciliation approaches:

• Temporal analysis: Examine IFI27 effects at different time points post-infection or treatment

• Context-specific studies: Investigate IFI27 function in different cell types and disease models

• Mechanistic investigations: Determine if different protein interactions occur in different contexts

• Dose-dependent analysis: Assess if IFI27 concentration affects its functional outcomes

How can IFI27 be used as a biomarker in viral infections and other diseases?

IFI27 has significant potential as a biomarker in several disease contexts:

COVID-19 prognosis:

• IFI27 transcription serves as an early predictor for COVID-19 outcomes

• Its expression is significantly upregulated in SARS-CoV-2 infected patients' blood cells and respiratory swabs

• Upregulated by 3-fold at 24 hpi and 40-fold at 48 hpi during SARS-CoV-2 infection in vitro

Influenza detection:

• IFI27 expression is upregulated after IAV infection in patient blood cells

• Dramatic upregulation (60-fold at 24 hpi and 55-fold at 48 hpi) during IAV infection in cell culture

Cancer prognosis:

• Low IFI27 expression correlates with poor prognosis in bladder cancer patients

• Could potentially serve as a predictive marker for immunotherapy response

Methodological approaches for biomarker development:

• RT-qPCR analysis of IFI27 mRNA expression in patient samples

• Western blot quantification of IFI27 protein levels

• Correlation analysis between IFI27 expression and clinical outcomes

• Development of standardized cutoff values for prognostic classification

• Combination with other biomarkers to improve predictive accuracy

What are the most effective methods for manipulating IFI27 expression in experimental settings?

Based on published research, several effective methods exist for manipulating IFI27 expression:

IFI27 Knockout via CRISPR-Cas9:

• Select appropriate sgRNA sequences (e.g., 5′-GTGCCATGGGCTTCACTGCGG-3′)

• Clone complementary cDNAs into pX330 plasmid (which expresses guides under U6 promoter and encodes CAS9)

• Anneal and phosphorylate paired oligonucleotides using T4 polynucleotide kinase

• Insert between BbsI restriction sites in the plasmid vector

• Transfect target cells and select with puromycin

• Validate knockout through Western blot and RT-qPCR

siRNA-mediated knockdown:

• Design multiple siRNAs targeting different regions of IFI27 mRNA

• Transfect cells using standard lipid-based transfection protocols

• Validate knockdown efficiency (>90% reduction at mRNA level has been achieved)

• Perform experiments 24-48 hours post-transfection for optimal knockdown

Overexpression systems:

• Use pCAGGS plasmid encoding IFI27 fused to an HA tag for detection

• Transfect using standard protocols (lipofection works well in 293T cells)

• Validate expression through Western blot analysis

• Optimal expression typically occurs 24-48 hours post-transfection

Induction of endogenous IFI27:

• Treat cells with recombinant IFN-α (typically induces 3.7-fold increase in mRNA and 4.3-fold increase in protein)

• Transfect cells with poly(I:C) (induces 4.5-fold increase in mRNA and 5.6-fold increase in protein)

• Infect cells with RNA viruses like IAV or SARS-CoV-2 (induces up to 60-fold increase in mRNA)

What are the critical parameters for accurately measuring IFI27 RNA binding activity?

The following protocol outlines critical parameters for accurately measuring IFI27 RNA binding:

Protocol 1: Poly(I:C)-agarose binding assay

• Transfect cells with pCAGGS-IFI27-HA (include appropriate controls like GFP and known RNA-binding proteins like PRKRA)

• Prepare cellular lysates in appropriate binding buffer

• Expose lysates to agarose beads conjugated to poly(I:C) as dsRNA analog and poly(C) as negative control

• Critical parameters:

  • Binding buffer composition (salt concentration affects specificity)

  • Washing conditions (stringency affects signal-to-noise ratio)

  • Bead type and quality

  • Incubation time and temperature

• Detect bound proteins by Western blot using anti-HA antibody

Protocol 2: Biotinylated poly(I:C) pulldown

• Transfect cells with IFI27 expression construct

• Transfect cells with biotinylated or non-biotinylated poly(I:C)

• Prepare cellular extracts

• Bind to streptavidin-conjugated agarose beads

• Critical parameters:

  • Biotin concentration and coupling efficiency

  • Cell lysis conditions to preserve protein-RNA interactions

  • Blocking conditions to prevent non-specific binding

  • Elution conditions

• Analyze bound IFI27 by Western blot

Validation approaches:

• Competition assays with unlabeled RNA

• Dose-dependency analysis

• RNA specificity testing (various RNA types)

• Mutational analysis of predicted RNA-binding residues (60-65, 68, 69, and 82-86)

How can researchers effectively study the impact of IFI27 on anti-tumor immunity?

A comprehensive approach to studying IFI27's impact on anti-tumor immunity should include:

In vitro studies:

• Establish cell models with IFI27 overexpression and knockdown in cancer cell lines

• Assess direct effects on cancer cell properties:

  • Proliferation and colony formation assays

  • Migration and invasion assays

  • Epithelial-mesenchymal transition marker analysis by Western blot

• Co-culture with immune cells to evaluate:

  • T cell activation and proliferation

  • Regulatory T cell development and function

  • Cytokine production profiles (particularly IFN-γ, IL-2)

In vivo models:

• Footpad-popliteal lymph node model to evaluate lymphatic metastasis :

  • Inoculate tumor cells into footpad of mice

  • Monitor tumor growth using bioluminescence imaging

  • Assess popliteal lymph node involvement at weeks 2-4

  • Measure volume and weight of dissected lymph nodes

• Tumor immunotherapy response model:

  • Establish subcutaneous tumors with IFI27-modified cancer cells

  • Treat with PD-1 antibody immunotherapy

  • Monitor tumor growth and immune infiltration

  • Analyze FOXP3+ regulatory T cell populations by flow cytometry

Analytical techniques:

• Flow cytometry to quantify:

  • CD4+/CD8+ T cell ratios

  • CD4+FOXP3+ regulatory T cells

  • Tumor-infiltrating lymphocytes

  • Activation markers (CD69, CD25)

• Immunohistochemistry of tumor sections to assess:

  • Immune cell infiltration patterns

  • IFI27 expression in tumor microenvironment

  • FOXP3 expression in infiltrating cells

• Cytokine profiling:

  • Multiplex assays for comprehensive cytokine analysis

  • ELISA for specific cytokines of interest

  • qRT-PCR for cytokine mRNA expression

This multi-dimensional approach will provide comprehensive insights into how IFI27 modulates anti-tumor immunity and enhances immunotherapy response.

What are the most promising therapeutic applications of IFI27 modulation?

Based on current research, several therapeutic applications of IFI27 modulation show promise:

Enhancing immunotherapy response in cancer:

• Upregulating IFI27 expression could potentially enhance response to PD-1 antibody immunotherapy in bladder cancer and possibly other cancers

• Combined therapy approaches targeting IFI27 and immune checkpoint inhibitors could provide synergistic effects

• Delivery mechanisms for IFI27 to tumor sites could include nanoparticle-based approaches or viral vectors

Antiviral therapy development:

• Since IFI27 knockdown decreases viral titers for IAV and SARS-CoV-2, inhibiting IFI27 could represent a novel antiviral strategy

• Small molecule inhibitors targeting IFI27's RNA-binding domains could potentially disrupt its proviral activities

• Targeting the interaction between IFI27 and viral components could provide virus-specific approaches

Modulating excessive inflammatory responses:

• IFI27's role in counteracting innate immune responses suggests potential applications in autoimmune disorders or hyperinflammation scenarios

• Controlled upregulation of IFI27 might help resolve excessive inflammatory states

• Therapeutic window determination would be critical, as complete suppression might compromise antiviral immunity

Biomarker development:

• Development of IFI27-based diagnostic tests for early detection of viral infections, particularly respiratory viruses

• Prognostic tests for predicting COVID-19 severity or immunotherapy responsiveness

• Companion diagnostics to guide treatment decisions in cancer and infectious diseases

Future research should focus on the development of specific modulators of IFI27 activity and rigorous clinical testing to establish efficacy and safety profiles.

What are the current limitations in IFI27 research and how might they be addressed?

Several significant limitations exist in current IFI27 research:

Mechanistic understanding gaps:

• The precise mechanism by which IFI27 binds RNA and counteracts innate immune responses remains incompletely understood

• Solution: Structural biology approaches including crystallography or cryo-EM of IFI27-RNA complexes would provide valuable insights

Tissue and context specificity:

• Most research has focused on respiratory epithelial cells or cancer cells, with limited understanding of IFI27 function in other tissues

• Solution: Systematic analysis across diverse cell types and tissue contexts using single-cell RNA-seq and spatial transcriptomics

Translational challenges:

• Bridging the gap between in vitro findings and clinical applications remains challenging

• Solution: Development of better in vivo models, including humanized mice and organoid systems

Technical limitations:

• Lack of high-quality, specific antibodies for IFI27 detection in various applications

• Solution: Development and validation of monoclonal antibodies with verified specificity

Regulatory network complexity:

• IFI27 functions within complex immunoregulatory networks that are incompletely mapped

• Solution: Systems biology approaches including network analysis and mathematical modeling

Species differences:

• Differences between human IFI27 and its murine orthologs complicate translation of animal studies

• Solution: Comparative studies and humanized mouse models expressing human IFI27

Therapeutic development challenges:

• Targeting an immunomodulatory protein like IFI27 risks unintended consequences

• Solution: Tissue-specific or context-dependent delivery systems and rigorous safety testing