IFIT5 Antibody

What is IFIT5 and what role does it play in antiviral immunity?

IFIT5 (Interferon-induced protein with tetratricopeptide repeats 5) is a critical component of the innate immune response with RNA-binding capabilities. IFIT5 functions as a sensor of viral single-stranded RNAs through its ability to specifically bind single-stranded RNA bearing a 5'-triphosphate group (PPP-RNA). This represents a molecular signature that distinguishes between self and non-self mRNAs during viral infection, as viral RNAs often lack 2'-O-methylation of the 5' cap and instead bear a 5'-triphosphate group .

IFIT5 has a remarkably broad and adaptable RNA structure recognition capability, which is important for RNA recognition specificity in antiviral defense. It can bind to:

• Precursor and processed tRNAs

• Poly-U-tailed tRNA fragments

• Single-stranded PPP-RNAs (in a non-sequence-specific manner)

• AT-rich dsDNA

Beyond RNA binding, IFIT5 also positively regulates IKK-NFKB signaling by enhancing the recruitment of IKK to MAP3K7, further contributing to the innate immune response .

What structural features enable IFIT5 to recognize viral RNA?

IFIT5 possesses eight tandem α-helix tetratricopeptide repeats (TPRs) arranged in an atypical TPR Eddy topology, creating a wide cleft with a basic surface generally favorable for RNA interaction. At the center of this cleft is a contrasting acidic pocket that plays a crucial role in RNA discrimination .

Key structural elements include:

• A central cavity with an acidic pocket that discriminates between different RNA 5' end structures

• Side chains that reorient upon RNA binding, including E33 and Y250 which coordinate with the triphosphate group

• A broad RNA-binding cavity that can accommodate both 5'-monophosphate and 5'-triphosphate RNA ends

• An adaptable binding site that can distinguish phosphate-containing 5' ends

Research has shown that mutations in the acidic pocket, particularly the E33A and D334A substitutions, affect the RNA binding specificity of IFIT5, especially in discriminating between cap0 and cap1 RNA structures .

What are the optimal protocols for using IFIT5 antibodies in different experimental applications?

Based on data from multiple antibody sources, the following protocols represent optimized conditions for various applications:

For more specific protocols:

• For IP applications: After preclearing cell lysate with protein A Sepharose, incubate with IFIT5 antibody overnight at 4°C. Precipitate immune complexes with protein A Sepharose for 1 hour at 4°C, then wash with RIPA buffer before analysis .

• For IF applications: After blocking with 3% BSA, incubate with primary IFIT5 antibody overnight at 4°C, followed by fluorescently-labeled secondary antibody incubation for 2 hours at room temperature. Counterstain nuclei with DAPI .

How should researchers design experiments to evaluate IFIT5 expression during viral infection?

To properly evaluate IFIT5 expression during viral infection, researchers should consider a time-course experimental design with appropriate controls. Based on existing research methodologies:

• Time-course design:

  • Expose cells to the virus of interest at different time points (0, 3, 6, 24, and 72 hours post-exposure) to capture the dynamics of IFIT5 expression

  • Include both transcriptional (mRNA) and protein level analyses to detect potential post-transcriptional regulation

• Cell selection:

  • Choose relevant cell types for your viral model; research has used various cell types including red blood cells (RBCs), nucleated RBCs, and cell lines like RTG-2 and HEK293T

  • Consider primary cells for physiological relevance and cell lines for experimental consistency

• Detection methods:

  • For transcriptional analysis: Use real-time RT-qPCR to measure ifit5 gene expression

  • For protein analysis: Use Western blot with validated IFIT5 antibodies

  • For localization studies: Use immunofluorescence to determine IFIT5 cellular distribution during infection

  • For comprehensive analysis: Consider supplementing with co-immunoprecipitation (Co-IP) to identify viral-host protein interactions

• Controls:

  • Include uninfected control cells at each time point

  • Use a virus-specific marker (e.g., viral N protein) to confirm infection

  • Include housekeeping genes/proteins (e.g., GAPDH) as loading controls

Research on rainbow trout RBCs exposed to VHSV showed correlation between highest IFIT5 expression levels at 6 hours post-exposure and decline in viral replication, providing a methodological template for similar studies .

How can researchers validate IFIT5 antibody specificity across different species?

Validating IFIT5 antibody specificity across species requires careful consideration of sequence homology and experimental verification:

• Sequence alignment analysis:

  • Compare IFIT5 protein sequences across target species to identify conserved regions

  • Determine the immunogen sequence of your antibody and assess homology (e.g., human IFIT5 antibody has ~37% sequence identity with mouse and ~39% with rat orthologs)

  • Focus on antibodies raised against highly conserved epitopes for cross-species applications

• Validation experiments:

  • Positive controls: Use recombinant IFIT5 proteins from each species or cell lines with confirmed IFIT5 expression

  • Negative controls: Employ IFIT5 knockout/knockdown samples or tissues known not to express IFIT5

  • Cross-reactivity testing: Test the antibody on samples from multiple species under identical conditions

  • Western blot analysis: Confirm expected molecular weight across species (typically 56-58 kDa for human IFIT5)

• Additional validation approaches:

  • Peptide competition assay: Pre-incubate antibody with the immunizing peptide to confirm signal specificity

  • Multiple antibody approach: Use antibodies targeting different IFIT5 epitopes to confirm results

  • Gene silencing: Validate antibody specificity using ifit5 siRNA or shRNA knockdown experiments

Research examples include successful use of rainbow trout anti-IFIT5 antibodies designed and produced specifically for cross-species research, and commercial antibodies have been validated in species including human, pig, and canine samples .

What are the key considerations for distinguishing between different IFIT family members in immunological assays?

Accurately distinguishing between IFIT family members (particularly IFIT1, IFIT2, IFIT3, and IFIT5) presents a significant challenge due to structural similarities. Researchers should consider:

• Sequence uniqueness:

  • Select antibodies raised against unique, non-conserved regions of IFIT5

  • Avoid antibodies targeting the TPR domains, which are highly conserved across IFIT family members

  • Consult sequence alignment data to identify IFIT5-specific epitopes

• Experimental validation approaches:

  • Western blot discrimination: IFIT5 typically appears at 56-58 kDa, while other IFIT family members have different molecular weights (IFIT1: ~55 kDa, IFIT2: ~54 kDa, IFIT3: ~49 kDa)

  • Co-expression analysis: In co-expression studies of multiple IFIT proteins, use tagged versions (e.g., FLAG-tagged IFIT5) to distinguish between family members

  • Knockdown controls: Use specific siRNA targeting individual IFIT members to validate antibody specificity

• Advanced verification methods:

  • Mass spectrometry validation: Follow immunoprecipitation with mass spectrometry to confirm the identity of the detected protein

  • Immunoprecipitation followed by Western blot: Use one antibody for IP and another targeting a different epitope for Western blot

  • Recombinant protein controls: Include purified recombinant proteins of each IFIT family member as controls

Research examining both IFIT3 and IFIT5 in porcine pulmonary microvascular endothelial cells demonstrates the importance of using specific antibodies that can distinguish between these family members during viral infection studies .

How can IFIT5 antibodies be used to investigate RNA-protein interactions in antiviral immunity?

IFIT5 antibodies can be powerful tools for investigating the complex RNA-protein interactions central to antiviral immunity through several advanced techniques:

• Immunoprecipitation coupled with RNA sequencing:

  • Use IFIT5 antibodies to immunoprecipitate IFIT5-RNA complexes from infected or interferon-stimulated cells

  • Extract and sequence bound RNAs to identify viral and cellular RNA targets

  • Implement TGIRT-seq (thermostable group II intron reverse transcriptase sequencing) for comprehensive profiling of IFIT5-bound cellular RNAs, especially for capturing tRNAs that are difficult to sequence with standard methods

• Proximity ligation assay (PLA):

  • Employ PLA to detect protein-protein colocalization between IFIT5 and viral proteins

  • This technique has successfully demonstrated colocalization between IFIT5 and the glycoprotein G of VHSV in rainbow trout red blood cells

  • PLA provides higher sensitivity than traditional colocalization studies and can detect transient interactions

• Structure-function analysis using mutants:

  • Generate structure-guided mutants (e.g., E33A, D334A, Q41A, T37A, Y250A) to disrupt the RNA-binding cavity

  • Use immunoprecipitation with wild-type and mutant IFIT5 antibodies to compare RNA binding profiles

  • This approach has revealed that the E33A and D334A mutations in the acidic pocket affect RNA binding specificity

• Electrophoretic mobility shift assays (EMSAs):

  • Use purified IFIT5 protein (detected by IFIT5 antibodies) with radiolabeled RNAs containing different 5' end structures (5'-p, 5'-ppp, cap0, cap1)

  • Quantify binding affinities to understand IFIT5's preference for different RNA structures

  • Research has shown that IFIT5 binds to both 5'-p and 5'-ppp RNAs with similar nanomolar affinity

These techniques have been instrumental in establishing IFIT5's role in binding viral RNA structures and discriminating between different cellular and viral RNAs as part of the antiviral response.

What methodologies can be employed to investigate IFIT5's role in different cellular compartments during infection?

To comprehensively investigate IFIT5's role across cellular compartments during infection, researchers can employ multi-faceted approaches:

• Subcellular fractionation coupled with immunoblotting:

  • Separate cells into cytoplasmic, nuclear, membrane, and cytoskeletal fractions

  • Use IFIT5 antibodies to detect the protein distribution across fractions before and during infection

  • Include markers for each compartment (e.g., GAPDH for cytoplasm, histone H3 for nucleus) to validate fractionation quality

  • This approach can reveal translocation of IFIT5 during the course of infection

• High-resolution microscopy techniques:

  • Confocal microscopy: Use fluorescently-labeled IFIT5 antibodies to visualize subcellular localization

  • Super-resolution microscopy: Employ techniques like STORM or PALM for nanoscale localization

  • Live-cell imaging: Use cell-permeable fluorescent tags to track IFIT5 movement during infection

  • Co-localization studies: Combine IFIT5 antibodies with markers for organelles (mitochondria, ER, Golgi) or viral components

• Proximity-based labeling methods:

  • BioID or APEX2: Fuse IFIT5 with a biotin ligase or peroxidase to identify proteins in close proximity across different compartments

  • PLA: Use proximity ligation assays to detect interactions between IFIT5 and compartment-specific proteins

  • These approaches can reveal previously unknown interactions in specific cellular locations

• Functional inhibition studies:

  • Use compartment-specific inhibitors in conjunction with IFIT5 functional assays

  • Employ cell-specific targeting sequences to direct IFIT5 to specific compartments and assess functional consequences

  • Correlate compartmental localization with viral replication inhibition using viral markers

Research in rainbow trout has demonstrated that IFIT5 plays a role in halting VHSV replication inside nucleated red blood cells, suggesting compartment-specific antiviral functions that can be further investigated using these methodologies .

How should researchers address inconsistent IFIT5 antibody staining patterns in different cell types?

Inconsistent IFIT5 antibody staining across cell types is a common challenge that requires systematic troubleshooting:

• Cell type-specific expression patterns:

  • IFIT5 expression varies naturally between cell types and can be induced by interferon treatment

  • Establish baseline expression in each cell type using RT-qPCR before antibody experiments

  • Include positive controls (interferon-treated cells) and negative controls (IFIT5 siRNA-treated cells)

  • Consider that subcellular localization may differ between cell types, affecting staining patterns

• Protocol optimization for each cell type:

  • Fixation method: Test multiple fixation protocols (paraformaldehyde, methanol, or combination) as IFIT5 epitope accessibility may differ

  • Permeabilization: Adjust detergent concentration (Triton X-100, saponin) based on cell membrane composition

  • Blocking conditions: Optimize blocking agent (BSA, serum) concentration to reduce background

  • Antibody concentration: Titrate antibody for each cell type (recommended ranges: 1:20-1:200 for IHC, 1:500-1:1000 for WB)

• Antigen retrieval optimization:

  • For tissue sections or fixed cells, test multiple antigen retrieval methods

  • Compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0) as recommended for IFIT5 antibodies

  • Adjust retrieval time and temperature based on cell type and fixation method

• Technical validation approaches:

  • Use different antibody clones targeting distinct IFIT5 epitopes to confirm staining patterns

  • Perform parallel Western blot analysis to confirm antibody specificity in each cell type

  • Include peptide competition controls to verify specificity of observed signal

  • Consider fluorophore selection to avoid cell-specific autofluorescence interference

Researchers studying IFIT5 in porcine pulmonary microvascular endothelial cells found that specific protocol adjustments were necessary to obtain consistent staining results compared to other cell types, highlighting the need for cell-specific optimization .

What strategies can help differentiate between specific and non-specific signals when studying IFIT5 in tissues with high background?

High background is a persistent challenge when studying IFIT5 in certain tissues. Implement these strategies to improve signal-to-noise ratio:

• Advanced blocking techniques:

  • Sequential blocking: Use protein-free blocking buffer followed by species-specific serum

  • Avidin/biotin blocking: Essential if using biotinylated secondary antibodies

  • Fc receptor blocking: Particularly important in immune tissues with high Fc receptor expression

  • Endogenous peroxidase quenching: Use hydrogen peroxide treatment before antibody incubation if using HRP-based detection systems

• Signal amplification with specificity controls:

  • Tyramide signal amplification: Enhances specific signal while maintaining signal-to-noise ratio

  • Fluorophore selection: Choose fluorophores outside the autofluorescence spectrum of the tissue

  • Multiplexed detection: Co-stain with markers of known IFIT5-expressing cells

  • Comparison detection methods: Compare DAB staining, fluorescence, and chemiluminescence to identify optimal detection method for each tissue

• Technical approaches to reduce background:

  • Extended washing steps: Increase number and duration of washes between antibody incubations

  • Detergent optimization: Adjust Tween-20 or Triton X-100 concentration in wash buffers

  • Temperature control: Perform antibody incubations at 4°C overnight rather than at room temperature

  • Antibody pre-adsorption: Pre-incubate antibody with tissues known not to express IFIT5

• Validation controls:

  • Isotype controls: Use matched isotype antibodies at the same concentration

  • Absorption controls: Pre-incubate antibody with recombinant IFIT5 protein

  • Genetic controls: When possible, use IFIT5 knockout or knockdown tissues

  • Multiple antibody approach: Use antibodies against different IFIT5 epitopes to confirm staining pattern

  • Secondary-only controls: Omit primary antibody to assess secondary antibody background

For tissues with particularly high background, researchers have successfully employed antigen retrieval with TE buffer pH 9.0 for IFIT5 detection in human skin cancer tissue, suggesting this approach may be beneficial for other challenging tissues .

How can researchers investigate the differential roles of IFIT5 in RNA sensing versus effector functions?

IFIT5 exhibits dual functionality as both a sensor of viral RNAs and an effector in antiviral responses. To dissect these functions:

• Structure-function mutational analysis:

  • Generate targeted mutations in distinct IFIT5 domains:

    • RNA-binding pocket mutations (E33A, D334A) to disrupt sensing function

    • TPR domain mutations to potentially affect protein-protein interactions

  • Compare mutants' abilities to bind viral RNA versus their antiviral activities

  • Use co-immunoprecipitation with IFIT5 antibodies to identify protein interaction partners affected by each mutation

• Domain-specific interaction studies:

  • Employ truncated IFIT5 constructs expressing specific domains

  • Use IFIT5 antibodies that recognize distinct domains to immunoprecipitate domain-specific interacting partners

  • Perform RNA-immunoprecipitation assays to map which domains bind which RNA structures

  • Correlate domain-specific interactions with antiviral activities

• Temporal dissection of IFIT5 functions:

  • Design time-course experiments to determine when IFIT5 functions primarily as a sensor versus an effector

  • Use IFIT5 antibodies to track protein localization and interaction partners at different time points post-infection

  • Correlate time-dependent changes with viral replication levels and interferon signaling

  • Research in rainbow trout RBCs showed correlation between peak IFIT5 expression at 6 hours post-exposure and decline in VHSV replication

• Pathway inhibition approaches:

  • Selectively inhibit downstream signaling pathways (e.g., IKK-NFKB) to distinguish direct RNA-binding effects from signaling effects

  • Use chemical modulators of the IFIT5 RNA-binding pocket (as identified in the SuperNatural II database) to specifically disrupt RNA sensing

  • Combine with viral replication assays to determine which function predominantly controls antiviral activity

These approaches have revealed that IFIT5's antiviral function involves both direct interaction with viral components (e.g., binding the glycoprotein G of VHSV) and signaling effects through IKK-NFKB pathway regulation .

What experimental designs best illuminate IFIT5's role in the discrimination between self and non-self RNA?

To investigate IFIT5's critical role in discriminating between self and non-self RNA:

• Comparative RNA-binding assays:

  • Design in vitro binding experiments comparing IFIT5 affinity for:

    • 5'-triphosphate RNAs (viral signature) versus 5'-monophosphate RNAs (typical cellular RNAs)

    • Uncapped RNAs versus cap0 (7-methylguanosine) versus cap1 (additional 2'-O-methylation) RNAs

    • AT-rich dsDNA versus GC-rich sequences

  • Use electrophoretic mobility shift assays (EMSAs) with purified recombinant IFIT5 and radiolabeled RNA substrates

  • Quantify binding affinities to understand preferences for different RNA structures

• Comprehensive IFIT5-bound RNA profiling:

  • Implement TGIRT-seq (thermostable group II intron reverse transcriptase sequencing) for unbiased analysis of IFIT5-bound RNAs

  • Compare RNA profiles between:

    • Uninfected versus virus-infected cells

    • Untreated versus interferon-treated cells

    • Wild-type versus mutant IFIT5 (E33A, D334A) expressing cells

  • Analyze both coding and non-coding RNAs, especially focusing on tRNAs which are known IFIT5 targets

• Single-molecule biophysical approaches:

  • Use fluorescence resonance energy transfer (FRET) to measure real-time binding kinetics

  • Implement surface plasmon resonance (SPR) or bio-layer interferometry (BLI) to determine association/dissociation constants

  • These techniques can reveal subtle differences in how IFIT5 interacts with different RNA structures

• Cellular RNA fate tracking:

  • Design reporter RNAs with different 5' structures (5'-ppp, 5'-p, cap0, cap1)

  • Track their stability, localization, and translation efficiency in cells with normal versus altered IFIT5 levels

  • Use IFIT5 antibodies for co-localization studies with these reporter RNAs

  • Correlate with antiviral responses to determine functional consequences of discrimination

Research has shown that IFIT5 binds to both 5'-p and 5'-ppp RNAs with similar nanomolar affinity but strongly discriminates against cap1 RNAs. This discrimination relies partly on an acidic pocket in the central cavity of IFIT5, with E33A and D334A mutations affecting this discriminatory capacity .

What are the recommended approaches for studying IFIT5 in different viral infection models?

Studying IFIT5 across diverse viral infection models requires tailored approaches depending on the virus and host system:

• Model-specific considerations:

  • RNA viruses (VHSV, influenza, coronavirus): Focus on IFIT5's interaction with viral 5'-triphosphate RNA

  • DNA viruses (herpesviruses, poxviruses): Investigate IFIT5's ability to bind AT-rich dsDNA

  • Retroviruses (HIV): Examine IFIT5's effect on reverse-transcribed viral intermediates

  • Each viral family may engage IFIT5 through distinct mechanisms requiring specific detection strategies

• Comparative time-course designs:

  • Implement standardized time points (0, 3, 6, 24, 72 hours post-infection) across different viral models

  • Measure both IFIT5 expression and viral replication markers simultaneously

  • Include parallel protein and RNA analyses to capture post-transcriptional regulation

  • Perform subcellular fractionation to track IFIT5 localization changes during infection progression

• Multi-level IFIT5 modulation:

  • Overexpression: Use plasmid-based or viral vector systems to express wild-type or mutant IFIT5

  • Knockdown/knockout: Implement siRNA, shRNA, or CRISPR-Cas9 to reduce IFIT5 levels

  • Chemical modulation: Apply compounds that target the IFIT5 RNA-binding pocket

  • Compare the impact on viral replication across different viral models using the same modulation strategy

• Host species considerations:

  • Select appropriate antibodies validated for your host species (human, rainbow trout, pig, etc.)

  • Consider species-specific differences in IFIT5 sequence and function

  • Implement cross-species comparisons to identify conserved versus species-specific mechanisms

  • Rainbow trout RBCs have been successfully used to study IFIT5's role in VHSV infection, while porcine studies have examined PRRSV

Research in rainbow trout demonstrated that silencing ifit5 resulted in significantly increased VHSV replication in RBCs, and chemical modulation of the IFIT5 RNA-binding pocket using compounds from the SuperNatural II database increased viral replication, providing methodological templates for similar studies in other viral models .

How should researchers design experiments to investigate potential therapeutic applications targeting IFIT5?

For researchers exploring IFIT5 as a therapeutic target, systematic experimental approaches are essential:

• Target validation experiments:

  • Establish clear correlation between IFIT5 modulation and antiviral outcomes across multiple viral models

  • Implement dose-dependent IFIT5 modulation using inducible expression systems or titrated siRNA

  • Determine therapeutic window by comparing antiviral efficacy versus potential cellular toxicity

  • Assess specificity by comparing effects on different virus families (RNA viruses, DNA viruses)

• Small molecule screening approaches:

  • Develop high-throughput screening assays based on:

    • IFIT5-RNA binding (fluorescence polarization assays)

    • IFIT5 protein-protein interactions (FRET-based assays)

    • Functional readouts (viral replication reporters)

  • Screen compound libraries (like SuperNatural II) for modulators of IFIT5 activity

  • Validate hits using orthogonal assays and structure-activity relationship studies

  • Previous research successfully identified compounds from the SuperNatural II database that modulate the IFIT5 RNA-binding pocket

• Structure-guided drug design:

  • Utilize the known crystal structure of IFIT5 for in silico screening

  • Focus on the RNA-binding cavity and acidic pocket as druggable sites

  • Design compounds that either enhance IFIT5's discrimination capabilities or modulate its interaction with specific viral RNAs

  • Test designed compounds in binding assays with purified recombinant IFIT5 before cellular studies

• Translational evaluation approaches:

  • Progress from cell lines to primary cells to relevant animal models

  • Compare prophylactic versus therapeutic administration timing

  • Assess potential interference with normal cellular IFIT5 functions

  • Evaluate combination approaches with established antivirals

  • Measure both direct antiviral effects and potential immunomodulatory outcomes