M2-1 Antibody

What is the RSV M2-1 protein and what is its structural organization?

RSV M2-1 is a 194-amino acid basic protein found in all known pneumoviruses that functions as an essential cofactor of the viral RNA polymerase complex. Structurally, M2-1 is predominantly α-helical and forms a 5.4S tetramer of approximately 89 kDa with a diameter of ~7.6 nm at micromolar concentrations. The protein contains a CCCH motif (a putative zinc-binding domain) at its N-terminus that is essential for M2-1 function. The oligomerization domain has been mapped to a putative α-helix consisting of amino acid residues 32 to 63, which is critical for the protein's activity . Phosphorylated forms of M2-1 exist in infected cells, with phosphorylation occurring primarily at Thr56, Ser58, and Ser61, although different research groups have reported slightly different phosphorylation patterns .

How does M2-1 function in RSV replication?

M2-1 serves as a transcriptional processivity and antitermination factor that prevents premature termination during viral transcription. It functions by enhancing readthrough of intergenic junctions, allowing the synthesis of complete mRNAs. During infection, M2-1 interacts with viral mRNA across the entire length of each gene, consistent with M2-1 contacting each nucleotide of nascent mRNA being extruded from the polymerase. This interaction is part of the transcription machinery rather than binding to specific RSV RNA sequences . Importantly, M2-1 has no effect on RNA replication, which distinguishes its role specifically in transcription. Deletion studies have demonstrated that the oligomerization of M2-1 is essential for its transcriptional activity, as M2-1 deletion mutants lacking the oligomerization domain (amino acids 32-63) show significant reduction in RNA transcription compared to wild-type M2-1 .

What cellular partners does M2-1 interact with during infection?

Mass spectrometry analyses have identified 137 potential cellular partners of M2-1, many of which are associated with mRNA metabolism, particularly mRNA maturation, translation, and stabilization. The cytoplasmic polyA-binding protein 1 (PABPC1) is a confirmed interaction partner that plays a major role in both translation and mRNA stabilization. This interaction has been validated using protein complementation assays and specific immunoprecipitation techniques. PABPC1 colocalizes with M2-1 from its accumulation in inclusion bodies associated granules (IBAGs) to its liberation in the cytoplasm . These findings suggest that M2-1 interacts with mRNA metabolism factors throughout the process from transcription to translation, implying a potential additional role in the fate of viral mRNA downstream of transcription.

What techniques are most effective for studying M2-1:RNA interactions?

Crosslinking immunoprecipitation with RNA sequencing (CLIP-Seq) has proven highly effective for mapping points of M2-1:RNA interactions in RSV-infected cells. This technique revealed that M2-1 interacts specifically with positive-sense RSV RNA but not negative-sense genome RNA. Studies employing CLIP-Seq analysis at 8 and 18 hours post-infection demonstrated that M2-1 makes contacts along the entire length of each viral mRNA, confirming its role as a component of the transcriptase complex . For biochemical studies of M2-1 RNA binding properties, researchers have used purified M2-1 protein and RNA oligonucleotides to show that M2-1 has greater affinity for A-rich oligonucleotides and sequences containing the complement of the RSV gene end signal sequence. These in vitro approaches should be complemented with cellular studies, as the RNA binding preferences observed in vitro do not fully explain M2-1's function in facilitating transcription elongation .

How can researchers differentiate between M2-1's roles in transcription versus potential post-transcriptional functions?

To distinguish between M2-1's established role in transcription and its potential post-transcriptional functions, researchers should employ a combination of approaches:

• Temporal analysis: Compare M2-1:RNA associations at different time points post-infection to track changes in M2-1's RNA binding profile.

• Subcellular fractionation: Isolate M2-1 from different cellular compartments to determine where interactions occur.

• Mutational analysis: Use M2-1 mutants with altered RNA binding or oligomerization properties to dissect functional domains.

• Comparative binding studies: Analyze M2-1 binding to both viral mRNAs and cellular mRNAs, as research has shown that M2-1 binds discrete sites within cellular mRNAs with a preference for A/U-rich sequences, suggesting additional roles beyond transcription elongation .

• Polysome profiling: Determine if M2-1 associates with translating ribosomes, which would support a role in translation.

This multi-faceted approach can help clarify whether M2-1's interaction with PABPC1 and other RNA-binding proteins reflects a genuine post-transcriptional regulatory function.

What are the critical considerations when using M2-1 antibodies for immunoprecipitation studies?

When designing immunoprecipitation (IP) experiments with M2-1 antibodies, researchers should consider:

• Antibody specificity: Validate antibody specificity using both positive controls (RSV-infected cells) and negative controls (uninfected cells) to ensure the antibody recognizes authentic M2-1 protein .

• Cross-reactivity assessment: Test for potential cross-reactivity with cellular proteins, particularly those with CCCH zinc finger motifs similar to M2-1.

• Native vs. denatured conditions: Since M2-1 forms tetramers and has both phosphorylated and unphosphorylated forms, the choice between native and denaturing conditions is critical. For studying M2-1 in its tetrameric form or for co-IP of interaction partners, use non-denaturing conditions .

• Salt concentration: M2-1 binds RNA, and GST-M2-1 fusion proteins co-purify with bacterial RNA that can be eliminated by high-salt wash . Therefore, optimize salt concentrations to maintain specific protein-protein interactions while reducing RNA-mediated associations.

• RNA digestion: Include RNase treatment controls to distinguish direct protein-protein interactions from RNA-mediated associations, as some M2-1-N interactions appear to be mediated by RNA .

• Phosphorylation state: Consider that M2-1 exists in both phosphorylated and unphosphorylated forms, which may affect antibody recognition and interaction properties .

How can researchers optimize detection of M2-1 in different experimental systems?

For optimal detection of M2-1 in various experimental systems, researchers should:

• Western blot optimization:

  • Use reducing conditions as M2-1 contains cysteine residues in its zinc finger domain

  • Select appropriate gel percentage (12-15%) for optimal resolution of the 22kDa M2-1 protein

  • For phosphorylation studies, consider Phos-tag gels to separate phosphorylated and unphosphorylated forms

• Immunofluorescence considerations:

  • Fixation method impacts M2-1 detection; paraformaldehyde preserves native structure while methanol provides better accessibility to certain epitopes

  • M2-1 localizes to cytoplasmic inclusion bodies associated with viral RNA synthesis in infected cells

  • Co-stain with markers for inclusion bodies (such as P protein) for proper interpretation

• Flow cytometry applications:

  • For native M2-1 detection, researchers have developed flow cytometric assays using 293FT transfected cell lines stably expressing full-length tetrameric forms of M2-1, which overcomes technical problems with high background encountered in cell surface ELISA

• Epitope masking: Be aware that M2-1's interactions with RNA or other viral proteins may mask epitopes, requiring optimization of sample preparation protocols .

How does M2-1 deletion impact live-attenuated RSV vaccine candidates?

Deletion or modification of M2-1's regulatory partner M2-2 (not M2-1 itself) has been used in live-attenuated RSV vaccine development, resulting in a highly desirable phenotype where virus growth is attenuated while gene expression is concomitantly increased. The M2-2 protein functions as a regulatory switch from transcription to RNA replication. When M2-2 is deleted:

• Growth characteristics: The resulting virus grows less efficiently than wild-type virus in vitro, with titers reduced 1,000-fold during initial days and 10-fold by days 7-8 .

• RNA synthesis balance: Intracellular genomic RNA accumulation is reduced 3-4 fold, while mRNA accumulation is increased 2-4 fold compared to wild-type virus .

• Antigen expression: Synthesis of F and G glycoproteins (major RSV neutralization and protective antigens) increases in proportion with mRNA levels .

• Transcription regulation: In wild-type RSV, mRNA accumulation increases dramatically up to approximately 12-15 hours post-infection and then levels off, whereas accumulation continues to increase in cells infected with M2-2 knockout viruses .

These findings make M2-2 deletion an attractive strategy for vaccine development, as it creates viruses with reduced replication capacity but enhanced immunogen production.

What immunological responses to M2-1 have been observed in clinical studies of RSV vaccines?

Clinical studies with RSV vaccine candidates incorporating M2-1 have shown promising immunological responses:

• Human antibody responses: A Phase I randomized study of a live-attenuated RSV vaccine candidate with deletion of RNA synthesis regulatory protein M2-2 (LIDΔM2-2) in RSV-seronegative children ages 6-24 months showed that 90% of vaccinees had ≥4-fold rise in serum neutralizing antibodies .

• Anamnestic responses: Eight of 19 vaccinees versus 2 of 9 placebo recipients showed substantially increased RSV antibody titers after RSV season without medically attended RSV disease, indicating anamnestic vaccine responses to wild-type RSV without significant illness .

• Viral vector vaccines: A Phase I randomized study evaluating an investigational vaccine against RSV (ChAd155-RSV) using chimpanzee-adenovirus-155 viral vector expressing RSV fusion (F), nucleocapsid, and transcription antitermination proteins (including M2-1) found that RSV-A neutralizing antibodies geometric mean titer ratios (post/pre-immunization) following a high dose were 2.6 at day 30 and 2.3 at day 60 .

These findings suggest that vaccines incorporating M2-1 or modifying the M2-1/M2-2 regulatory system can effectively induce neutralizing antibody responses.

How should researchers interpret conflicting data on M2-1 RNA binding specificity?

Several studies have reported seemingly contradictory results regarding M2-1 RNA binding specificity. To properly interpret these findings:

• Consider methodological differences: Some studies used purified recombinant M2-1 with short RNA oligonucleotides, while others examined M2-1:RNA interactions in infected cells. In vitro binding studies showed greater affinity for A-rich oligonucleotides and gene end sequences, while CLIP-Seq analysis of infected cells revealed binding along the entire length of viral mRNAs .

• Distinguish between in vitro and in vivo contexts: M2-1's RNA binding preferences in vitro may not fully reflect its biological behavior in the context of the viral transcription complex where additional factors may influence RNA interactions.

• Recognize dual RNA-binding modes: Evidence suggests M2-1 may have distinct modes of RNA interaction:

  • Non-specific interactions with viral mRNAs as part of the transcription complex

  • Specific binding to discrete sites within cellular mRNAs (preferentially A/U-rich sequences)

• Consider protein context: M2-1's RNA binding properties may be influenced by its phosphorylation state and interactions with other viral proteins. The region encompassing amino acid residues 59-178 binds to both P protein and RNA in a competitive manner .

• Apply multiple methodologies: To resolve discrepancies, researchers should employ complementary approaches including biochemical binding assays, CLIP-Seq, and functional transcription assays.

What factors influence the detection of anti-M2-1 antibodies in human serum samples?

When analyzing anti-M2-1 antibody responses in human serum samples, researchers should consider:

• Age-dependent responses: Studies have shown that antibodies to viral proteins like M2 are found at higher percentages and levels in adults aged ≥40 years compared to younger donors, suggesting cumulative exposure effects .

• Assay sensitivity and specificity: Traditional assays for measuring antibodies to native M2 presented technical challenges with high background in cell surface ELISA. More sensitive flow cytometric assays using 293FT transfected cell lines stably expressing full-length tetrameric forms of M2 have improved detection .

• Threshold definition: The choice of positivity threshold is critical. Some researchers have defined negative samples as those with similar binding to M2-expressing cells and control cells (≤3% difference), but this threshold choice can affect interpretation of kinetic differences in antibody responses .

• Response kinetics: Anti-M2 antibody responses may appear with different kinetics than other antibody responses (such as hemagglutination inhibiting antibodies), potentially due to previous priming to M2 .

• Age-specific induction: Induction of antibodies to M2 appears difficult to achieve in very young children, even upon infection with pandemic viruses, while a single infection leads to robust responses to other viral proteins in this age group. This suggests boosting may be required for strong responses to M2 .

• Strain differences: There may be differences in the ability of pandemic versus seasonal strains to induce anti-M2 antibodies, though this remains speculative and would be difficult to assess in human populations due to confounding variables .

What are the most reliable methods for producing and purifying recombinant M2-1 for research purposes?

Based on published research, the following approaches have proven effective for producing and purifying recombinant M2-1:

• Expression system: M2-1 has been successfully produced in E. coli as a recombinant protein using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins are often copurified with bacterial RNA, which can be eliminated by high-salt wash .

• Purification considerations:

  • Include zinc in buffers to maintain the integrity of the CCCH zinc finger domain

  • Use high salt concentrations (≥500 mM NaCl) to remove bound RNA if protein-RNA interactions are not the focus of study

  • Consider tag removal with appropriate proteases if the native structure is critical

• Tetrameric state: Recombinant M2-1 naturally forms tetramers at micromolar concentrations, which can be verified through chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses .

• Protein quality assessment: Circular dichroism analysis can confirm that purified M2-1 maintains its predominantly α-helical structure .

• Functional validation: Verify the activity of purified M2-1 in an RSV minigenome replicon system using a luciferase gene as a reporter. Wild-type M2-1 should enhance transcription compared to oligomerization-deficient mutants .

What advanced imaging techniques are most informative for studying M2-1 localization and dynamics?

Advanced imaging approaches for studying M2-1 localization and dynamics include:

• Live-cell imaging with fluorescent protein tags: Fusion of M2-1 with fluorescent proteins allows real-time tracking of its localization and movement during infection, particularly its association with and liberation from inclusion bodies.

• FRAP (Fluorescence Recovery After Photobleaching): This technique can measure the mobility of M2-1 within inclusion bodies and the cytoplasm, providing insights into the dynamics of M2-1's interactions with viral and cellular components.

• Super-resolution microscopy: Techniques such as STORM, PALM, or STED can overcome the diffraction limit to visualize M2-1's precise localization relative to other viral proteins and cellular structures.

• FRET (Förster Resonance Energy Transfer): By tagging M2-1 and potential interaction partners with appropriate fluorophores, FRET can detect direct protein-protein interactions in living cells.

• Correlative light and electron microscopy (CLEM): This approach combines the specificity of fluorescence microscopy with the ultrastructural detail of electron microscopy to precisely localize M2-1 within viral structures.

• Two-photon FLIM (Fluorescence Lifetime Imaging Microscopy): This technique can detect changes in the local environment of fluorescently labeled M2-1, potentially revealing conformational changes or interaction states.

Research has shown that M2-1 colocalizes with PABPC1 from its accumulation in inclusion bodies associated granules (IBAGs) to its liberation in the cytoplasm , making these advanced imaging techniques particularly valuable for investigating the dynamic nature of these interactions during the viral life cycle.

What are the most promising therapeutic targets within the M2-1 structure or function?

Several aspects of M2-1 structure and function present promising therapeutic targets:

• Zinc finger domain: The CCCH zinc finger motif at the N-terminus is essential for M2-1 function. Small molecules that disrupt zinc coordination or alter the conformation of this domain could inhibit M2-1 activity .

• Oligomerization interface: Since M2-1 tetramerization is critical for its function, compounds that interfere with the interaction between monomers (targeting amino acids 32-63) could disrupt viral transcription .

• RNA binding domain: The region encompassing amino acid residues 59-178 binds to RNA. Molecules that compete for RNA binding or alter the RNA binding properties of M2-1 could impair viral transcription .

• M2-1:P protein interface: M2-1 interacts with the viral phosphoprotein (P), and this interaction is competitive with RNA binding. Compounds that stabilize either the M2-1:P or M2-1:RNA complex, disrupting the normal dynamic exchange, could interfere with viral transcription .

• Phosphorylation sites: M2-1 phosphorylation at specific residues (Thr56, Ser58, Ser61) affects its function. Kinase inhibitors targeting the responsible cellular kinases or compounds that mimic phosphorylated/dephosphorylated states could modulate M2-1 activity .

• Interaction with host factors: The recently identified interactions between M2-1 and cellular proteins involved in mRNA metabolism, such as PABPC1, present novel therapeutic targets that could disrupt viral replication while potentially minimizing the development of resistance .

How might advances in structural biology further our understanding of M2-1 function?

Advances in structural biology techniques could significantly enhance our understanding of M2-1 function:

• Cryo-electron microscopy (cryo-EM): High-resolution structures of the M2-1 tetramer in complex with RNA and other viral proteins could reveal crucial interaction interfaces and conformational changes that occur during viral transcription.

• Single-molecule techniques: Methods such as smFRET (single-molecule FRET) could provide insights into the dynamics of M2-1's interactions with RNA and proteins, capturing transient intermediates during the transcription process.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique could map regions of M2-1 that undergo conformational changes upon RNA binding or during interactions with other viral and cellular proteins.

• Integrative structural biology: Combining multiple structural techniques (X-ray crystallography, NMR, cryo-EM, HDX-MS) with computational modeling would provide a more complete picture of M2-1's structural dynamics.

• In situ structural biology: Emerging techniques for determining protein structures within cells could reveal how M2-1's conformation and interactions are influenced by the cellular environment.

• Time-resolved structural studies: Capturing structural snapshots of M2-1 during different stages of viral transcription would illuminate its mechanism of action as a transcription processivity factor.