id: influenza-ha-membrane-signaling title: Influenza HA Antagonism of Membrane Signaling short_title: HA Membrane Signaling visibility: confidential status: Active stage: validation primary_lane: Mechanistic pathogenesis secondary_lanes: - Programmable immune instruction family: Programmable influenza pathogenesis ownership_model: primary-owner primary_owner: Fiorella Chacon contributors: - Ben tenOever - Mark Chua - Ryla Cantorgenali - Christina Higgins - Lucia Carrau manuscript_type: discovery evidence_status: supported last_reviewed: 2026-07-10 reviewed_by: Ben tenOever

Influenza HA Antagonism of Membrane Signaling

One-sentence thesis

Influenza A virus hemagglutinin suppresses the ability of infected cells to respond to multiple extracellular ligands, potentially by reorganizing the plasma membrane and thereby limiting receptor-proximal signaling independently of canonical viral antagonism of pathogen sensing.

Biological question

Does accumulation of influenza HA at the plasma membrane broadly diminish extracellular receptor signaling, and if so, what membrane-level mechanism explains this effect?

Working model

HA is necessary for efficient suppression of several plasma-membrane-initiated signaling pathways during IAV infection. Because signaling through IFN, TNF, and IL-4 is restored when HA expression is reduced, whereas signaling through the intracellular glucocorticoid receptor is comparatively preserved, the current model is that HA alters membrane organization, receptor mobility, clustering, or local protein interactions rather than targeting one cytokine pathway specifically.

A remaining alternative is that another product or property of segment 4 contributes to the phenotype; this must be excluded through HA-only sufficiency experiments and segment-4/domain dissection.

Experimental strategy

The project uses miRNA-targeted IAVs to silence individual viral ORFs after infection while preserving production in eggs. Exogenous ligand is added after infection to separate pathogen sensing and endogenous cytokine production from the cell's capacity to respond to a defined signal.

Core systems include:

• PR8 wild-type, HA-5T, NA-5T, and HANA-5T viruses
• broader ORF-targeted IAV libraries
• wild-type and RIG-I-knockout A549 cells
• doxycycline-inducible A549 lines expressing HA, NA, NP, or NS1
• IFN-beta/MX1, TNF-alpha/IL-8, IL-4/pSTAT6, and dexamethasone/FKBP5 readouts
• single-cell RNA-seq comparisons of PR8 and HANA infection
• FLOW-FRET and proposed FRAP/membrane-imaging assays

Evidence map

Established or strongly supported observations

1. Reducing HA restores IFN responsiveness during infection. In the ORF-targeting screen, HA silencing produced a stronger MX1 response after exogenous IFN-beta than wild-type PR8. The phenotype was reproduced with alternative targeting cassettes and HA-5T/HANA configurations rather than relying on one construct.

2. The effect is downstream of pathogen sensing. RIG-I-knockout A549 cells retained the differential response between wild-type and HA-silenced virus after exogenous IFN-beta, arguing that the central phenotype reflects signaling competence rather than altered RIG-I sensing.

3. The phenotype extends beyond IFN signaling. Cells infected with HA-silenced virus responded more strongly to TNF-alpha, measured by IL-8 production, and to IL-4, measured by STAT6 phosphorylation. This argues against a mechanism specific only to IFNAR/JAK-STAT signaling.

4. An intracellular receptor pathway is comparatively spared. Dexamethasone-induced FKBP5 expression did not show the same clear HA-dependent suppression, supporting a distinction between plasma-membrane-initiated and intracellular-receptor signaling.

5. Single-cell data support a signaling rather than production defect. HANA infection produces broader/higher ISG expression without a commensurate increase in IFN transcripts, consistent with altered responsiveness to cytokine rather than simply increased cytokine production.

6. Inducible HA-expressing A549 clones have been generated and stratified. Low-, intermediate-, and high-surface-HA populations have been identified by flow cytometry, although clone classification still requires protein-level confirmation and functional testing.

Supported interpretation

HA is necessary for broad suppression of extracellular ligand signaling during IAV infection.

Working model

HA accumulation changes plasma-membrane organization or receptor dynamics, reducing signaling efficiency across otherwise distinct receptor systems.

Unresolved questions

• Is HA alone sufficient outside infection?
• Is the phenotype attributable to HA protein rather than another segment-4 product, peptide, RNA feature, or replication consequence?
• Which HA domain is required: signal peptide/trafficking, ectodomain, transmembrane region, or cytoplasmic tail?
• Does HA alter receptor abundance, mobility, nanoscale clustering, membrane domains, or downstream adaptor recruitment?
• Is the effect conserved across H1, H3, and H5 HAs?
• Does enhanced signaling in HA-silenced infection feed back to reduce viral replication?
• Are type I and type III IFN affected equivalently?

Current workstreams

1. HA sufficiency in inducible cell lines

Doxycycline-inducible A549-HA clones are being screened and categorized by surface expression. The decisive experiment is to test whether induced HA, in the absence of infection, suppresses IFN, TNF, and IL-4 responses. NA, NP, and NS1 lines provide localization and viral-protein controls.

Decision rule:

• If HA-high cells reproduce the phenotype, prioritize membrane mechanism experiments in induced versus uninduced cells.
• If HA expression alone does not reproduce the phenotype, determine whether infection-specific trafficking, HA maturation, segment-4 context, or another segment-4 product is required.

2. Membrane organization and receptor proximity

FLOW-FRET constructs for CD80, CD86, and CD8a fluorescent pairs have been obtained and are being validated. These constructs can assess changes in membrane-protein proximity or organization. FRAP remains planned through the microscopy core to test membrane mobility.

3. Segment-4 and HA-domain dissection

Planned constructs include codon-optimized HA, signal-sequence alteration, and cytoplasmic-tail deletion. Additional H3 and H5 PR8 7:1 viruses with and without 5T targeting could test whether the phenotype is subtype-conserved.

4. Kinetics and replication consequences

Planned studies compare wild-type and HA-5T infections across early and late time points, in wild-type and RIG-I-knockout A549 cells, with type I versus type III IFN treatment. Viral proteins, MX1, and replication should be measured together to determine whether restored signaling constrains HA-silenced virus.

Experiments not currently prioritized

• The initial comparison using limited 6:2 avian-virus stocks is not currently prioritized because X31 did not infect under the tested conditions, available stocks are limiting, and the experiment is not required to establish the core mechanism.

Publication trajectory

Proposed paper type

Discovery/mechanism paper.

Provisional central claim

Influenza HA broadly suppresses plasma-membrane receptor signaling by altering the organization or function of the infected-cell surface.

Existing figure backbone

1. Single-cell observation: infected cells show poor ISG induction; HANA increases ISGs without proportionate IFN production.
2. ORF-targeting screen separates sensing from signaling and identifies HA as necessary for reduced IFN responsiveness.
3. Independent HA-targeting constructs reproduce the phenotype.
4. RIG-I knockout confirms that the phenotype is downstream of sensing.
5. HA dependence extends to TNF and IL-4 signaling.
6. Intracellular glucocorticoid signaling is comparatively preserved.
7. HA sufficiency in inducible cell lines.
8. Membrane mechanism through FLOW-FRET, FRAP, receptor imaging, or domain mutants.
9. Optional conservation/physiologic extension across HA subtypes or in a second cellular system.

What is already publication-enabling

• A coherent biological claim rather than only a technology observation
• Independent viral constructs supporting HA necessity
• Multiple extracellular signaling pathways affected
• A negative/control pathway supporting membrane specificity
• A mechanistically informative RIG-I-knockout experiment
• Single-cell data that provide the originating observation and broader context

What is still required

1. HA sufficiency outside infection, or a clear explanation for why infection context is required.
2. A direct membrane-level mechanism demonstrating altered mobility, organization, clustering, or receptor function.
3. Protein-level and kinetic validation of key observations, with consistent replicate structure.
4. Segment-4 specificity/domain logic sufficient to exclude alternate products or indirect replication effects.

Publication-readiness assessment

Figure-building, approaching manuscript formation. The conceptual paper is already visible. The project is not waiting for discovery of a story; it is waiting for two causal closures: HA sufficiency and the membrane mechanism.

Immediate next decision

Determine whether the HA-high inducible clone suppresses IFN signaling in the absence of infection.

Immediate actions

1. Confirm low/intermediate/high HA clone expression by surface flow cytometry and western blot.
2. Test induced versus uninduced HA clones with exogenous IFN-beta using MX1 protein as the primary readout.
3. If positive, repeat TNF-alpha and IL-4 signaling in the same system.
4. Validate FLOW-FRET plasmid expression and optimize infection/induction order.
5. Re-engage the microscopy core to initiate FRAP.

Current blockers

• HA sufficiency has not yet been established.
• Clone categories require protein-level confirmation.
• The membrane assay has not yet been operationalized.
• Some earlier experiments have inconsistent RNA-versus-protein readouts.
• The project could expand excessively unless experiments are ranked by their necessity for the central claim.

Dependencies and connections

• IAV ORF TARGETING LIBRARY — discovery and validation platform
• SINGLE CELL IAV HOST STATE — originating observation and transcriptional context
• MIRNA GATING — enabling viral perturbation strategy
• INFLUENZA PATHOGENESIS FAMILY — broader program connection
• Mark Chua — viral stocks, reverse genetics, HA subtype plasmids
• Microscopy core — FRAP and membrane imaging

De-risking / secondary dissertation direction

A smaller related project should be defined within the same conceptual space so that the dissertation does not depend entirely on resolving a technically difficult membrane mechanism. Candidate directions should use existing viruses, inducible lines, or signaling assays and be capable of yielding a self-contained result.

Source provenance

This record was reconstructed from Fiorella Chacon's May 22, 2026 departmental seminar and two July 8, 2026 project-meeting documents. Interpretive statements are labeled according to evidence maturity and should be revised as primary datasets are linked.