SIRPA suppresses integrin-dependent virus endocytosis

This study reveals that Signal Regulatory Protein Alpha (SIRPA) suppresses the endocytosis of New World arenaviruses and other pathogenic RNA viruses by inhibiting the integrin-dependent signaling axis involving SHP2, FYN, and FAK, thereby identifying this pathway as a potential therapeutic target for blocking viral entry.

The Gist

The Big Picture: A Viral Heist and a Security Guard

Imagine your body is a high-security city, and your cells are the buildings inside. Viruses are like master thieves trying to break into these buildings to steal resources and start a crime spree (infection).

To get inside, these viruses usually have to sneak through the front door or trick the building's security system into opening the gates. This process is called endocytosis (the cell swallowing the virus).

The scientists in this paper discovered a specific "Security Guard" in our cells called SIRPA. Usually, this guard's job is to stop the cell from eating things it shouldn't (like healthy neighboring cells). But the researchers found that SIRPA is also a fantastic virus-fighter. It stops the viruses from getting inside by shutting down the very mechanism they need to enter.

The Cast of Characters

1. The Thieves (New World Arenaviruses): These are dangerous viruses (like Junín virus) that cause severe hemorrhagic fevers. They need to get inside cells to survive.
2. The Security Guard (SIRPA): A protein on the cell surface that says, "Stop! Don't eat that!" It normally prevents the cell from phagocytosis (eating) other cells.
3. The Gatekeepers (Integrins): These are like the heavy-duty hydraulic doors and the machinery that opens them. The viruses hijack these doors to pull themselves inside.
4. The Foreman (FAK & FYN): These are the managers who tell the Gatekeepers to open the doors.
5. The Saboteur (SHP2): This is the guard's assistant. When the Security Guard (SIRPA) is activated, it calls in the Saboteur to break the machinery so the doors can't open.

How the Heist Usually Works (Without the Guard)

1. The Approach: The virus bumps into the cell and grabs onto a handle (a receptor).
2. The Signal: This grab sends a signal to the Foreman (FAK/FYN).
3. The Action: The Foreman shouts, "Open the gates!" The Gatekeepers (Integrins) activate, rearranging the cell's skeleton to form a "cup" that swallows the virus.
4. The Result: The virus is inside, and the infection begins.

How the Security Guard (SIRPA) Stops the Heist

The researchers found that when a virus tries to enter, it accidentally triggers the Security Guard (SIRPA). Here is the step-by-step breakdown of how SIRPA stops the infection:

1. The Trigger: When the virus touches the cell, it accidentally activates the Foreman (FYN), a type of kinase (a protein that adds "tags" to other proteins).
2. The Call to Arms: The Foreman puts a "flag" (a phosphate tag) on the Security Guard (SIRPA).
3. The Saboteur Arrives: Seeing the flag, the Saboteur (SHP2) jumps out of hiding and attaches to the Security Guard.
4. The Sabotage: The Saboteur (SHP2) goes to work on the Gatekeepers (Integrins) and the Foreman (FAK). It removes their "open" tags.
5. The Lockdown: Without those tags, the Gatekeepers refuse to open. The "cup" never forms. The virus is left outside, unable to get in.

The "Aha!" Moment of the Study

The scientists wanted to know exactly how SIRPA does this. They tested it by:

• Removing the Guard: When they took SIRPA out of the cells, the viruses got in much easier.
• Removing the Saboteur: When they took away SHP2, the viruses got in easier, proving SHP2 is the one actually breaking the machinery.
• Using "Brakes": They used drugs that act like "brakes" on the Gatekeepers (Integrin inhibitors) and the Foreman (FAK inhibitors). These drugs stopped the viruses from entering, even in cells that didn't have the Security Guard.

Why This Matters (The Takeaway)

This study is like finding a new way to lock the front door of a bank.

• New Weapons: Currently, there are very few drugs to treat these specific viruses. This paper shows that we can use drugs that block the Integrin pathway (the Gatekeepers) to stop the virus.
• Double Duty: These drugs work because they mimic the body's natural defense (SIRPA). They essentially tell the cell, "Don't open the door for the virus."
• Broad Protection: Since many different viruses use this same "swallowing" method to enter cells, this strategy could work against a wide range of viral threats, not just the ones studied here.

In short: The virus tries to trick the cell into swallowing it. The cell has a security system (SIRPA) that detects the trick, calls in a saboteur (SHP2), and jams the gears of the swallowing machine (Integrins/FAK), keeping the virus locked out. The researchers found that we can use drugs to jam those gears ourselves, offering a new hope for treating these dangerous infections.

Technical Summary

1. Problem Statement

New World arenaviruses (NWAs), such as Junín virus (JUNV) and Machupo virus (MACV), cause severe viral hemorrhagic fevers with high mortality rates. Currently, there are no FDA-approved therapeutics for these infections, and the only effective vaccine is a live-attenuated strain for JUNV. While the cellular receptor for NWAs (Transferrin Receptor 1, TfR1) is known, the downstream host mechanisms facilitating viral entry and endocytosis remain poorly defined.

The authors previously identified Signal Regulatory Protein Alpha (SIRPA) as a host restriction factor that inhibits the entry of NWAs and other RNA viruses trafficking to acidic compartments. SIRPA is a transmembrane protein known to inhibit macrophage phagocytosis via the CD47-SIRPA axis. The central question of this study is: What is the molecular mechanism by which SIRPA inhibits viral endocytosis, and can this pathway be targeted therapeutically?

2. Methodology

The study employed a combination of cell biology, virology, and pharmacological approaches across multiple cell lines (U2OS, A549, THP-1 macrophages) and mouse models:

• Genetic Manipulation:
  • Knockdown (KD): Used siRNA and shRNA to deplete SIRPA, SHP2, Src-family kinases (SFKs: SRC, LYN, FYN, YES), Focal Adhesion Kinase (FAK), and integrin components in U2OS and THP-1 cells.
  • Knockout (KO): Utilized SIRPA-deficient mice and fibroblasts.
  • Overexpression: Transfected cells with wild-type and mutant SIRPA (lacking cytoplasmic tail or ITIM tyrosine mutations) and SHP2 expression vectors.
• Viral Assays:
  • Infection Assays: Measured viral RNA levels (RT-qPCR) and infectious titers (plaque/TCID50 assays) for TCRV, JUNV (C1 and Romero strains), LCMV, VSV, and pseudoviruses (H5N1, MACV, ZIKV).
  • Virus Internalization Assays (VIAs): Quantified internalized viral RNA after proteinase K treatment to distinguish entry from post-entry replication.
• Biochemical Analysis:
  • Co-immunoprecipitation (Co-IP) & Western Blot: Analyzed protein-protein interactions (SIRPA-SHP2, SIRPA-SFKs) and phosphorylation states (SIRPA tyrosine, SHP2 Y542, FAK Y397, PYK2 Y402).
  • Proximity Ligation Assay (PLA): Visualized SIRPA-SHP2 interactions in situ.
  • Deglycosylation: Used to resolve SIRPA phosphorylation bands for accurate quantification.
• Pharmacological Inhibition:
  • Treated cells with PP1 (pan-SFK inhibitor), BTT-3033 (integrin inhibitor), and FIB-14 (FAK inhibitor).
  • In Vivo Model: Used human TfR1 transgenic mice (susceptible to JUNV) pre-treated with anti-IFNAR antibody. Mice received daily IP injections of BTT-3033 or Favipiravir (control) prior to and during JUNV C1 challenge.
• Macrophage Differentiation: Differentiated THP-1 cells into M0, M1, and M2 phenotypes to assess SIRPA expression and viral susceptibility in immune-relevant cells.

3. Key Contributions & Results

A. SHP2 is a Critical Restriction Factor Downstream of SIRPA

• Depletion of SHP2 (Src homology region 2-containing protein tyrosine phosphatase 2) in non-hematopoietic cells increased infection by TCRV, JUNV, LCMV, and VSV, mimicking the phenotype of SIRPA knockdown.
• Mechanism: SIRPA recruits SHP2 upon virus binding. Co-IP and PLA showed that SIRPA and SHP2 interact physically, and this interaction increases significantly within 5–15 minutes of viral infection.
• Activation: Virus infection triggers phosphorylation of SHP2 at tyrosine 542 (activation site) and SIRPA at its ITIM tyrosines.

B. FYN Kinase Drives SIRPA Activation

• Among Src-family kinases (SFKs), FYN was identified as the primary kinase responsible for SIRPA phosphorylation during viral entry.
• FYN knockdown reduced SIRPA phosphorylation and increased viral infection/internalization to levels similar to SIRPA KD.
• Overexpression of FYN enhanced the SIRPA-SHP2 interaction, confirming FYN's role in initiating the inhibitory cascade.

C. The Integrin Signaling Pathway is Essential for Viral Entry

• The study established that SIRPA inhibits viral entry by suppressing the integrin signaling pathway.
• Integrin Inhibition: Treatment with BTT-3033 (an integrin inhibitor) significantly reduced infection and internalization of arenaviruses, VSV, and ZIKV. Crucially, BTT-3033 remained effective even in SIRPA-knockdown cells, indicating it targets the pathway downstream of SIRPA's regulatory point.
• FAK Inhibition: FIB-14, an inhibitor of FAK autophosphorylation (Y397), also blocked viral entry. FAK phosphorylation was observed to increase upon virus binding.
• Macrophage Relevance: In THP-1 macrophages, differentiation to M1/M2 phenotypes increased SIRPA expression and reduced infection. Inhibition of integrin signaling (BTT-3033) or knockdown of PYK2 (a FAK family member in macrophages) reduced viral internalization, confirming the pathway's relevance in immune cells.

D. Therapeutic Potential In Vivo

• In a mouse model of JUNV infection (huTfR1 Tg mice), daily treatment with BTT-3033 significantly reduced viral loads in serum and spleen.
• The efficacy of BTT-3033 was comparable to Favipiravir, a known antiviral, demonstrating that targeting the host integrin entry pathway is a viable therapeutic strategy.

4. Proposed Mechanism Model

The authors propose a unified model for SIRPA-mediated antiviral activity:

1. Virus Binding: RNA viruses bind to cell surface receptors.
2. SIRPA Activation: This binding triggers FYN kinase to phosphorylate the ITIM tyrosine residues of SIRPA.
3. SHP2 Recruitment: Phosphorylated SIRPA recruits and activates SHP2.
4. Integrin Suppression: Activated SHP2 dephosphorylates key components of the integrin signaling pathway (e.g., FAK, PYK2, NMII).
5. Inhibition of Endocytosis: The suppression of integrin signaling prevents the cytoskeletal rearrangements (actin polymerization) required for the formation of the "phagocytic cup" or endocytic vesicle, thereby blocking viral internalization.

5. Significance

• Novel Entry Mechanism: This study defines a specific host signaling axis (SIRPA-FYN-SHP2-Integrin/FAK) that regulates viral endocytosis, linking the "don't-eat-me" immune checkpoint (SIRPA/CD47) directly to viral restriction.
• Broad Antiviral Potential: The mechanism is not limited to arenaviruses; it also restricts VSV, ZIKV, and Influenza, suggesting a broad-spectrum host dependency on integrin-mediated endocytosis.
• Therapeutic Strategy: The study validates integrin and FAK inhibitors as potent antiviral agents. Since these drugs target host factors rather than viral proteins, they may reduce the likelihood of viral resistance.
• Drug Repurposing: The success of BTT-3033 in vivo suggests that existing integrin-targeting drugs could be repurposed for treating hemorrhagic fevers and other RNA viral infections.

In conclusion, the paper elucidates how SIRPA acts as a gatekeeper against viral entry by hijacking the integrin signaling pathway and demonstrates that pharmacological blockade of this pathway offers a promising therapeutic avenue for RNA virus infections.