# Genome-wide CRISPR screening identifies Annexin A1 as a facilitator of porcine astrovirus entry

## TL;DR

This study identifies Annexin A1 as a key host protein that helps porcine astrovirus enter pig cells, offering a new target for controlling the virus.

## Contribution

The study is the first to identify Annexin A1 as a facilitator of porcine astrovirus entry using a genome-wide CRISPR screen.

## Key findings

- ANXA1 deficiency reduces PAstV binding, early viral signals, and progeny production.
- ANXA1 directly interacts with the viral capsid and supports attachment and uptake.
- Pharmacological targeting of ANXA1 lowers PAstV replication in cell culture.

## Abstract

Porcine astrovirus (PAstV) is an important and widespread pathogen in swine, linked to diarrheal outbreaks and extraintestinal disease. How PAstV enters host cells has remained unclear, and no cellular factor has been defined for PAstV entry. Here, a genome-wide CRISPR–Cas9 loss-of-function screen in porcine epithelial cells identifies Annexin A1 (ANXA1) as a host factor that facilitates PAstV entry. Genetic ablation or pharmacological/antibody blockade of ANXA1 reduces binding, lowers early viral RNA and capsid signals, and delays the rise of progeny, whereas re-expression restores susceptibility. Biochemical assays and surface plasmon resonance indicate a direct interaction between ANXA1 and the acidic C-terminal domain of the PAstV ORF2 capsid protein, and imaging shows ANXA1 co-localizes with incoming particles at the cell surface and supports attachment and uptake. Loss of ANXA1 does not alter infection by the non-astrovirus panel tested, indicating selectivity for PAstV under our conditions. Notably, infection is reduced but not abolished in ANXA1-deficient cells, consistent with additional entry factors acting alongside ANXA1. These findings position ANXA1 as an entry cofactor for PAstV and provide a mechanistic basis to refine models of astrovirus host-cell recognition.

Porcine astroviruses are common causes of diarrhea in piglets, yet how these viruses enter host cells is still unclear. Here, we used a genome-wide CRISPR–Cas9 knockout screen in porcine kidney cells to search for host genes that support porcine astrovirus type 1 (PAstV1) infection. We identified the membrane-associated protein Annexin A1 (ANXA1) as an entry cofactor. Cells lacking ANXA1 bound fewer viruses, showed reduced early viral RNA and capsid protein signals, and produced fewer progeny virions, whereas restoring or overexpressing ANXA1 increased susceptibility. Biochemical and imaging experiments indicate that ANXA1 directly binds the acidic C-terminal region of the viral capsid and co-localizes with incoming particles at the cell surface to support attachment and uptake. Loss of ANXA1 did not alter infection by several unrelated RNA viruses, suggesting selectivity for porcine astroviruses under our conditions. We further show that ANXA1 influences host responses, contributing to RIG-I–IRF3 signalling and virus-induced apoptosis. Pharmacological or antibody-mediated targeting of ANXA1 lowered PAstV replication in cell culture, highlighting the virus–ANXA1 interface as a potential, though context-dependent, target for antiviral intervention.