Genome-wide CRISPR/Cas9 knockout screen identifies host factors essential for Bovine Parainfluenza Virus Type 3 replication

This study is reported by Guojun Wang’s group from the College of Life Sciences, Inner Mongolia University, Hohhot, China. Bovine Parainfluenza Virus Type 3 (BPIV3) is a leading cause of respiratory illness in cattle and a primary component of Bovine Respiratory Disease Complex (BRDC), resulting in significant economic losses to the global cattle industry. To decode the host-dependent mechanisms of BPIV3 infection, the team led by researchers in IMU established a genome-wide CRISPR/Cas9 screening system in bovine cells.

Using unmodified MDBK cells to avoid phenotypic interference, the team optimized screening conditions to ensure library integrity. Multiple rounds of lethal BPIV3 infection enriched key host genes, and validation confirmed drastically reduced viral replication in SLC35A1 and LSM12 knockout cells, verifying the screen’s robustness.

“Identifying these key host factors advances BPIV3 research and highlights new antiviral targets,” noted Guojun Wang, the study’s corresponding author. “Our established CRISPR system lays a solid foundation for reliable virus-host interaction studies.

SLC35A1, a sialic acid transporter regulating cell surface glycosylation, was the most highly enriched candidate factor in this screen. This study confirms its association with BPIV3 infection—knockout of this gene significantly inhibits BPIV3 infection, as BPIV3 preferentially binds to α2,3-linked sialic acids, and treatment with sialyltransferase inhibitors further suppresses BPIV3 infection.

LSM12, an NAADP receptor that regulates calcium homeostasis via TPC channels, is essential for BPIV3 internalization. Mechanistic investigations further confirmed the key role of intracellular Ca²⁺ homeostasis in BPIV3 internalization—this link aligns with LSM12’s known function in mediating TPC channel activation, as its interaction with NAADP enables regulation of TPC-dependent processes supporting viral entry. Additional experiments showed that viral fusion with the endosomal membrane was blocked in LSM12 knockout cells. Decreased endocytic cargo degradation in knockout cells supports this phenotype, suggesting that impaired endosomal acidification disrupts pH-dependent viral fusion with the endosomal membrane in the endo-lysosomal pathway.

In summary, the study identifies two critical BPIV3 host factors, SLC35A1  and LSM12. These findings unlock novel antiviral strategies (modulating sialic acid levels, targeting LSM12-TPC signaling) to combat BPIV3.

---

---