# The B169L protein of African swine fever virus functions as a viroporin that activates the calcium-mediated inflammasome

## TL;DR

This study shows that a protein from African swine fever virus disrupts calcium balance in cells, triggering inflammation and aiding viral replication.

## Contribution

The study is the first to show that the B169L protein is a viroporin that activates the inflammasome through calcium signaling in African swine fever virus.

## Key findings

- The B169L protein forms calcium-permeable channels that disrupt cellular calcium homeostasis.
- B169L activates the NLRP3 inflammasome, leading to proinflammatory cytokine production.
- Mutations in key residues (P29, K55, K57) abolish B169L's function and reduce inflammation.

## Abstract

African swine fever (ASF) is a highly contagious and often fatal viral disease caused by African swine fever virus (ASFV), which poses a significant economic burden on the global pig industry. ASFV infection triggers a robust production of proinflammatory cytokines, leading to severe inflammation that contributes significantly to the high mortality rate associated with ASF. However, the underlying mechanisms remain incompletely understood. Here, we identified the ASFV B169L protein (pB169L) as a viroporin that exerts dual functions in viral replication and proinflammatory responses. We demonstrated that pB169L formed oligomeric calcium (Ca2+)-permeable channels in vitro by bilayer lipid membrane assay. The ectopically expressed pB169L significantly altered Ca2+ homeostasis in cells and induced robust proinflammatory responses. Mutagenesis revealed critical residues—including P29, K55, and K57—that are indispensable for channel function and proinflammatory signaling. Importantly, the B169L gene knockdown during ASFV infection reduced inflammasome activation and viral replication, highlighting its dual role as both a structural component of virus and an inflammatory mediator. These findings provide the first direct evidence that ASFV encodes a functional viroporin and uncover a novel mechanism by which ASFV manipulates Ca2+ homeostasis to drive inflammasome activation, offering new insights into ASFV pathogenesis and potential antiviral targets.

African swine fever (ASF) is a devastating infectious disease of domestic pigs and wild boar caused by African swine fever virus (ASFV), leading to great socioeconomic losses to the pig industry worldwide. The robust inflammatory responses elicited by ASFV infection is a critical contributor to mortality in pigs. However, the mechanism underlying the proinflammatory responses induced by ASFV infection is not yet fully understood. In this study, we demonstrated that the ASFV B169L protein (pB169L) is a Ca2+-permeable cation channel protein that disrupts Ca2+ homeostasis. This disruption activates the NLRP3 inflammasome, a key trigger of proinflammatory cytokine secretion. Importantly, P29A, K55A, and K57A mutations abolished the pB169L-mediated NLRP3 inflammasome activation by inhibiting pB169L oligomerization and reducing calcium flux, respectively. Furthermore, B169L knockdown inhibits the production of the proinflammatory cytokine interleukin1beta (IL-1β) and IL-18 during ASFV infection. Our findings reveal a previously unrecognized role of B169L in ASFV pathogenesis and highlight the viroporin as potential targets for therapeutic intervention against ASF.