Cyclophilin A Stabilizes the Capsid Protein p72 to Facilitate African Swine Fever Virus Replication

African swine fever virus (ASFV) is a large DNA virus that poses a major threat to the global swine industry. Its virion is encapsulated by an icosahedral capsid predominantly composed of the structural protein p72, which constitutes approximately one-third of the total virion mass. Despite its abundance, the mechanisms regulating p72 stability remain poorly understood. Here, we demonstrate that host-mediated stabilization of p72 is essential for efficient ASFV replication. Mass spectrometry of p72 co-precipitates identified host cyclophilin A (CypA, also known as PPIA) as a key binding partner of p72. CypA interacts with p72 both in vitro and in vivo, specifically engaging exposed regions of p72 via its hydrophobic cavity. CypA interaction stabilizes p72 by reducing K63-linked ubiquitination and preventing proteasomal degradation, whereas cyclic CypA inhibitors destabilize p72 by disrupting this interaction and promoting its ubiquitination. Importantly, genetic or pharmacological inhibition of CypA markedly impairs ASFV replication. Mechanistically, CypA inhibition disrupts viral factory formation and virion assembly by decreasing p72 protein accumulation without affecting its transcription. Together, our findings uncover a previously unrecognized host-dependent mechanism regulating capsid protein stability and highlight host CypA as a promising target for antiviral strategies against ASFV. Overall design: RNA-seq profiling of porcine alveolar macrophages to compare transcriptomic profiles under ASFV infection and NIM811 treatment conditions, including four experimental groups: (1) uninfected with DMSO control, (2) uninfected with NIM811, (3) ASFV-infected with DMSO, and (4) ASFV-infected with NIM811.