PEDV Nsp8 alanine-53 promotes viral replication in a partially DTX3L-dependent manner

Abstract Porcine epidemic diarrhea virus (PEDV) employs multiple strategies to interact with the host and promote its replication, with nonstructural proteins playing critical roles; however, the underlying mechanisms remain largely unclear. In this study, we found that PEDV infection significantly downregulated DTX3L expression both in vivo and in vitro, whereas knockdown of DTX3L markedly enhanced viral replication. Using a DTX3L knockout (DTX3L-KO) cell model combined with transcriptomic analysis, we further demonstrated that DTX3L knockout negatively regulates interferon expression and downstream signaling. Mechanistically, PEDV Nsp8 downregulates DTX3L expression. Sequence alignment revealed that while most PEDV strains harbor an alanine (A) at position 53 of Nsp8, the G2b strain AH2012/12 has a valine (V) substitution. Notably, compared with valine-53 (Nsp8-V53), Nsp8 with alanine-53 (Nsp8-A53) had a stronger downregulatory effect on DTX3L. Using reverse genetics, we generated the recombinant virus rNsp8-V53A (the V53A substitution of Nsp8 in the AH2012/12 strain), which replicated more efficiently and downregulated DTX3L more significantly in wild-type cells than the parental rAH2012/12 strain, whereas this increase was partially abolished in DTX3L-KO cells. Collectively, our findings demonstrate that PEDV promotes replication by downregulating DTX3L expression, with alanine‑53 in Nsp8 playing a critical role in a partially DTX3L‑dependent manner. This study reveals a novel immune evasion mechanism and highlights the importance of nonstructural protein variation in PEDV evolution.