The neddylation of the RNA-dependent RNA polymerase 3D of Coxsackievirus B3 promotes viral replication

The pathogenesis of Coxsackievirus B3 (CVB3), the causative pathogen for severe diseases such as myocarditis, pancreatitis, and meningitis, remains largely unknown. Neddylation, the covalent modification of proteins through the conjugation of NEDD8, a ubiquitin-like molecule, plays critical roles in regulating cellular activities. Our previous study demonstrated that 3D^pol, the RNA-dependent RNA polymerase of CVB3, was modified by ubiquitin. Since NEDD8 is highly homologous to ubiquitin in both the sequence and structure, we hypothesized that 3D^pol may also undergo neddylation. Here, we demonstrated that 3D^pol of CVB3 is modified by NEDD8. Proteomics and immunoprecipitation studies identified that NEDD8 expression was upregulated in CVB3-infected cells, and 3D^pol was neddylated. Through the overexpression or knockdown of the subunit of NEDD8-activating enzyme or NEDD8, we show that neddylation promoted CVB3 replication. Furthermore, the overexpression of NEDD8-specific protease 1 completely blocked 3D^pol neddylation and suppressed viral replication. The neddylation sites of 3D^pol, K261, and K457 were identified by mutagenesis studies. We further showed that neddylation enhanced 3D^pol stability. In contrast, when the neddylation sites were mutated (K261R, K457R, or K261/457R), the 3D^pol degradation rate was significantly accelerated. Moreover, the growth kinetics of the recombinant viruses containing the mutated 3D^pol at the neddylation sites was markedly decreased. Through analyzing the 3D^pol interacting proteins with mass spectrometry, we identified that TRIM4 is the E3 ligase, which upregulates 3D^pol neddylation. TRIM4 knockdown markedly suppressed viral replication and almost completely blocked 3D^pol neddylation. In contrast, TRIM4 overexpression promoted CVB3 replication. Taken together, this study demonstrated that 3D^pol neddylation facilitates CVB3 replication.IMPORTANCECVB3 infection is commonly related to the inflammatory disease of the heart, which may develop to dilated cardiomyopathy and heart failure. Neddylation is a process in which the ubiquitin-like molecule NEDD8 is covalently linked to the specific lysine residues of the target proteins. Increasing evidence has shown that the neddylation of either host or viral proteins may alter viral replication. In this study, we demonstrated that 3D^pol, the RNA-dependent RNA polymerase of CVB3, is modified by NEDD8 at its lysine residues 261 and 457, and the neddylation process is mediated by the E3 ligase TRIM4. Neddylation enhances the stability of 3D^pol and facilitates viral replication, while viruses with mutated 3D^pol, which cannot be neddylated, showed decreased replication capacity. Our findings not only add novel insights for understanding the pathogenesis of CVB3 but also identify that targeting neddylation could be a potential antiviral strategy for the treatment of CVB3 infection.