The K247 ubiquitination site of the Newcastle disease virus M protein enhances viral budding to facilitate rapid exploitation of host cell sphingolipid metabolites

Abstract

Newcastle disease virus (NDV), a significant avian paramyxovirus, depends on the acquisition of host-derived membranes for viral envelope assembly during budding. However, the relationship between NDV budding efficiency and host metabolic reprogramming remains incompletely understood. Our previous research demonstrated that the highly virulent strain Herts/33 and the non-virulent strain LaSota differ significantly in budding efficiency, which is attributed to the difference in ubiquitination levels at the K247 site of the M protein. In this study, using recombinant viruses generated in our previous study, we investigated the effects of the K247 ubiquitination site on host cell metabolism. Through RNA sequencing and LC–MS/MS, systematically profiled the transcriptional and metabolic alterations induced by these viruses in HeLa cells. We also compared organ pathology in three-week-old SPF chicks infected with Herts/33 versus LaSota. The results showed that NDV infection broadly reprograms host sphingolipid metabolism. Compared with rLaSota-WT, rLaSota-R247K exhibited higher budding efficiency, linked to accelerated ceramide depletion and a stronger innate immune response. Specifically, rLaSota-R247K infection upregulated interferon- and interleukin-related components such as ISG15, CXCL8, TNF-α, CXCL10, NOD2, CD274, OAS, and IFNB1. Furthermore, we confirmed that blocking the ceramide synthesis pathway significantly suppresses NDV-M protein-mediated budding of VLPs and virions. Pathologically, Herts/33 induced more severe tissue damage than LaSota. Together, these findings indicate that the K247 residue of the NDV M protein enhances viral budding and promotes rapid utilization of host sphingolipids. This work provides mechanistic insight into NDV budding and highlights virus–host metabolic interactions that may inform future antiviral strategies.