Deficient of glycosylation site in the Envelop protein attenuated Zika virus replication in mosquito cells

Jie Tong^1*Wen-Jing Wang¹Zi-Han Wang¹Jing Li¹Sai-Ya Ma¹Mei He¹Meng-Xuan Liu¹Yu - Fei Zhan¹Feng Jin¹Guosheng Qu¹Chunhong Yin²

• ¹College of Life Sciences, Hebei University, Baoding, China
• ²Shandong Center for Disease Control and Prevention, Shandong, China

The Zika virus (ZIKV) envelope (E) protein is critical for viral replication and host interactions.Although glycosylation of the E protein is known to influence viral infectivity and immune evasion, the specific functional roles of E protein glycosylation in ZIKV infectivity in mosquito cells remain unclear. In this study, we generated a deglycosylation mutant ZIKV with a T156I substitution in the E protein and investigated its effects on viral replication and viral-host interactions in mosquito C6/36 cells. Our results demonstrated that the T156I mutant exhibited attenuated replication compared to the wild-type virus during the early stages (0-24 hours) post-virus infection in mosquito C6/36 cells. This attenuation was associated with reduced E protein expression, which was regulated at the posttranscriptional level. RNA sequencing further revealed that the T156I mutation significantly altered virus-host interactions, particularly affecting the extracellular matrix (ECM) signaling pathway.Notably, several genes involved in the ECM signaling pathway, including THBS1, ITGAL, IL-1A, and CXCL8, were found to inhibit the T156I mutant but not the wild-type ZIKV. Structural analysis and in silico molecular docking suggested that the T156I mutation impaired the stability of the E protein dimer rather than its interactions with neutralizing antibodies. Collectively, these findings provide novel insights into the role of E protein glycosylation in ZIKV infection, and may have significant implications for anti-ZIKV strategies.