Glycosphingolipids Is Critical For Dengue Virus Replication, But Not Virus Binding And Entry

Dengue virus(DENV), a member of the flavivirus genus of the Flaviviridae family, causes the most prevalent arthropod-borne viral disease of humans worldwide. Currently, there is no approved vaccine, nor any antiviral drugs for the treatment of dengue. Glycosphingolipids(GSLs), amphipathic molecules consisting of a ceramide and sugar residues, are ubiquitous cellular components of eukaryotic membrane structures. Recently, GSLs were reported to involve in virus infection by regulating various steps of viral-host cell interaction. However, the distinct role of GSLs during DENV infection remains unclear. In this study, we used mouse melanoma B16 cells and their GSL-deficient mutant counterpart GM95 cells to study the influence of GSLs on DENV infection. We have found that GSL-deficient GM95 cells were highly resistant to DENV infection when compared with B16 wild type cells. Pretreatment of B16 cells with GSLs synthesis inhibitor or impairing the critical enzymes(mt3gal5, ma4 Gal T or mb3 Gn T) required for the synthesis of three major groups of GSLs by RNAi both significantly inhibited dengue virus infection. Instead, the decreased DENV infection was partially rescued by addition of exogenous GSLs into GM95 cells. DENV binding and entry, as determined by q PCR of viral genes or TRITC-labeled virus imaging, were not impaired in GM95 cells when compared with B16 wt cells. We further confirmed that GLSs are critical for DENV RNA replication by directly transfecting a DNA-based dengue virus replicon into B16 and GM95 cells. DENV replicon showed a 50 fold-increase in B16 cells when compared to GM95 cells. Furthermore, GSLs were co-localized with DENV nonstructural protein 4A(NS4A), a known component of viral replication complex(RC) inside the cytoplasm of B16 cells. Taken together, these data indicated that GSLs are not required for DENV virus attachment and endocytosis, however, participate in virus replication. Targeting GSLs could be a novel strategy to inhibit DENV infection.