Bluetongue Virus Nonstructural Protein 3 Orchestrates Virus Maturation and Drives Non-Lytic Egress via Two Polybasic Motifs

Abstract

Bluetongue virus (BTV) is an arthropod-borne virus that infects domestic and wild ruminants. The virion is a non-enveloped double-layered particle with an outer capsid that encloses a core containing the segmented double-stranded RNA genome. Although BTV is canonically released by cell lysis, it also exits non-lytically. In infected cells, the BTV nonstructural glycoprotein 3 (NS3) is found to be associated with host membranes and traffics from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane. This suggests a role for NS3 in BTV particle maturation and non-lytic egress. However, the mechanism by which NS3 coordinates these events has not yet been elucidated. Here, we identified two polybasic motifs (PMB1/PMB2), consistent with the membrane binding. Using site-directed mutagenesis, confocal and electron microscopy, and flow cytometry, we demonstrated that PBM1 and PBM2 mutant viruses retained NS3 either in the Golgi apparatus or in the endoplasmic reticulum, suggesting a distinct role for each motif. Mutation of PBM2 motif decreased NS3 export to the cell surface and virus production. However, both mutant viruses produced predominantly inner core particles that remained close to their site of assembly. Together, our data demonstrates that correct trafficking of the NS3 protein is required for virus maturation and release.

Differential Localization of Structural and Non-Structural Proteins during the Bluetongue Virus Replication Cycle

“…Viruses were titrated using a plaque assay. NS3 PBM mutant viruses (NS3 PBM1 and NS3 PBM2 ) were generated as previously described [9].…”

“…Since NS3 has been shown to be associated with VP5 previously during virus assembly and egress [21,29], we wanted to examine the localization of NS3 in relation to VIBs. NS3 is a membrane-associated glycosylated protein involved in virus trafficking and budding [9,16]. During the virus egress, NS3 interacts with VP5 to mediate intracellular trafficking to the plasma membrane [21].…”

“…One characteristic of BTV infection is the formation of large globular structures called viral inclusion bodies (VIBs) in the infected cell cytoplasm, which lack a membrane [8,9]. The major component of the VIBs is the non-structural protein 2 (NS2).…”

“…While NS2 is indispensable for virus replication and core assembly, the non-stuctural protein 3 (NS3) protein is associated with intracellular virus trafficking [16]. During virus infection, NS3 is glycosylated and associates with intracellular membranes as well as the plasma membrane [9,17,18]. The carboxy-terminal of NS3 interacts with several cellular proteins, such as a subunit of the calpactin complex of the exocytic pathway, the cellular component p11 (S100A10), Tsg101, and the E3 ubiquitin-protein ligase NEDD4 family of proteins [16,19,20].…”

“…BTV has been found to require multi-vesicular-body (MVB) components and exocytic pathway proteins for infectious virus production [20]. NS3 also interacts with the outer capsid proteins VP5 and VP2, through its cytoplasmic N-terminal domain, hence hypothesized to be involved in virus maturation [9,16,21].…”