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Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

London School of Hygiene and Tropical Medicine

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.

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

Members of the  family assemble virus factories within the cytoplasm of infected cells to replicate and assemble virus particles. Bluetongue virus (BTV) forms virus inclusion bodies (VIBs) that are aggregates of viral RNA, certain viral proteins, and host factors, and have been shown to be sites of the initial assembly of transcriptionally active virus-like particles. This study sought to characterize the formation, composition, and ultrastructure of VIBs, particularly in relation to virus replication. In this study we have utilized various microscopic techniques, including structured illumination microscopy, and virological assays to show for the first time that the outer capsid protein VP5, which is essential for virus maturation, is also associated with VIBs. The addition of VP5 to assembled virus cores exiting VIBs is required to arrest transcriptionally active core particles, facilitating virus maturation. Furthermore, we observed a time-dependent association of the glycosylated non-structural protein 3 (NS3) with VIBs, and report on the importance of the two polybasic motifs within NS3 that facilitate virus trafficking and egress from infected cells at the plasma membrane. Thus, the presence of VP5 and the dynamic nature of NS3 association with VIBs that we report here provide novel insight into these previously less well-characterized processes.

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

Bluetongue virus (BTV) is an arthropod-borne virus infecting livestock. Its frequent emergence in Europe and North America had caused significant agricultural and economic loss. BTV is also of scientific interest as a model to understand the mechanisms underlying non-enveloped virus release from mammalian and insect cells. The BTV particle, which is formed of a complex double-layered capsid, was first considered as a lytic virus that needs to lyse the infected cells for cell to cell transmission. In the last decade, however, a more in-depth focus on the role of the non-structural proteins has led to several examples where BTV particles are also released through different budding mechanisms at the plasma membrane. It is now clear that the non-structural protein NS3 is the main driver of BTV release, via different interactions with both viral and cellular proteins of the cell sorting and exocytosis pathway. In this review, we discuss the most recent advances in the molecular biology of BTV egress and compare the mechanisms that lead to lytic or non-lytic BTV release.

Multiple Routes of Bluetongue Virus Egress.

Recent developments on extracellular vesicles (EVs) containing multiple virus particles challenge the rigid definition of non-enveloped viruses. However, how non-enveloped viruses hijack cell machinery to promote non-lytic release in EVs, and their functional roles, remain to be clarified. Here we used Bluetongue virus (BTV) as a model of a non-enveloped arthropod-borne virus and discovered that the majority of viruses are released in EVs. Based on the cellular proteins detected in these EVs, and use of inhibitors targeting the cellular degradation process, we demonstrated that these extracellular vesicles are derived from secretory lysosomes, in which the acidic pH is neutralized upon the infection. Moreover, we report that secreted EVs are more efficient than free-viruses for initiating infections, but that they trigger super-infection exclusion that only free-viruses can overcome.

Thomas Labadie

Department of Infection Biology

London School of Hygiene and Tropical Medicine

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Thomas Labadie

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Infection Biology

London School of Hygiene and Tropical Medicine

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Department of Infection Biology