Trafficking of Bluetongue Virus Visualized by Recovery of Tetracysteine-Tagged Virion Particles

Abstract

Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a double-capsid insect-borne virus enclosing a genome of 10 double-stranded RNA segments. Like those of other members of the family, BTV virions are nonenveloped particles containing two architecturally complex capsids. The two proteins of the outer capsid, VP2 and VP5, are involved in BTV entry and in the delivery of the transcriptionally active core to the cell cytoplasm. Although the importance of the endocytic pathway in BTV entry has been reported, detailed analyses of entry and the role of each protein in virus trafficking have not been possible due to the lack of availability of a tagged virus. Here, for the first time, we report on the successful manipulation of a segmented genome of a nonenveloped capsid virus by the introduction of tags that were subsequently fluorescently visualized in infected cells. The genetically engineered fluorescent BTV particles were observed to enter live cells immediately after virus adsorption. Further, we showed the separation of VP2 from VP5 during virus entry and confirmed that while VP2 is shed from virions in early endosomes, virus particles still consisting of VP5 were trafficked sequentially from early to late endosomes. Since BTV infects both mammalian and insect cells, the generation of tagged viruses will allow visualization of the trafficking of BTV farther downstream in different host cells. In addition, the tagging technology has potential for transferable application to other nonenveloped complex viruses. IMPORTANCELive-virus trafficking in host cells has been highly informative on the interactions between virus and host cells. Although the insertion of fluorescent markers into viral genomes has made it possible to study the trafficking of enveloped viruses, the physical constraints of architecturally complex capsid viruses have imposed practical limitations. In this study, we have successfully genetically engineered the segmented RNA genome of bluetongue virus (BTV), a complex nonenveloped virus belonging to the Reoviridae family. The resulting fluorescent virus particles could be visualized in virus entry studies of both live and fixed cells. This is the first time a structurally complex capsid virus has been successfully genetically manipulated to generate virus particles that could be visualized in infected cells.

Elucidating virus entry using a tetracysteine-tagged virus

“…This suggested that the 100kDa protein fragment lacked the first 94 amino acids. With an exposed loop identified, the TC tag was inserted, for subsequent determination of whether its introduction still preserved overall folding and biological activity (14).…”

“…The power of this technology was demonstrated through the enabling of the delineation of the dynamics of the two outer capsid proteins, which were shown to dissociate from one another during the early stages of virus entry into mammalian cells (14). Here we describe the prerequisite methodology that led to this observation.…”

“…Recently, it was shown that such limitations could be overcome by the successful genetic engineering of the segmented RNA genome of Bluetongue virus (BTV), a complex non-enveloped Orbivirus belonging to the Reoviridae family. This facilitated the generation of fluorescent virus particles that could be visualized in virus entry studies for both live and fixed cells (14). BTV, with multiple serotypes (27 recognized to date) is endemic in most parts of the world, often resulting in high morbidity and mortality in ruminants.…”

Visualization of hepatitis B virus entry – novel tools and approaches to directly follow virus entry into hepatocytes

“…One well characterized small tag is the 6‐amino acid tetracysteine (TC) tag, which can be labelled with biarsenical dyes (FLAsH). Successful visualization using the TC tag has been shown for HIV , alphavirus , bluetongue virus and HCV . For HBV, the TC tag has been incorporated into the core protein, and virus particles labelled with a biarsenical dye were secreted into the medium .…”

Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged μNS for Live-Cell Visualization of Viral Factories

“…FlAsH-EDT2 was first described in 1998 and was shown to fluoresce when bound to two pairs of cysteines separated by two amino acids (CCXXCC), referred to as a tetracysteine (TC) tag (22). This technique has been utilized in several recombinant viruses, including HIV-1, to visualize TC-Gag protein during infection, and in bluetongue virus, a member of the Reoviridae family, to label viral protein VP2 to visualize virus particle entry (23,24). Once the TC tag is added to a protein, FlAsH-EDT2, which contains two 1,2-ethanedithiol motifs, creates covalent bonds with the TC motif, resulting in fluorescence.…”