Role of an arbovirus nonstructural protein in cellular pathogenesis and virus release

Abstract

The insect-borne Bluetongue virus (BTV) is considered the prototypic Orbivirus, a member of the Reovirus family. One of the hallmarks of Orbivirus infection is the production of large numbers of intracellular tubular structures of unknown function. For BTV these structures are formed as the polymerization product of a single 64-kDa nonstructural protein, NS1, encoded by the viral double-stranded RNA genome segment 6. Although the NS1 protein is the most abundant viral protein synthesized in infected cells, its function has yet to be determined. One possibility is that NS1 tubules may be involved in the translocation of newly formed viral particles to the plasma membrane, and NS1-specific monoclonal antibodies have been shown to react with viral particles leaving infected cells. In the present study we generated a mammalian cell line that expresses a recombinant single-chain antibody fragment (scFv) derived from an NS1-specific monoclonal antibody (10B1) and analyzed the effect that this intracellular antibody has on BTV replication. Normally, BTV infection of mammalian cells in culture results in a severe cytopathic effect within 24 to 48 h postinfection manifested by cell rounding, apoptosis, and lytic release of virions into the culture medium. However, infection of scFv-expressing cells results in a marked reduction in the stability of NS1 and formation of NS1 tubules, a decrease in cytopathic effect, an increased release of infectious virus into the culture medium, and budding of virions from the plasma membrane. These results suggest that NS1 tubules play a direct role in the cellular pathogenesis and morphogenesis of BTV.

FIG. 1.

Construction of a double stable BSR cell line for coexpression of scFv-αNS1 and EGFP. (A) The scFV-αNS1 gene fragment was rescued from phage and ligated into the Tet-Off-responsive plasmid pB1-EGFP (Clonetech) and cotransfected into BSR Tet-Off cells along with pTK-Hyg (Clonetech) carrying a selectable marker (Hyg^r) for isolation of clones in the presence of hygromycin. MCS, multiple cloning site. (B) One clone, designated BSR-scFv-αNS1 (shown in all four panels; light microscopy was used to produce the images shown in the top panels, and fluorescence microscopy was used to produce the images of the same clone shown in the bottom panels), was selected for its tight regulation and high-level expression of the EGFP reporter gene in the absence (−) of Dox, as determined by fluorescence microscopy (bottom right panel). There was no EGFP fluorescence observed in the presence of Dox (bottom left panel). (C) Radioimmunoprecipitation and SDS-PAGE analysis of BSR-scFv-αNS1 cells in the presence (+) and absence (−) of Dox, showing expression of scFv-αNS1 in the absence of Dox (right lane).

FIG. 2.

CPE of BTV-10 infection in the presence and absence of scFv-αNS1. Light microscopy of uninfected BSR-scFv-αNS1 cells (left panels) or infected with BTV-10 at 1 day p.i. (middle panels) and 2 days p.i. (right panels) in the presence (top panels) or absence (bottom panels) of Dox. Little-to-no CPE was observed in cells expressing the scFv-αNS1 molecule (bottom middle and bottom right panels), whereas extensive CPE (typical of BTV infection in BSR cells) was observed in the presence of Dox (top middle and top right panels).

FIG. 3.

Velocity gradient analysis of NS1-containing subcellular structures. BSR-scFv-αNS1 cells were infected with BTV-10 in the presence (A) or absence (B) of Dox, radiolabeled with [³⁵S]Met-Cys, and analyzed by sucrose gradient centrifugation (20 to 60%), immunoprecipitation, and SDS-PAGE for the formation of NS1 tubules. A typical pattern of multimeric NS1 distribution about two-thirds of the way into the gradient consistent with tubule formation was observed in the presence of Dox (A). In contrast, no NS1 protein was detected in this portion of the gradient in the absence of Dox (B), indicating that tubules were not formed.

FIG. 4.

Pulse chase analysis of NS1 expression in the presence and absence of scFv-αNS1. BSR-scFv-αNS1 cells were infected with BTV-10 in the presence (+) or absence (−) of Dox, pulse labeled (P) with [³⁵S]Met-Cys for 15 min, chased (C) with unlabeled medium for 4 h, and analyzed by immunoprecipitation with RaαNS1 antiserum and SDS-PAGE. Uninfected cells (uninf) were used as controls. NS1 protein was stable after the 4-h chase in the presence of Dox (fourth lane from the left), whereas NS1 appeared to be degraded after 4 h in the absence of Dox when scFv-αNS1 was being expressed (asterisk in lane 8). Under these conditions all of the BTV structural proteins are often coprecipitated with NS1 due to a weak interaction between virions and the NS1 protein.

FIG. 5.

Ultrastructural analysis of BTV-10 infection in the presence and absence of scFv-αNS1. BSR-scFv-αNS1 cells were infected with BTV-10 in the presence (+) or absence (−) of Dox and prepared for electron microscopy at 24 h p.i. as described in Materials and Methods. (A) Typical effects of BTV infection in mammalian cells; these effects include the presence of cytoplasmic viral inclusion bodies (VIBs), tubules (TUBs), and mature virus particles and areas of severe morphological destruction near the plasma membrane consistent with lytic release of virus. (B) During expression of scFv-αNS1, no cytoplasmic tubules were detected and virus particles were seen exiting cells by budding from the plasma membrane. Bar, 500 nm.