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

Abstract

Members of the Reoviridae 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.

Keywords: bluetongue virus; viral inclusion bodies; virus egress; virus maturation.

Figure 1

Both inner and outer viral capsid proteins can be found within VIBs. (a) Immunofluorescence analysis of the intracellular localization of the non-structural protein NS2 (red), the outer capsid protein VP5 (green), and the inner capsid protein VP6 (yellow) from 4h to 8 h.p.i., in cells infected at an MOI 5. Scale bar = 5 µm. n, nucleus. (b) Enlargement of section from (a) showing NS2, VP5, and VP6 individually. Scale bar = 1 µm. (c) Quantification of Pearson’s correlation coefficient between VP6 and VP5 with VIBs (NS2). The data are depicted as means with SE, n = 30. (d) Densitometry analysis of Western blot detection of the expression of the viral proteins NS2, VP5, VP6, and host cell GAPDH from 5 to 10 h.p.i. (Supplementary Figure S1). BSR cells were infected with BTV1 as above. The Western blots are expressed as a percentage, normalized to GAPDH. Error bars represent the SD values from three independent experiments. (e) Western blot detection of the viral proteins NS2, VP5, VP6, and host cell GAPDH at 8 h.p.i from Co-IPs using control antibody and GP-anti-NS2 antibody bound Protein A beads. BSR cells were infected with BTV1 as above. (f) Western blot detection of the viral proteins NS2, VP5, VP6, and host cell GAPDH at 8 h.p.i. from centrifugal enrichment of VIBs from cells treated as above.

Figure 2

The dynamic nature of NS3 association with VIBs during the BTV replication cycle. Immunofluorescence analysis of the intracellular localization of the VIB-specific non-structural protein NS2 (red), the non-structural protein NS3 (magenta), and the outer capsid protein VP5 (green) from 4 h.p.i. to 9 h.p.i. Scale bar = 10 µm. (b) Enlargement of section from (a) showing NS2, VP5, and VP6 individually. Scale bar = 1 µm. (c) Quantification of Pearson’s correlation coefficient between VP5 and NS3 with VIBs (NS2). The data are depicted as means with SE, n = 30, * p < 0.01. (d) Densitometry analysis of the Western blots (Supplementary Figure S2) expressed as a percentage, normalized to GAPDH. Error bars represent the SD values from three independent experiments.

Figure 3

NS3 associates with the periphery of VIBs during the BTV replication cycle and outer capsid protein VP5. Immunofluorescence analysis of the localization of the VIB-specific non-structural protein 2 (NS2) and the non-structural protein 3 (NS3) and the outer capsid protein VP5. (a) VIB localization of NS2 (red) and NS3 (magenta) in BTV infected cells 9 h.p.i. (scale bar = 5 µm). Right panel: 3D projection of the z-stack. (b) VIB localization of VP5 (green) and NS3 (magenta) in BTV infected cells 8.5 h.p.i. (scale bar = 5 µm). Right panel: 3D projection of the z-stack. (c) VIB localization of the non-structural protein NS3 (magenta) and the outer capsid protein VP5 (red) at 5 h.p.i and 8 h.p.i. as observed by SIM (scale bar = 5 µm). (d) VIB localization of the non-structural protein NS3 (magenta), NS2 (red), and VP5 (green) at 8.5 h.p.i. as observed by SIM (scale bar = 5 µm). White arrows indicate peripheral association of NS3 with VIB.

Figure 4

NS3 association with VIBs decreases during the BTV replication cycle. Analysis of cell lysates of mock and infected BSR cells. (a) Western blot detection of the viral proteins NS2, VP5, VP6, NS3, and host cell GAPDH at 5 and 8 h.p.i from differential velocity centrifugation for VIBS from cells infected at an MOI of 5. (b) Densitometry analysis of the Western blots (a) expressed as a percentage, normalized to NS2. Error bars represent the SD values from three independent experiments. * p < 0.01.

Figure 5

NS3 trafficking mutants affect VP5 export to the plasma membrane. (a) Confocal microscopy images of VP5 (green) and NS3 (purple) in BSR cells infected by either WT BTV (top row), BTV NS3PBM1 (middle row), and BTV NS3PBM2 (bottom row) viruses 24 h.p.i, and labelled with the membrane fluorescent marker Wheat germ agglutinin (WGA, white). The nucleus is shown in blue, and the scale bar represents 10 μm (white line, bottom left corner). The squares represent the region of interest magnified and shown in (b), where the scale bar represents 2 µm (white line). The colocalization between WGA and VP5 in whole cells was quantified for each virus using (c) the Pearson’s correlation coefficient. The data are depicted as means and SD; n = 30; ** p < 0.01; **** p < 0.0001).