Article Figures & Data
Figures
- Fig. 1.
Schematic representation of NS3. The two cytoplasmic domains (N terminal and C terminal), the two transmembrane domains (TM), and the extracellular domain (EC) are indicated. Numbers above indicate amino acid position according to the NS3 amino acid sequence. The glycosylation site at asparagine position 150 is also shown (arrow). The changes in either the first or the second methionine residue introduced by site-directed mutagenesis are shown in the lower panel; dots indicate no change, and dashes indicate absence of expressed residues.
- Fig. 2.
Characterization of BTVM1 and BTVM14 mutant viruses. (A) The total titer at different time points postinfection for either mutant or WT viruses in complementary BSR/NS3 cells (left panel) or normal BSR cells (right panel) was determined, expressed as PFU/ml, and plotted on a logarithmic scale. (B) The expression of NS3 was assessed in cell lysates from mammalian BSR cells at 48 h postinfection with mutant virus BTVM1 (lane 2) or BTVM14 (lane 3); cell lysates from mock-infected cells (lane 4) or WT-infected cells (lane 1) were included. Lane M, molecular mass standard in kDa. Western blotting was performed using an antibody against BTV NS3. (C) Genomic dsRNA from BSR cells infected with WT virus (lane 1) or mutant virus BTVM1 (lane 2) or BTVM14 (lane 3) was purified and analyzed on a nondenaturing polyacrylamide gel.
- Fig. 3.
Interaction of BTV NS3 with S100A10/p11 in infected mammalian cells. (A) Colocalization of NS3 (green) with S100A10/p11 (red). BSR cells were infected with WT or mutant viruses as indicated and at 24 h postinfection were fixed, permeabilized, and processed for confocal microscopy. (B) Coimmunoprecipitation of NS3 and S100A10/p11. BSR cells were infected with WT virus (lane 1) or mutant viruses BTVM1 (lane 2) and BTV M14 (lane 3) and at 36 h postinfection were harvested and processed as described in Materials and Methods. Expression of NS3 in cell lysates was assayed by Western blotting using an antibody against NS3 (left panel). As control, a lysate from noninfected cells (lane 4) was included. The coimmunoprecipitation of NS3 WT virus (right panel, lane 1) or mutants M1 (lane 2) and M14 (lane 3) with S100A10/p11 was performed using a monoclonal antibody that recognized S100A10/p11 and analyzed by Western blotting with an antibody against NS3. As controls, a lysate from mock-infected cells (lane 4) and a lysate from cells infected with WT virus but without antibody against S100A10/p11 (lane 5) were included.
- Fig. 4.
Schematic representation of the substitution mutations introduced in the N-terminal end of NS3. Specific changes in the first 13 residues in each mutant (Q7P, R8E, and E10A) are indicated. Dots indicate no change.
- Fig. 5.
Characterization of mutant BTVQ7P, BTVR8E, and BTVE10A viruses. (A) Complementary BSR/NS3 cells (left panel) or normal BSR cells (right panel) were infected with mutant virus BTV Q7P, R8E, or E10A or with the WT and harvested at different time points postinfection as indicated. Total titer was determined by plaque assay, expressed as PFU/ml, and plotted on a logarithmic scale. (B) The expression of NS3 was assessed by Western blotting in BSR cell lysates processed at 48 h postinfection with mutant virus Q7P (lane 1), R8E (lane 2), or E10A (lane 3). Lysate from cells infected with WT BTV (lane 4) was included as control. The number on the left indicate the molecular mass standard in kDa. (C) Genomic dsRNA purified from BSR cells infected with BTVQ7P (lane 2), BTVR8E (lane 3), or BTVE10A (lane 4) was purified and analyzed in a nondenaturing polyacrylamide gel. As a control, dsRNA from WT virus was included (lane 1).
- Fig. 6.
Effects of single substitutions in NS3 on NS3-S100A10/p11 interaction. (A) Colocalization of NS3/NS3A (green) with S100A10/p11 (red) proteins. BSR cells were infected with mutant viruses as indicated and at 24 h postinfection were processed for confocal microscopy as described in Materials and Methods. (B) Interaction of NS3 with S100A10/p11. BSR cells were infected with WT or mutant viruses and at 36 h postinfection cells were processed as described in Materials and Methods. The expression of NS3 in lysate from cells infected with BTVQ7P (lane 1), BTVR8E (lane 2), or BTVE10A (lane 3) was verified by Western blotting (left panel). The coimmunoprecipitation of NS3 using a monoclonal antibody that recognizes S100A10/p11 was analyzed by Western blotting using the antibody against NS3 (right panel).
- Fig. 7.
Ultrastructural analysis of mammalian cells infected with mutant or WT viruses. BSR cells were infected with WT, BTVM1, BTVM14, or BTVQ7P virus, and at 24 h postinfection cells were processed for sectioning. Arrows in the left panels indicate particles. An area of each section is amplified in the right panels to show the detail. Bars, 500 nm.
- Fig. 8.
Virus growth and virus release from Culicoides-derived cells. (A) KC cells were infected with mutant BTVM1, BTVM14, BTVQ7P, BTVR8E, or BTVE10A or with WT virus, and at 1, 2, and 3 days postinfection cells and supernatant were harvested and the total titer was determined by plaque assay. (B) KC cells were infected with mutant or WT viruses, and at 4 days postinfection cell-free and cell-associated fractions were collected. The titer of each fraction was determined and the relative release calculated as the ratio of cell-free to total virus titer, normalized for the control virus, and plotted on a logarithmic scale. Bars represent the averages and standard deviations from at least two experiments.
- Fig. 9.
Cell sectioning analysis of infected insect Culicoides cells. (A) KC cells were infected with WT virus, harvested at 2, 4, and 6 days postinfection, and processed for cell sectioning. White arrows indicate virus particles and black arrows membrane structures. (B) KC cells were infected with mutant BTVM1, BTVM14, or BTVQ7P viruses as indicated and processed at 6 days postinfection. Bar, 500 nm.
