Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus

Constructs used to generate stable cell lines expressing T. ni KHC and KLC that contain epitope tags or EGFP fusions. (A) Schematic diagrams of the expressed KHC or KLC proteins in the stable cell lines indicated on the left. The expressed proteins were tagged at either the N or C terminus with a Myc tag (gray) or an HA tag (black). In addition, two cell lines were generated that had EGFP fused to the C-HA-KHC and C-HA-KLC constructs at the C terminus (green). The relative locations of the motor, stalk, and tail domains in KHC and the heptad repeat and TPR domains in KLC are also indicated. (B) Total protein from polyclonal stably transformed cells was subjected to SDS-PAGE, and the expression of tagged KLC and KHC was analyzed by Western blotting. The HA and Myc tags and the EGFP fusion were detected with the corresponding antibodies, indicated below each blot. Numbers to the left of the gel are molecular masses (in kilodaltons). (C) (Top) Total protein from polyclonal stable cell lines expressing tagged KLC were subjected to SDS-PAGE, and the expression of endogenous KLC and engineered KLC was analyzed by Western blotting with a polyclonal antibody against T. ni KLC. (Bottom) The loading control, which is the same blot shown at the top of panel C probed with anti-actin antibody.

Coimmunoprecipitation of AC141 with C- or N-HA-KLC or C- or N-HA-KHC expressed in stably transformed cells. Cells were infected with WT virus and harvested at 24 hpi; coimmunoprecipitated protein complexes were subjected to Western blotting (WB) and probed with the antibodies shown on the left. Input lanes were loaded with 0.25% of the total input for every blot. (A) Cells of the C- or N-HA-KLC cell line were infected with WT virus, and protein complexes were coimmunoprecipitated with anti-AC141 rabbit polyclonal antibody or the control preimmune serum. Eluent lanes contained 4% of the total eluent for the AC141 blot and 25% of the total eluent for the HA-tagged KLC and β-tubulin blots. IP, immunoprecipitation. (B) Cells of the C-HA-KLC- or N-HA-KLC cell line or control Tn5B1 cells were infected with WT virus, and protein complexes were coimmunoprecipitated with anti-HA beads. Eluent lanes contained 4% of the total eluent for the C- and N-HA-KLC blots and 25% of the total eluent for the AC141 blot. (C) Cells of the C-HA-KHC- or N-HA-KHC cell line or Tn5B1 cells were infected with WT virus, and protein complexes were coimmunoprecipitated with anti-HA beads. The eluent lanes contained 4% of the total eluent for the C- and N-HA-KHC blots and 25% of the total eluent for the AC141 blot. Actin (B) or β-tubulin (C) was used as a loading control (not shown).

Colocalization analysis of HA-AC141 and T. ni Myc-KLC at 20, 24, and 48 hpi. (A) Cells of the N-Myc-KLC cell line were infected with ac141KO-HA-AC141 and fixed at 20, 24, and 48 hpi. Nuclei were stained with DAPI (blue), and HA-AC141 was detected with rabbit polyclonal anti-HA and goat anti-rabbit immunoglobulin antibodies conjugated to Alexa Fluor 488 (green). N-Myc-KLC was detected with mouse monoclonal anti-Myc and goat anti-mouse immunoglobulin antibodies conjugated to Alexa Fluor 635 (red). (B) The Rr and R values for 30 ROIs from 5 different cells were calculated at each time point for N-Myc-KLC and HA-AC141.

Colocalization analysis of HA-AC141 and T. ni Myc-KHC at 20, 24, and 48 hpi. (A) Cells of the N-Myc-KHC cell line were infected with ac141KO-HA-AC141 and fixed at 20, 24, and 48 hpi. Nuclei were stained with DAPI (blue), and HA-AC141 was detected with rabbit polyclonal anti-HA and goat anti-rabbit immunoglobulin antibodies conjugated to Alexa Fluor 488 (green). N-Myc-KHC was detected with mouse monoclonal anti-Myc and goat anti-mouse immunoglobulin antibodies conjugated to Alexa Fluor 635 (red). (B) The Rr and R values for 30 ROIs from 5 different cells were calculated at each time point for N-Myc-KHC and HA-AC141.

Colocalization analysis of AC141, KLC- or KHC-EGFP, and microtubules. KLC-EGFP (A) or KHC-EGFP (B) cells were infected with WT virus at an MOI of 10, and cells were fixed at 24 hpi. AC141 was detected with rabbit polyclonal anti-AC141 and goat anti-rabbit immunoglobulin conjugated to Alexa Fluor 647 (red). β-Tubulin was detected with anti-β-tubulin mouse monoclonal antibody and goat anti-mouse immunoglobulin antibody conjugated to Alexa Fluor 405 (cyan). KLC- or KHC-EGFP is shown in green. The colocalization of β-tubulin with KLC- or KHC-EGFP gives blue pixels, and the colocalization of all three proteins results in pink pixels. (Top) Single two-dimensional images; (bottom) three-dimensional representations of a merged z-stack of the same cells in the top panels.

siRNA downregulation of T. ni HA-KLC expression and the impact on AcMNPV BV production. (A) Cells of the N-HA-KLC cell line were transfected with two KLC siRNAs (siRNA) or two nonspecific control siRNAs (control siRNA) or were mock transfected (mock). At 24 and 48 hpt, cells were harvested and total cell lysates were subjected to Western blotting and probed with anti-HA to detect the expression of N-HA-KLC. Actin was detected with an anti-actin antibody as a loading control. The percentage above each lane shows the levels of N-HA-KLC relative to that in untreated mock-transfected cells, which were given a value of 100%. The levels were determined using Image Lab (v5.1) software. (B) Cells of the N-HA-KLC cell line were transfected with KLC-specific siRNA (lanes +) or nonspecific control siRNA (lanes −) or were untreated and mock transfected (lanes *). At 4, 12, 24, and 48 h after siRNA transfection, the cells were infected with ac141KO-HA-AC141. Supernatants and cells were harvested at 24 hpi. Total cell lysates of infected cells were subjected to Western blotting and probed with anti-HA to analyze the expression levels of N-HA-KLC and HA-AC141. Membranes were reprobed for actin as a loading control. (C) BV levels in supernatants were determined by TCID₅₀ endpoint dilution assay and are presented as a percentage of the value for the untreated control, which was given a value of 100%. BV titers are the averages from two biological replicates, each of which was performed with two technical replicates.

Association of nucleocapsid proteins VP39, FP25, and BV/ODV-C42 with C-HA-KLC or N-HA-KLC. Analysis of coimmunoprecipitation of VP39, FP25, and BV/ODV-C42 with N- or C-HA-KLC. Tn5B1 and C- or N-HA-KLC cells were infected with WT virus at an MOI of 10, the cells were harvested at 24 hpi, and protein complexes were coimmunoprecipitated with anti-HA beads and analyzed by Western blotting by probing with the antibodies indicated on the left. The VP39 and BV/ODV-C42 blots were reprobed with anti-β-tubulin as a loading control. The input lanes contained 0.25% of the total lysate, and the eluent lanes contained 25% of the total eluent.

Colocalization of VP39-3×mCherry nucleocapsids with microtubules during entry and egress. (A) Tn5B1 cells infected with VP39-3×mCherry showing mCherry-labeled nucleocapsids (red) during entry at 0.5 hpi (top) and during egress at 24 hpi (bottom). Bars = 5 μm. Each panel is a z-stack maximum projection of 6 to 7 sections that encompasses the entire fluorescence of each NC. The microtubules were detected with mouse monoclonal antibody against β-tubulin and the corresponding secondary antibody, goat anti-mouse immunoglobulin antibody conjugated to Alexa Fluor 488 (green). The nucleus is stained with DAPI (blue). The enlarged region (center) shows potential regions of colocalization by the presence of yellow pixels (white arrows). White pixels (right) show regions of maximum colocalization, as determined by Pearson's coefficients of overlap (Leica confocal LAS X software). DAPI staining is not shown in the enlarged images. (B) Comparison of the average percentage of cytoplasmic VP39-3×mCherry nucleocapsids (NCs) that colocalize with microtubules (MTs) at 0.25 and 0.5 hpi and 20 and 24 hpi. Each bar of the graph represents the results for nucleocapsids from a total of 10 cells. Error bars represent standard errors of the means. (C) Confocal microscopy of Tn5B1 cells infected with VP39-3×mCherry at an MOI of 10 and fixed at 30 hpi. The image is a z-stack maximum projection through the entire cell showing the extensive ribbon structure of VP39-3mXCherry through the nuclear virogenic stroma. Arrows in enlarged panels 1 and 2, locations of cytoplasmic nucleocapsids. Staining of microtubules and the nucleus is the same as that described in the legend to panel A.