Article Figures & Data
Figures
- FIG. 1.
Description of VP1/2-null virus and VP1/2-complementing cell line. (A) Illustration of the PRV genome with characteristic internal and terminal repeats (IR and TR, respectively) shown as white rectangles. BamHI sites are indicated by vertical lollipops. The region of the genome relevant to this report is expanded to show the UL36 and UL37 ORFs, as well as the neighboring UL35 and UL38 ORFs (arrowheads indicate gene orientation). Below, dotted lines indicate regions deleted in the ΔUL36 (PRV-GS678) and ΔUL37 (PRV-GS993) viruses. (B) Ethidium bromide-stained agarose gel of BamHI-digested infectious clone DNAs isolated from E. coli and corresponding viral DNA isolated from nucleocapsids. The UL36 gene overlaps the two largest BamHI fragments of the viral genome, whereas the UL37 gene is exclusively on the second largest fragment. The FRT:kan:FRT insertion is ∼1.5 kbp and encodes two BamHI sites within the kanamycin resistance cassette, resulting in the truncation of the two large fragments during the ΔUL36 construction, and split of the second largest fragment into two smaller fragments during the ΔUL37 construction. Removal of the kanamycin resistance cassette and one FRT equivalent results in fusion of the remainders of the two largest fragments to produce the final ΔUL36 allele, and the reunion of the remainders of the second largest fragment to produce the final ΔUL37 allele. Additional fragment variations seen between the infectious clone plasmids and viral DNA result from loss of the E. coli vector backbone (as described in reference 36).
- FIG. 2.
Initial characterization of VP1/2- and UL37-null viruses and complementing cell lines. (A) Images of living PK15 cells 2 days posttransfection with infectious clones encoding fluorescent capsids (pGS575; “parent”) and clones carrying additional ΔUL36 (pGS678) or ΔUL37 (pGS993) mutations. (B) Images of living PK15 or complementing cells (as indicated) 2 days postinfection with viruses derived from the above three infectious clones (PRV-GS575, PRV-GS678, and PRV-GS993).
- FIG. 3.
Single-step growth kinetics of fluorescent-capsid viruses. (A) Comparison of the growth of the parent virus (PRV-GS575; circles) with the UL36 revertant virus (PRV-GS678R; squares). The ΔUL36 virus was not viable and therefore could not be examined. (B) Comparison of the growth of the parent virus (PRV-GS958; circles) with the ΔUL37 virus (PRV-GS993; triangles) and the UL37 revertant virus (PRV-GS993R; squares). Virions were harvested from the media (dashed lines; open symbols) and cells (solid lines; filled symbols) at the indicated times.
- FIG. 4.
Nuclear egress of capsids into the cytoplasm. The percentage of living cells displaying cytoplasmic capsids 11 to 15 h postinfection is shown. Vero cells were infected with PRV-GS575, having intact UL36 and UL37 genes (“WT”), PRV-GS678 (ΔUL36), PRV-GS993 (ΔUL37), the PRV-GS678R revertant virus (UL36R), or the PRV-GS993R revertant virus (UL37R) at an MOI of ≤0.1. Because capsids near the nuclear rim could not be scored easily in this assay, cells were only counted as positive if at least 10 fluorescent-capsid punctae were observed in the cytoplasm. Error bars are standard error of the proportions (SEp). PRV-GS678 and PRV-GS993 were significantly different from each other and from each revertant virus (z < 0.01).
- FIG. 5.
Transport of capsids in the cytoplasm. Individual moving fluorescent capsids were tracked in the cytoplasm of Vero cells at 18 hpi. Tracking was performed on time-lapse fluorescence recordings collected from a single focal plane at five frames/s. A profile of transport for each infection is shown by plots of the individual capsids tracked as distance from starting position in the first frame (y axis) versus time (x axis). Vero cells were infected with the following viruses: PRV-GS575 (A), PRV-GS678 (B), PRV-GS678R (C), PRV-GS993 (D), and PRV-GS993R (E). (F) Summary of data shown as percentage of tracked capsids that moved greater than 3, 4, 5, 6, 7, 8, and 9 μm from the origins for each infection (shown from left to right, respectively).
- FIG. 6.
Vero cell cytoskeleton susceptibility to cytochalasin D and nocodazole. Uninfected Vero cells were treated for 1 h with cytochalasin D, nocodazole, or both drugs together (or DMSO alone as control). Cells were fixed and processed for immunofluorescence imaging of filamentous actin and microtubules (image pairs).
- FIG. 7.
Dependence of cytoplasmic capsid transport on cytoskeleton integrity. Infected Vero cells were treated with cytochalasin D and nocodazole (or DMSO alone as control) at 17 hpi as indicated. (A to D) Individual moving cytoplasmic fluorescent capsids were tracked at 18 hpi and analyzed as described in the legend of Fig. 5. (E) Summary of data shown as a percentage of tracked capsids that moved greater than 3, 4, 5, 6, 7, 8, and 9 μm from the origins for each infection (shown from left to right). (F) Diffusion profiles of moving cytoplasmic capsids. The mean squared displacement of the capsids from origin is shown over the first 1.6 s of the recordings. Symbols are as follows: ▪, PRV-GS575 (“wild-type”); •, PRV-GS678: ΔUL36; □, PRV-GS575 + DMSO; ▿, PRV-GS575 + cytochalasin D; ▵, PRV-GS575 + nocodazole; ○, PRV-GS575 + cytochalasin D + nocodazole; ⋄, PRV-GS678 + cytochalasin D + nocodazole; ×, PRV-GS575 + cytochalasin D + nocodazole + azide. The error shown is 95% confidence.
Additional Files
HTML Page - index.htslp
Files in this Data Supplement:
- Supplemental file 1
-
Movie S1. Live cell imaging of Vero cells infected with GFP-capsid virus encoding wild-type VP1/2. Although some cytoplasmic
capsids display random motion, many transport directionally along curvilinear trajectories. The large fluorescent structures
demarcate the nucleus, where capsids are assembled.
QuickTime video, 2.6MB. - Supplemental file 2
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Movie S2. Live cell imaging of a Vero cell infected with VP1/2-null, GFP-capsid virus. Although all capsids move, the processive
motion frequently observed with wild-type VP1/2 virus is absent here. Instead, capsids are limited to random motion.
QuickTime video, 2.6MB.
- Supplemental file 1
-
Movie S1. Live cell imaging of Vero cells infected with GFP-capsid virus encoding wild-type VP1/2. Although some cytoplasmic
capsids display random motion, many transport directionally along curvilinear trajectories. The large fluorescent structures
demarcate the nucleus, where capsids are assembled.
