Recombinant bovine papillomavirus (BPV) L1 virus-like particles (VLPs) and BPV virions isolated from cow warts (15
) were purified by density ultracentrifugation in CsCl. CsCl was removed by dialysis into gradient buffer (20 mM Tris-Cl [pH 7.2], 5% glycerol, 180 mM NaCl, 10 mM KCl, 1 mM CaCl2
, 1 mM MgCl2
), and the VLPs or virions were then treated with either no chaperones or the purified recombinant prokaryotic chaperones DnaK, DnaJ, and GrpE (KJE) (Stressgen) (7
). These reactions were carried out by dialysis against reaction buffer (50 mM Tris-Cl [pH 7.2], 5% glycerol, 180 mM NaCl, 10 mM KCl, 1 mM CaCl2
, 20 mM MgCl2
, 20 mM ATP) or against reaction buffer with 20 mM ADP instead of ATP overnight at room temperature with 4 μM KJE.
Reaction mixtures were absorbed to glow-discharged, carbon, and Formvar-coated copper grids (G400 copper; EM Sciences), rinsed with buffer lacking phosphates, stained with uranyl acetate, and examined by transmission electron microscopy (TEM) (Philips CM10) at 80 kV. No morphological differences between the starting samples in CsCl and the dialyzed particles were seen (Fig. ). The addition of prokaryotic chaperones resulted in a decreased number of assembled particles per field and a concomitant increase in the amount of pentamers and background amorphous material (Fig. ). In the BPV-plus-KJE samples, capsid proteins may be present as aggregates on the viral genome (Fig. , arrow). No disassembly was observed when chaperone activity was inhibited by excess ADP (Fig. ).
FIG. 2. Prokaryotic chaperones disassemble BPV virions and VLPs (BPVLP). In vitro reactions were performed by dialysis overnight against disassembly buffer (no dithiothreitol, with calcium and ATP or ADP where indicated). (A) TEM showing disassembled particles (more ...)
The products of the in vitro reactions were analyzed by sucrose gradient sedimentation to separate VLPs or virions from the disassembly intermediates and to quantify the extent of disassembly. Ten to 50% discontinuous sucrose gradients were prepared with gradient buffer (20 mM Tris-Cl [pH 7.2], 5% glycerol, 180 mM NaCl, 10 mM KCl, 1 mM CaCl2, 1 mM MgCl2) in 2.2-ml tubes (Beckman). Samples were sedimented in an RP55S-351 swinging bucket rotor in a Sorvall RC M120 ultracentrifuge at 50,000 rpm for 30 min at 4°C. Fractions were trichloroacetic acid precipitated, and capsid proteins were assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gradients demonstrated that the assembled particles seen in the samples without chaperones were shifted to slower-sedimenting products in the reactions with chaperones (Fig. ). The products from the BPV VLP-plus-KJE reactions sedimented at the position of capsomere controls, while the BPV-plus-KJE reactions had a wider sedimentation profile, consistent with the genome-plus-capsid-protein aggregates seen by TEM. The presence of ADP inhibited chaperone disassembly of particles and also appeared to stabilize the inherent background dissociation seen in samples without chaperones. A similar stabilization was seen for polyomavirus particles, but to a lesser extent (see below). The assembled particles (Fig. , shaded regions) were quantitated (Fig. ), and the number of VLPs or virions was significantly reduced by chaperone-mediated disassembly in an energy-dependent manner. The relatively low percentage of assembled particles in the no-chaperone samples may represent a compromise between the need to stabilize assembled particles and the optimization of buffer conditions for chaperone activity. All of the disassembly reactions were repeated multiple times with similar results; however, only the data from the optimized buffer conditions are shown.
In vitro disassembly reactions were also performed with recombinant polyomavirus VLPs or polyomavirus virions purified, respectively, from recombinant baculovirus-infected Sf9 cells or virus-infected NIH 3T3 cells (12
). In vitro disassembly reactions were carried out as described for papillomavirus VLPs and virions. TEM demonstrated disassembly in the presence of prokaryotic chaperones, and the chaperone-mediated disassembly was inhibited by ADP (Fig. ). Again, capsid proteins appeared to form aggregates with the viral genome in the virion-plus-KJE samples (Fig. , arrow).
FIG. 3. Prokaryotic chaperones disassemble murine polyomavirus virions (PyV) and VLPs (PyVLP). In vitro reactions were performed by dialysis overnight against disassembly buffer (no dithiothreitol, with calcium and ATP or ADP where indicated). (A) TEM showing (more ...)
The products of the polyomavirus reactions were analyzed by sucrose gradient sedimentation (Fig. ). The polyomavirus virion sedimentation profile, although broadened in this experiment, was similar to that observed for BPV, with a shift to slower-sedimenting products in the presence of chaperones that was inhibited by ADP. However, the polyomavirus VLP reactions did not demonstrate disassembly as complete as that seen for BPV VLPs. Polyomavirus VLP disassembly may have been partially inhibited by calcium in the reaction buffer, which both stabilizes and promotes VLP assembly. Additionally, chaperones assemble VP1 capsomeres into capsids in the presence of ATP, although very few of these assemblies were identified. Reassembly probably did not occur in the polyomavirus virion disassembly reactions because the dissociated capsomeres likely formed aggregates with the viral minichromosomes. Nevertheless, quantitation of the assembled fractions revealed chaperone-mediated disassembly that was energy dependent (Fig. ).
Previous studies have demonstrated that disulfide bond-reducing agents expand BPV (15
) and that disulfide bonds and calcium bridges stabilize polyomavirus virions (4
). However, chelating and reducing agents did not enhance chaperone-mediated disassembly of either papilloma- or polyomavirus virions (data not shown).