Intercapsomeric disulfide bonds in papillomavirus assembly and disassembly

Abstract

In order to analyze bonding contacts that stabilize the virion or promote capsid assembly, bovine papillomavirus (BPV) virions were subjected to buffer conditions known to disrupt polyomavirus virions. At physiologic ionic strength, incubation with dithiothreitol (DTT), EGTA, or DTT plus EGTA did not disrupt BPV virions as determined by electron microscopy. However, incubation of virions with DTT rendered the BPV L1 protein susceptible to trypsin cleavage at its carboxy terminus and rendered the genome susceptible to digestion with DNase I. When DTT-treated BPV virions were analyzed by analytical ultracentrifugation, they sedimented at 230S compared with 273S for untreated virions, suggesting a capsid shell expansion. Incubation with EGTA had no effect on trypsin or DNase I sensitivity and only a small effect upon the virion S value. A single cysteine residue conserved among BPV and human papillomavirus (HPV) L1 proteins resides within the trypsin-sensitive carboxy terminus of L1, which is required for capsid assembly. A recombinant HPV type 11 L1 protein, which was purified after expression in Escherichia coli and which has a Cys-to-Gly change at this position (Cys424), formed pentamers; however, unlike the wild-type protein, these mutant pentamers could no longer assemble in vitro into capsid-like structures. These results indicate an important role for interpentamer disulfide bonds in papillomavirus capsid assembly and disassembly and suggest a mechanism of virus uncoating in the reducing environment of the cytoplasm.

FIG. 1

Trypsin digestion of BPV virions. Immunoblot of BPV L1 after virion digestion with 1 μg of trypsin for 15 min at 37°C (see Materials and Methods). Lanes: 1, no pretreatment; 2, 5 mM EGTA; 3, 10 mM DTT; 4, 5 mM EGTA plus 10 mM DTT.

FIG. 2

Electron micrographs of BPV virions digested with trypsin. Samples were incubated for 2 h at 20°C in dialysis buffer with 10 mM EGTA (A), 10 mM EGTA plus trypsin (B), 10 mM DTT (C), or 10 mM DTT plus trypsin (D).

FIG. 3

Trypsin digestion of BPV virions. BPV virions were preincubated with 10 mM DTT and then digested with trypsin at the indicated concentrations for 15 min at 37°C. Lanes: 1, no trypsin; 2 to 5, 1, 0.1, 0.01, and 0.001 μg of trypsin, respectively.

FIG. 4

BPV virions digested with DNase I; agarose gel electrophoresis of viral DNA extracted after digestion of BPV virions with DNase I. Virions were preincubated in BPV buffer (see Materials and Methods) with 15 mM MgCl₂ (no DNase I) (lane 1), 5 mM EGTA (followed by DNase I) (lane 2), 5 mM EGTA plus 10 mM DTT (followed by DNase I) (lane 3), 5 mM MgCl₂ (no DNase I) (lane 4), 5 mM MgCl₂ (followed by DNase I) (lane 5), and 10 mM DTT (followed by DNase I) (lane 6).

FIG. 5

Electron micrographs of BPV virions disrupted at low ionic strength. (A) Virions in buffer containing 10 mM Tris-HCl (pH 7.9); (B and C) the same buffer incubated for 15 min at 37°C with 1 mM EGTA and 10 mM DTT, respectively.

FIG. 6

Trypsin digestion of BPV virions in low-ionic-strength buffer. Immunoblot of BPV virions (lane 1) incubated (as in Fig. 5) with DTT (lane 2) or EGTA (lane 3) and then digested with trypsin for 15 min at 37°C.

FIG. 7

Sedimentation analysis of BPV virions. BPV virions in BPV buffer plus 10 mM DTT (A) or buffer alone (B) were analyzed by analytical ultracentrifugation (see Materials and Methods). The DTT-treated virions sedimented at 230S, compared with 273S for untreated virions. w2t, product of the square of the angular velocity and time; r, radius of boundary; rm, radius of the meniscus; wt. avg., weight average.

FIG. 8

Electron micrograph of L1 mutant protein Cys424Gly. The Cys424Gly mutant L1 protein was purified after expression in E. coli and incubated under assembly conditions with no reducing agent and 0.5 M NaCl (13). No capsid-like aggregates are seen, although the capsomeres cluster into small aggregates of five to six capsomeres around one.

FIG. 9

Trypsin digestion of the Cys424Gly recombinant protein; immunoblot of HPV11 L1. Wild-type (lanes 1 to 4) or Cys424Gly (lanes 5 to 8) purified recombinant proteins were digested with trypsin (weight ratio, 10:1) at 20°C for 0 (lanes 1 and 5), 30 (lanes 2 and 6), 60 (lanes 3 and 7), and 90 (lanes 4 and 8) min.