Intercapsomeric disulfide bonds in papillomavirus assembly and disassembly.
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.
Li
M
M
Department of Pediatrics, University of Colorado School of Medicine, Denver 80262, USA.
Beard
P
P
Estes
P A
PA
Lyon
M K
MK
Garcea
R L
RL
D000818:Animals; D052688:Bovine papillomavirus 1 / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D002213:Capsid / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D002417:Cattle; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009856:Oncogene Proteins, Viral / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014461:Ultracentrifugation; D019065:Virus Assembly
D036022:Capsid Proteins; D004220:Disulfides; C085217:L1 protein, Bovine papillomavirus; C094776:L1 protein, Human papillomavirus type 11; D009856:Oncogene Proteins, Viral; D003545:Cysteine; D005998:Glycine