Calcium is required in reassembly of bovine papillomavirus in vitro

Papillomaviruses are small DNA viruses which infect and induce benign warts and sometimes malignant tumours in the epithelium of the skin or mucosa. The viruses do not replicate in conventional tissue culture systems and little is known about the requirements for virus assembly. We investigated the effect of ethylene glycol-bis(aminoethyl ether)-tetraacetic acid (EGTA) and dithiothreitol (DTT) treatment on the stability of bovine papillomavirus type 1 (BPV-1) particles in vitro. Removal of calcium ions by 11 mM EGTA at pH 8.0 together with reduction of disulfide bonds by 15 mM DTT destabilized BPV particles. Electron microscopy examination of treated particles showed that the BPV particles had been disrupted to capsomeres. Addition of exogenous calcium ions to the disruption buffer prevented virus destabilization. Adding calcium to the disrupted BPV particles resulted in the reassembly of disrupted particles. The reassembled particles were morphologically similar to intact BPV virions. We further quantified the efficiency of reassembly by focus formation assay. We recorded 500-fold less infectivity for reassembled BPV and 4-fold less haemagglutination activity compared to untreated BPV, pointing towards a decrease in the amount of reassembled particles recovered.