In vitro reassembly of infectious polyoma virions

Abstract

Initial experiments in our laboratory have successfully reassembled infectious polyoma virions from dissociated virion products. Virions treated with ethyleneglycol-bis-N,N'-tetraacetic acid and the reducing agent ,B-mercaptoethanol at pH 7.5 were dissociated to a 48S DNA-protein complex and capsomere subunits. The virion dissociation products were not infectious by plaque assay and lacked hemagglutination activity. These virion dissociation products were reassembled to intact virions by overnight dialysis against a reassembly buffer containing CaCl2, dimethyl sulfoxide, and Triton X-100 in phosphate-buffered saline at pH 7.4. The biophysical characteristics of the reassembled virions were identical to those of untreated virions in that the reassembled virions had a sedimentation value of 240S in sucrose gradients and a buoyant density of 1.315 g/cm: in CsCl isopycnic gradients. The reassembled virions were intact as determined by electron microscopy and were found to be 60% resistant to DNase I treatment. Biologically, the reassembled purified virions were found to partially regain both hemagglutinating activity and plaque-forming ability.

Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

“…We have postulated that the action of a calcium-binding protein(s) in this cellular compartment, together with a microenvironment which provides the necessary reducing conditions, may contribute to viral uncoating. The available information suggests that calcium is also involved in capsid assembly, a late event in the virus life cycle (8,32,38,39,48). The findings presented in the present study demonstrate that mutations in the putative calcium-binding domain affect capsid assembly both in vitro and in vivo.…”

“…Studies employing purified polyomavirus virions (7)(8)(9)(10)47) have implicated calcium as having a role in maintaining the virion structure as well as in viral uncoating and assembly. In addition, calcium was shown to contribute to the assembly of recombinant polyomavirus VP1 expressed in E. coli into capsid-like structures (38,39).…”

“…A structural role for calcium was demonstrated in studies by Brady et al (9, 10) which showed that chelation of calcium by ethylene glycol-bis-N,N,N',N'-tetraacetic acid (EGTA), in conjunction with disulfide bond disruption by the reducing agent dithiothreitol, results in the breakdown of the virion into its capsomere subunits and a DNA-protein complex. Interestingly, addition of exogenous CaCl2 to this dissociated mixture permitted reassembly into intact virions which partially regained both infectivity and hemagglutination activity (8,47). Calcium binding appears to contribute to the stability of a number of plant viruses as well (1, 14-16, 22, 34, 37), possibly by helping to ensure that these viruses release their RNAs only in the host-cell cytoplasm.…”

Localization of calcium on the polyomavirus VP1 capsid protein

“…10) have shown that chelation of this cation with ethyleneglycol-bis-N,N'tetraacetic acid, in conjunction with disulfide bond disruption with the reducing agent dithiothreitol, leads to the dissociation of the virus into capsomere subunits and a DNA-protein complex. This cation also appears to be crucial for in vitro reassembly of infectious polyomavirus particles (8,31). The present report demonstrates that the major polyomavirus-coded capsid protein VP1 binds calcium.…”

Evidence that SV40 VP1–DNA interactions contribute to the assembly of 40‐nm spherical viral particles

“…1977(Brady et al . , 1978(Brady et al . , 1979, and VP1 pentamers can be reassembled into VLPs under appropriate conditions (Salunke et al .…”