The concentration of Ca2+ that solubilizes outer capsid proteins from rotavirus particles is dependent on the strain.
It has been previously shown that rotavirus maturation and stability of the outer capsid are calcium-dependent processes. More recently, it has been hypothesized that penetration of the cell membrane is also affected by conformational changes of the capsid induced by Ca2+. In this study, we determined quantitatively the critical concentration of calcium ion that leads to solubilization of the outer capsid proteins VP4 and VP7. Since this critical concentration is below or close to trace levels of Ca2+, we have used buffered solutions based on ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and Ca-EGTA. This method allowed us to show a very high variability of the free [Ca2+] needed to stabilize, at room temperature, the outer capsid of several rotavirus strains. This concentration is about 600 nM for the two bovine strains tested (RF and UK), 100 nM for the porcine strain OSU, and only 10 to 20 nM for the simian strain SA11. Titration of viral infectivity after incubation in buffer of defined [Ca2+] confirmed that the loss of infectivity occurs at different [Ca2+] for these three strains. For the bovine strain, the cleavage of VP4 by trypsin has no significant effect on the [Ca2+] that solubilizes outer shell proteins. The outer layer (VP7) of virus-like particles (VLP) made of recombinant proteins VP2, VP6, and VP7 (VLP2/6/7) was also solubilized by lowering the [Ca2+]. The critical concentration of Ca2+ needed to solubilize VP7 from VLP2/6/7 made of protein from the bovine strain is close to the concentration needed for the corresponding virus. Genetic analysis of this phenotype in a set of reassortant viruses from two parental strains having the phenotypes of strains OSU (porcine) and UK (bovine) confirmed that this property of viral particles is probably associated with the gene coding for VP7. The analysis of VLP by reverse genetics might allow the identification of the region(s) essential for calcium binding.
Ruiz
M C
MC
Laboratorio de Fisiologia Gastrointestinal, Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela.
Charpilienne
A
A
Liprandi
F
F
Gajardo
R
R
Michelangeli
F
F
Cohen
J
J
D000818:Animals; D000956:Antigens, Viral; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins; D002417:Cattle; D012401:Rotavirus / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000378:metabolism
D000956:Antigens, Viral; D036022:Capsid Proteins; C060339:VP4 protein, Rotavirus; C050779:VP7 protein, Rotavirus; D002118:Calcium