A zinc ion controls assembly and stability of the major capsid protein of rotavirus.
The recent determination of the crystal structure of VP6, the major capsid protein of rotavirus, revealed a trimer containing a central zinc ion coordinated by histidine 153 from each of the three subunits. The role of the zinc ion in the functions of VP6 was investigated by site-directed mutagenesis. The mutation of histidine 153 into a serine (H153S and H153S/S339H) did not prevent the formation of VP6 trimers. At pH <7.0, about the pK of histidine, wild-type and mutated VP6 proteins display similar properties, giving rise to identical tubular and spherical assemblies. However, at pH >7.0, histidine 153 mutant proteins did not assemble into the characteristic 45-nm-diameter tubes, in contrast to wild-type VP6. These observations showed that under conditions in which histidine residues are not charged, the properties of VP6 depended on the presence of the centrally coordinated zinc atom in the trimer. Indeed, wild-type VP6 depleted of the zinc ion by a high concentration (100 mM) of a metal-chelating agent behaved like the H153 mutant proteins. The susceptibility of wild-type VP6 to proteases is greatly increased in the absence of zinc. NH(2)-terminal sequencing of the proteolytic fragments showed that they all contained the beta-sheet-rich VP6 head domain, which appeared to be less sensitive to protease activity than the alpha-helical basal domain. Finally, the mutant proteins assembled well on cores, as demonstrated by both electron microscopy and rescue of transcriptase activity. Zinc is thus not necessary for the transcription activity. All of these observations suggest that, in solution, VP6 trimers present a structural flexibility that is controlled by the presence of a zinc ion.
Erk
Inge
I
Virologie Moléculaire et Structurale, UMR CNRS-INRA 2472, F-91198 Gif-sur-Yvette, France.
Huet
Jean-Claude
JC
Duarte
Mariela
M
Duquerroy
Stéphane
S
Rey
Felix
F
Cohen
Jean
J
Lepault
Jean
J
D000818:Animals; D000956:Antigens, Viral; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral; D008854:Microscopy, Electron; D009154:Mutation; D017924:Nucleopolyhedroviruses / Q000235:genetics; D012401:Rotavirus / Q000378:metabolism; D018411:Spodoptera; D014158:Transcription, Genetic; D019065:Virus Assembly; D015032:Zinc / Q000378:metabolism*
D000956:Antigens, Viral; D036022:Capsid Proteins; C061332:VP6 protein, Rotavirus; D015032:Zinc