Localization of calcium on the polyomavirus VP1 capsid protein

Abstract

Our laboratory has previously shown that the divalent cation Ca2+ is an integral part of the polyomavirus and plays a major role in stabilizing the intact virion structure. In this report, we show that calcium is sequestered on the major capsid protein VP1 of polyomavirus. The virion structural proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before being transferred to nitrocellulose and probed with 45CaC12. Autoradiography revealed 45Ca binding exclusively to VP1. Increasing the amount of VP1 transferred to the nitrocellulose resulted in a concomitant increase in 45Ca binding. 45Ca binding to VP1 could be reduced by competition with an excess of unlabeled CaC12. Separation of the species of VP1 by two-dimensional gel electrophoresis before electroblotting and probing with 45CaCl2 revealed that all six species (A to F) bind the radiolabeled calcium. Formic acid cleavage of the 43-kilodalton (kDa) VP1 protein into 29-, 18-, and 16-kDa fragments before 45Ca-binding analysis revealed that only the 18-and 16-kDa carboxylterminal fragments of this protein bind 45Ca.

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

“…Analysis of the 45Ca-binding abilities of proteins derived from purified virions (lane 2) reveals a major signal as well as several minor signals. The corresponding Western blot (lane 1) demonstrates that the principal 45Ca-binding protein seen in lane 2 is VP1, consistent with previous studies (27). These studies had also shown that the lowmolecular-weight43 Ca-binding entities represent C-terminal VP1 fragments resulting from breakdown of the protein (27) and that the minor capsid proteins (detected by the polyclonal antisera in lane 1) do not bind 45Ca.…”

“…Generation of polyomavirus genomes encoding site-directed and deletion mutations. A previous study (27), together with similarities in amino acid sequence to the calcium-binding EF hand structure, suggested that amino acids Asp-266 to Glu-277 of polyomavirus VP1 may have a role in calcium binding by this virus. Oligonucleotide-directed mutagenesis of Asp-266 was performed to determine the effects that substitution of this residue with a neutral amino acid (alanine) would have on the virus structure and function(s).…”

“…A 16-kDa fragment of the polyomavirus major capsid protein VP1, which is generated by formic acid cleavage and extends from Pro-232 to Asp-364, was shown to have calcium-binding capabilities (27). This fragment contains a sequence of 12 amino acids (Asp-266 through Glu-277) that is quite similar to the 12-amino-acid loops which make up part of the EF hand structures found in many calcium-binding proteins, including parvalbumin, calmodulin, thrombospondin, and troponin C (13,23,42).…”

Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

“…Studies with the murine polyomavirus system have localized the Ca2+-binding domain to a 16-kDa fragment of VPI which was generated by formic acid cleavage of VP1. This fragment, which extended from Pro-232 to Asp-364, was shown to have in vitro Ca2'-binding capabilities (30). This 16-kDa fragment was also found to have a sequence of 12 amino acids (Asp-266 to Glu-277) that was quite similar to the 12-amino-acid loops which make up part of the EF hand structures found in many calcium-binding proteins, including parvalbumin, calmodulin, thrombospondin, and troponin C (13,26,46).…”

Hydroxyproline in the major capsid protein VP1 of polyomavirus

“…The divalent cation calcium also appears to be involved in stabilizing the intact virus, as well as being crucial for in vitro reassembly of infectious polyomavirus particles (6)(7)(8)32) and stabilization of purified VP1 (29). Recently, we have localized the site of this calcium association to the carboxyl-terminal portion of VP1 (21). In keeping with our ongoing investigation into the modification of polyomavirus capsid proteins and their function(s) in the virus life cycle, the present report demonstrates that VP1 is also modified by hydroxylation of the amino acid proline.…”