Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: in vitro interactions with recombinant VP1 capsomeres

Abstract

The polyomavirus VP2 and VP3 capsid proteins were expressed in Escherichia coli. The majority of the expressed proteins were in an insoluble fraction, and they were extracted and initially purified in 8 M urea before renaturation. Soluble VP2 and VP3 were mixed with purified recombinant VP1 capsomeres, and their interactions were assayed by immunoprecipitation and ion-exchange chromatography. Coimmunoprecipitation could be demonstrated with antibodies to either VP1 or VP2/VP3. Mixing recombinant VP1 with VP2 and VP3 modified the recognition of VP1 by domain-specific antipeptide antibodies and altered the chromatographic behavior of the individual proteins. Similar results were observed when a truncated VP1 protein, ⌬NCOVP1, with 62 amino acids deleted from the carboxy terminus was mixed with VP2/VP3. After the mixing, equilibrium dissociation constants for their binding to either VP1 or ⌬NCOVP1 were determined to be 0.37 ؎ 0.23 M for VP2 and 0.18 ؎ 0.21 M for VP3. These studies demonstrate that the recombinant VP2 and VP3 proteins interact with VP1 to affect the biochemical properties of VP1 capsomeres and to change the epitope accessibility of VP1 pentamers. These changes may reflect conformational alterations in VP1 capsomeres which are necessary for viral genome encapsidation.

In vivo and in vitro association of hsc70 with polyomavirus capsid proteins

“…Immunoprecipitation. The antibodies used for immunoblots and immunoprecipitations were rabbit polyclonal anti-polyomavirus VP1 (I58) (8,27,29); mouse monoclonal anti-VP1 (Fd9) (7); an anti-peptide antibody raised against the common carboxy-terminal 24 residues of polyomavirus VP2 and VP3, designated R4 (7,20); and an anti-peptide antibody raised against amino acids 91 to 115 of VP2, a domain not shared by VP3, designated R3 (7). The anti-heat shock protein antibodies were SPA810 (mouse monoclonal anti-hsp70), SPA815 (rat monoclonal anti-hsc70), SPA820 (mouse monoclonal antibody raised against a common epitope of hsp70 and hsc70), and SPA804 (rabbit anti-hsp60; Stressgen, Victoria, Canada).…”

“…Proteins were eluted by boiling in SDS sample buffer and subjected to SDS-10 to 15% polyacrylamide gel electrophoresis (PAGE). Gels were fixed as previously described (7). For immunoblots, gels were transferred to polyvinylidene difluoride paper (Millipore Corp., Bedford, Mass.)…”

“…Alkaline phosphatase-linked secondary antibodies (anti-mouse or anti-rabbit) were used, and immunoblots were developed with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Sigma). The biotinylated antibodies used for enhanced chemiluminescence blots were prepared as previously described (7).…”

“…These results also demonstrate that this interaction is independent of those with other viral proteins, does not require posttranslational modification of VP1 (VP1 expressed in E. coli is unmodified) (19), and has been preserved across species. Some fraction of these proteins, particularly VP2, which becomes associated with cytoplasmic membranes (7), may be improperly folded, but recombinant VP1 appears to be structurally and functionally intact (37,38). Although 25 to 30% of the VP1 pentamers isolated after expression in E. coli copurified with DnaK, it is possible that more pentamers were once bound to DnaK within bacteria.…”

Identification of a DNA encapsidation sequence for human polyomavirus pseudovirion formation

“…The murine polyoma VP1 is the only structural protein that has DNA binding activity Chang et al, 1993]. The minor capsid proteins VP2 and VP3 are associated with the major capsid VP1 [Gharakhanian et al, 1988;Forstova et al, 1993;Barouch and Harrison, 1994;Cai et al, 1994;Delos et al, 1995;Chen et al, 1998]. The complex of the capsid proteins may interact subsequently with the viral genome for virion maturation.…”