Formation of transitory intrachain and interchain disulfide bonds accompanies the folding and oligomerization of simian virus 40 Vp1 in the cytoplasm

Abstract

The covalent oligomer formation is blocked in the presence of a sulfhydryl-modifying reagent. We propose that there are two stages in this Vp1 disulfide bonding. First, the newly synthesized Vp1 monomers acquire intrachain bonds as they fold and begin to interact. Next, these bonds are replaced with intermolecular bonds as the monomers assemble into pentamers. This sequential appearance of transitory disulfide bonds is consistent with a role for sulfhydryldisulfide redox reactions in the coordinate folding of Vp1 chains into pentamers. The cytoplasmic Vp1 does not colocalize with marker proteins of the endoplasmic reticulum. This paper demonstrates in vivo disulfide formations and exchanges coupled to the folding and oligomerization of a mammalian protein in the cytoplasm, outside the secretory pathway. Such disulfide dynamics may be a general phenomenon for other cysteine-bearing mammalian proteins that fold in the cytoplasm. H ow proteins fold into functional, three-dimensional structures has been under intense study (1), and the folding pathways for a number of eukaryotic proteins have been characterized in vitro (2, 3) or in vivo (4-9). In the secretory pathway, protein folding is coupled to the formation and reshuffling of disulfide bonds. These redox conversions, leading to native, disulfide-bonded proteins, are catalyzed by prokaryotic Dsb proteins in the periplasm (10-12) and eukaryotic protein disulfide isomerase (PDI) in the endoplasmic reticulum (ER) (13-16). Proteins that fold and assemble in the reducing environment of the cytoplasm generally do not harbor native disulfides, owing to the activities of thioredoxins and glutaredoxins. Transitory disulfide bonding, though, is required for the folding of bacteriophage P22 tailspike protein in the cytoplasm (17, 18). Whether disulfide bond-coupled folding pathways exist for nonsecretory proteins in the mammalian cytoplasm is not known.The structure of simian virus 40 (SV40), known at the atomic resolution, is determined by the major capsid protein Vp1 (19). Seventy-two pentamers of Vp1 form the outer shell of SV40, with each monomer making contact with its four intrapentamer neighbors via interdigitating secondary structural elements. The Vp1 pentamer is expected to form in the cytoplasm of SV40-infected cells during or soon after the monomers' synthesis (20, 21). There are seven cysteine residues in one Vp1 chain. No intrapentamer disulfide bridges, either between or within the monomers, are observed in the mature particle (22). Certain cysteine residues do lie in close proximity of one another, such as the Cys-49-Cys-87 and Cys-87-Cys-207 pairs within one monomer and the Cys-49-Cys-207 pair between two monomers within a pentamer (19). Each cysteine pair conceivably can become juxtaposed during the folding process and form a transient disulfide bond.In this study, we show that in the virus-infected cytoplasm, the newly synthesized Vp1 chain is an intramolecularly disulfidebonded monomer and is a precursor for intermolecularly disulfidebonded Vp1 oligome...

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