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

Abstract

Pentamer formation by Vp1, the major capsid protein of simian virus 40, requires an interdigitation of structural elements from the Vp1 monomers [Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L. & Harrison, S. C. (1991) Nature (London) 354, 278-284]. Our analyses reveal that disulfide-linked Vp1 homooligomers are present in the simian virus 40-infected cytoplasm and that they are derived from a 41-kDa monomeric intermediate containing an intrachain disulfide bond(s). The 41-kDa species, emerging within 5 min of pulse labeling with [(35)S]methionine, is converted into a 45-kDa, disulfide-free Vp1 monomer and disulfide-bonded dimers through pentamers. 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 sulfhydryl-disulfide 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.

Figure 1

Discrete, disulfide-bonded Vp1 oligomers exist in the cytoplasm. (A) SV40-infected cells at 48–50 h postinfection (lanes 3–6) or mock-infected cells (lanes 1 and 2) were labeled for 1 h and fractionated as described in Materials and Methods. The cytosolic fraction from 2.5 × 10⁵ cells was immunoprecipitated with anti-Vp1 serum (αVp1, lanes 1, 3, and 5) or monoclonal anti-Vp1 antibody α597 (lanes 2, 4, and 6) in the absence (lanes 1–4) or presence (lanes 5–7) of SDS, eluted under reducing (lanes 1–6) or nonreducing (lane 7) conditions, and analyzed by 12.5% (lanes 1–6) or 7% (lane 7) SDS/PAGE and autoradiography. The positions for Vp1, Vp2, and Vp3 (Left) and the apparent molecular masses of labeled bands in lane 7 (Right) are indicated. Arrows mark the positions for dimeric, trimeric, tetrameric, and pentameric Vp1s, and an empty arrowhead marks the position for the 78-kDa species. (B and C) The same cytosolic fraction as in A, prepared from 2.5 × 10⁶ cells, was sedimented through a 5–20% sucrose gradient under nondenaturing (B) or denaturing (C) conditions. Eighteen fractions were collected from the bottom of the gradient, immunoprecipitated with anti-Vp1 serum in the absence of SDS (even fractions 2–18) or with α597 in the presence of SDS (odd fractions 7–13), and analyzed by reducing (Red) or nonreducing (Nonred) 7% SDS/PAGE, respectively, followed by autoradiography. The nonreducing gels represent a three-times-longer autoradiographic exposure relative to the reducing gels. Solid arrowheads point to positions for sedimentation markers determined in parallel gradients: (from left to right) 13S, β-galactosidase; 7S, goat IgG; and 5S, ovalbumin.

Figure 2

Cytoplasmic Vp1 oligomers are homooligomers. (A) Anti-Vp1 Western blot of infected cytosolic fraction from 5 × 10⁴ non-DTSP-treated (lane 1) or DTSP-treated (lane 2) cells. The cytosolic sample was incubated in 120 mM Hepes, pH 6.8/3% SDS/10% glycerol/4 mM NEM at 50°C for 30 min, clarified by centrifugation, and resolved by 8%/12.5% step-gradient SDS/PAGE before being electrotransferred to membrane and blotted as described (21). (B) Coomassie blue staining of cytosolic proteins from 2.5 × 10⁵ non-DTSP-treated infected cells after 2D SDS/PAGE. Spots on the diagonal line corresponding to the presumed locations of Vp1, Vp2, and Vp3 are circled. (C and D) Anti-Vp1 Western blots of the same cytosolic fraction as in A, lane 1 (C) and lane 2 (D), after 2D SDS/PAGE. The positions for monomeric (V₁), dimeric (V₂), trimeric (V₃), tetrameric (V₄), and pentameric (V₅) Vp1s are marked beneath respective spots. (E) The same cytosolic fraction as in Fig. 1A, lane 3, prepared from 8 × 10⁵ cells, was immunoprecipitated with anti-Vp1 serum and analyzed by 2D SDS/PAGE and autoradiography. For A– E, an open circle or an open rectangle indicates the origin or the end, respectively, of the resolving gel; a solid diamond, a point two-thirds of the way down; and a solid circle, the boundary for the 8%/12.5% step. The positions for molecular mass standards of 30–200 kDa are indicated in A and E. Note that Vp1 dimers and trimers migrated at the boundary of the step gradient in A and C– E.

Figure 3

Kinetics of formation of disulfide-bonded Vp1 monomer and oligomers. (A) The Vp1 homooligomers are formed from intramolecularly disulfide-linked monomer in the cytoplasm. Infected cells were pulse-labeled for 5 min and then either harvested immediately (lanes b, g, and L) or chased in the absence of NEM for 10 min (lanes c and h), 20 min (lanes d and i), or 40 min (lanes a, e, f, and j) or chased in NEM-containing medium for 40 min (lanes m and n). The cytosolic fraction from 5 × 10⁵ cells (Sol, lanes a–f and L and m) or the cytoskeletal fraction from 1 × 10⁶ cells (Csk, lanes g–j and n) was immunoprecipitated in the presence of SDS with anti-Vp1 serum (αVp1 IP, lanes b–j and L–n) or with preimmune serum (ctrl, lane a), eluted under nonreducing conditions (Nonred), and analyzed by 8%/12.5% step-gradient SDS/PAGE and autoradiography. Lanes f and L are longer exposures of lanes e and b, respectively. In lane k, 2 × 10⁴ infected, unlabeled nuclei (Nuc) were lysed with SDS and a reducing agent (Red) and analyzed by anti-Vp1 Western blot. The positions for Vp1 pentamer (band 1), tetramer (band 2), trimers (bands 3 and 4), dimers (bands 5 and 6), monomers (bands 7 and 8), and monomer-like species (bands 9–11, lane L) are marked, as are the positions for molecular mass standards of 30–200 kDa (Left). The apparent molecular masses of bands 7, 8, 9, 10, and 11 are 45, 41, 38, 36, and 34 kDa, respectively. (B) The 41-kDa Vp1 monomer is converted into the 45-kDa monomer upon reduction. The same cytosolic sample as in A, lane b, prepared from 1 × 10⁶ cells, was immunoprecipitated with anti-Vp1 serum and analyzed by 2D SDS/PAGE and autoradiography. The 41-kDa Vp1 (spot 8) is positioned off the diagonal, whereas the 45-kDa Vp1 (spot 7) lies on the diagonal.

Figure 4

Localization of Vp1 and ER marker proteins in infected cells. SV40 infected cells at 60 h postinfection were fixed, doubly stained either with rabbit anti-Vp1 (A) and mouse anti-PDI (B) antibodies or with rabbit anti-Vp1 (C) and mouse anti-calnexin (D) antibodies, followed by fluorescein-labeled (A and C) or rhodamine-labeled (B and D) secondary antibodies. The same cells are shown in A and B and in C and D.

Figure 5

Model of cytoplasmic Vp1 folding and oligomerization. The newly synthesized Vp1 in the cytoplasm is an intrachain disulfide-linked monomer (a), which is converted into a disulfide-free monomer (b) and then into interchain disulfide-linked oligomers (c through d). The last species is transported into the nucleus, where it participates in virus particle assembly. A disulfide linkage is denoted by “S-S,” and a free sulfhydryl group of cysteine residue is denoted by “SH.”