Kinetics and intracellular location of intramolecular disulfide bond formation mediated by the cytoplasmic redox system encoded by vaccinia virus

Abstract

Poxviruses encode a redox system for intramolecular disulfide bond formation in cytoplasmic domains of viral proteins. Our objectives were to determine the kinetics and intracellular location of disulfide bond formation. The vaccinia virus L1 myristoylated membrane protein, used as an example, has three intramolecular disulfide bonds. Reduced and disulfide-bonded forms of L1 were distinguished by electrophoretic mobility and reactivity with monoclonal and polyclonal antibodies. Because disulfide bonds formed during 5 min pulse labeling with radioactive amino acids, a protocol was devised in which dithiothreitol was present at this step. Disulfide bond formation was detected by 2 min after removal of reducing agent and was nearly complete in 10 min. When the penultimate glycine residue was mutated to prevent myristoylation, L1 was mistargeted to the endoplasmic reticulum and disulfide bond formation failed to occur. These data suggested that viral membrane association was required for oxidation of L1, providing specificity for the process.

Fig. 1

Specificities of L1 MAb and PAb for reduced and oxidized L1. (A) Immunoaffinity purification. BS-C-1 cells were infected with VACV and labeled from from 2 to 16 h with [³⁵S]methionine/cysteine mixture. Lysates were prepared in the presence of DTT (R) or NEM (NR) and incubated with L1 MAb 7D11 or L1 PAb R180 complexed to protein G agarose. The proteins were eluted with sample buffer lacking reducing agent and analyzed by SDS-PAGE and autoradiography. Bands corresponding to reduced (red) and oxidized (ox) L1 are indicated. (B) Western blotting. Lysates were prepared as described in panel A and analyzed by SDS-PAGE and Western blotting with MAb 7D11 and PAb R180. Proteins were detected by chemiluminescence.

Fig. 2

Kinetics of disulfide bond formation. BS-C-1 cells were infected with VACV for 16 h, incubated with methionine- and cysteine-free medium for 15 min, and then pulsed with 100 μCi of [³⁵S]methionine/cysteine-labeling mix for 5 min. Following the pulse, cells were incubated in medium containing excess methionine and cysteine for 2 to 30 min as indicated, lysed, affinity purified with L1 MAb (A) or L1 PAB (B) and analyzed by SDS-PAGE and autoradiography. Bands corresponding to reduced (red) and oxidized (ox) L1 are indicated on the right. Numbers on the left indicate the masses of the L1 proteins in kDa determined by co-electrophoresis of marker proteins.

Fig. 3

Kinetics of disulfide bond formation following DTT removal. The pulse-chase protocol was similar to that described in the legend to Fig. 2 except that the 15 min pre-incubation in methionine- and cysteine-free medium was followed by an additional 5 min incubation in the same medium containing 5 mM DTT and then pulsed with 100 μCi of [³⁵S]methionine/cysteine-labeling mix for 5 min in the continued presence of DTT. After the chase of 2 to 30 min in the absence of DTT, the L1 protein was affinity purified with L1 MAb (A) or L1 PAB (B) and analyzed by SDS-PAGE and autoradiography. The protocol for the experiment in panel C was similar except that 5 mM DTT (+ DTT) was maintained during the chase in one sample. Abbreviations are the same as in the legend to Fig. 2.

Fig. 4

Effect of mutation of the myristoylation site of L1 on disulfide bond formation. BS-C-1 cells were infected with vL1Ri in the presence (+) or absence (−) of IPTG and transfected 1 h later with pL1wt or pL1G2A or left untransfected (UnT). After 8, 10 and 12 h the cells were lysed and analyzed by SDS-PAGE and Western blotting with L1 PAb. The blot was reprobed with antibody to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. Numbers on the left refer to position and mass of marker protein in kDa.

Fig. 5

Intracellular localization of myristoylated and unmyristoylated L1. HeLa cells were infected with vL1Ri in the presence (top row) or absence (middle and bottom rows) of IPTG and 1 h later transfected with pL1wt (middle row) or pL1G2A (bottom row). After 16 h the cells were stained with L1 PAb and L1 MAb followed by Alexa Fluor 488 anti-rabbit antibody and Alexa Fluor 568 anti-mouse, respectively. Cells were subsequently stained with DAPI and visualized by confocal microscopy. Red, L1 MAb; green, L1 PAb; blue, DNA. N, nucleus.

Fig. 6

Co-localization of unmyristoylated L1 with ER resident protein. HeLa cells infected with vL1Ri in the presence (1^st column) or absence (2^nd and 3^rd columns) of IPTG were transfected with pL1wt (2^nd column) or pL1G2A (3^rd column). Cells were stained with L1 PAb (top row), anti-D8 MAb (2^nd row), and anti-calreticulin chicken PAb (4^th row) followed by Alexa Fluor 488 anti-rabbit, Alexa Fluor 568 anti-mouse and Alexa Fluor 647 anti-chicken antibody and analyzed by confocal microscopy. DNA was stained with DAPI. Pink, anti-D8 MAb; green, L1 PAb; red, anti-calreticulin PAb; blue, DNA. N, nucleus.