Vaccinia virus G4L glutaredoxin is an essential intermediate of a cytoplasmic disulfide bond pathway required for virion assembly

Abstract

Our previous studies provided evidence that E10R, a vaccinia virus protein belonging to the ERV1/ALR family, has a redox function and is required for virion assembly. Repression of E10R prevented the formation of intramolecular disulfide bonds of the G4L glutaredoxin, the L1R membrane protein, and the structurally related F9L protein. Here, we demonstrate an oxidation pathway (E10R(SS) --> G4L(SS) --> L1R(SS), F9L(SS)) in which G4L occupies an intermediate position. By reacting free thiols with 4-acetamido-4'-malemideylstilbene-2,2'-disulfonic acid, alkylated and nonalkylated disulfide-bonded forms of G4L could be resolved from each other by polyacrylamide gel electrophoresis. The cysteines of intracellular G4L were in both disulfide and reduced forms, whereas those of E10R, L1R, and F9L and virion-associated G4L were mostly disulfide bonded. Repression of G4L expression prevented the formation of disulfide bonds in both L1R and F9L but not E10R. Both cysteines of G4L were required for L1R and F9L disulfide bond formation or for trans-complementation of virus infectivity when G4L expression was repressed. No role in the E10R-G4L redox pathway was found for O2L, a nonessential glutaredoxin encoded by vaccinia virus. We suggest that cytoplasmic G4L is a redox shuttle between membrane-associated E10R and L1R or F9L.

FIG. 1.

Redox status of G4L and O2L. (A) BS-C-1 cells were infected at a multiplicity of 5 with vaccinia virus WR. After 24 h, the cells were TCA precipitated, and the pelleted proteins were solubilized with SDS, treated or not with TCEP, and alkylated with NEM or AMS. Proteins were resolved by electrophoresis in a 10 to 20% polyacrylamide gradient Tricine gel and detected by Western blotting with G4L-specific rabbit antiserum, followed by an anti-rabbit MAb conjugated to peroxidase. Sucrose gradient-purified virions (approximately 10⁸ PFU) were solubilized in 50 mM Tris-HCl (pH 8.0)-0.1% SDS containing NEM or AMS and analyzed as above. The upper arrow points to G4L alkylated with AMS; the lower arrow points to reduced G4L or G4L alkylated with NEM. (B) BS-C-1 cells were infected and the proteins were alkylated as in panel A. Proteins were resolved on a 12% polyacrylamide NuPAGE gel with MOPS buffer and detected as in panel A. The upper arrow points to G4L alkylated with AMS; the lower arrow points to G4L alkylated with NEM. (C) BS-C-1 cells were infected with standard vaccinia virus (WR), vA9Li, vE10Ri, or vG4Li in the presence (+) or absence (−) of IPTG. After 24 h, the cells were TCA precipitated and the solubilized proteins were alkylated with NEM or AMS or reduced with β-mercaptoethanol (2me). Proteins were resolved as in panel B, and O2L was detected with a rabbit antiserum and an anti-rabbit HRPO-conjugated secondary antibody. The upper arrow points to the O2L protein alkylated with AMS; the lower arrow points to reduced O2L or O2L alkylated with NEM. The positions of 14- and 6-kDa protein markers are indicated on the right.

FIG. 2.

Effect of G4L repression on formation of disulfide bonds in L1R and F9L proteins. BS-C-1 cells were infected with vG4Li in the presence (+) or absence (−) of inducer. Cells were transfected with plasmids containing C-terminal HA epitope-tagged copies of the L1R or F9L ORF regulated by late vaccinia virus promoters. After 24 h, the cells were TCA precipitated and the proteins were solubilized in SDS and alkylated with NEM or reduced with TCEP. The proteins were resolved on 10 to 20% polyacrylamide gradient Tris-glycine gels. L1R and F9L were detected by Western blotting using MAb HA.11 and an anti-mouse Ig HRPO-conjugated secondary antibody. The reduced (red) and oxidized (ox) forms of L1R and F9L are indicated.

FIG. 3.

Effect of G4L repression on the formation of disulfide bonds in E10R. (A) BS-C-1 cells were infected with standard vaccinia virus (WR) and transfected with a plasmid containing the E10R gene with a C-terminal HA epitope tag under the control of the vaccinia virus P11 promoter. At 24 h after infection, cells were lysed directly in 10 mM Tris-HCl (pH 7.5)-10 mM KCl-0.5 mM EDTA-1 mM CaCl₂-0.2% (vol/vol) NP-40-40 mM NEM or AMS-1 μg of micrococcal nuclease per μl. Cells were allowed to lyse on ice for 15 min, and an equal volume of nonreducing loading buffer was added. Proteins were resolved by electrophoresis on a 16% polyacrylamide-Tricine gel and detected by Western blotting with an anti-HA MAb and anti-mouse Ig HRPO-conjugated secondary antibody. Arrows point to E10R proteins alkylated with three residues of AMS (upper), one residue of AMS (middle), or NEM (lower). (B) BS-C-1 cells were infected with standard vaccinia virus (WR) or vG4Li in the absence of IPTG and transfected with a plasmid containing the E10R gene as described for panel A. Proteins were alkylated and detected as described for panel A. The bands corresponding to modification with three AMS residues (upper) and one AMS residue (lower) are indicated.

FIG. 4.

Expression of G4L with cysteine-to-serine mutations. BS-C-1 cells were infected with vG4Li in the absence of inducer and transfected with the pUC19 vector plasmid or plasmids containing an unmutated or mutated G4L ORF under its natural promoter. The mutations consisted of Cys₁₃Ser (C13-S), Cys₁₆Ser (C16-S), or Cys_13,16Ser (C13,16-S). At 24 h after infection, the cells were treated with TCA and the proteins were solubilized with SDS and NEM (A) or AMS (B), resolved on a 10 to 20% polyacrylamide gradient Tricine gel, and detected by Western blotting with G4L rabbit antiserum and an anti-rabbit Ig HRPO-conjugated secondary antibody.

FIG. 5.

Effect of G4L cysteine-to-serine mutations on the formation of disulfide bonds in the L1R protein. BS-C-1 cells were uninfected (U) or infected with vG4Li in the presence (+) or absence (−) of IPTG and cotransfected with plasmids containing HA-tagged L1R and either pUC19, wild-type G4L, or mutated G4L as described in the legend to Fig. 4. At 24 h after infection, proteins were TCA precipitated, solubilized with SDS, alkylated with NEM, and resolved by electrophoresis on a Tris-glycine-10 to 20% polyacrylamide gradient gel. Proteins were detected by Western blotting with MAb HA.11. The reduced (red) and oxidized (ox) forms of L1R are indicated.

FIG. 6.

trans-Complementation of virus infectivity. BS-C-1 cells were infected with vG4Li in the presence (+) or absence (−) of 50 μM IPTG and mock transfected with Lipofectamine 2000 alone (mock) or with pUC19 vector plasmid or plasmids containing the wild-type or mutated G4L ORF under its natural (nat) or the P11 promoter as described in the legend to Fig. 4. At 24 h after infection, the cells were harvested and the virus titers were determined by plaque assay in the presence of 50 μM IPTG.

FIG. 7.

Relationship of E10R, G4L, L1R, and F9L proteins in a redox pathway. E10R_SS reacts directly or indirectly with G4L_2SH, resulting in the transfer of a disulfide bond. Three molecules of G4L_SS then interact directly or indirectly through another redox protein with 1 molecule of L1R_6SH or F9L_6SH to form L1R_3S-S or F9L_3S-S. Arrows indicating oxidation are solid, and arrows indicating reduction are open.