Disulfide bonds and membrane topology of the vaccinia virus A17L envelope protein

Abstract

The envelope protein encoded by the vaccinia virus A17L open reading frame is essential for virion assembly. Our mutagenesis studies indicated that cysteines 101 and 121 form an intramolecular disulfide bond and that cysteine 178 forms an intermolecular disulfide linking two A17L molecules. This arrangement of disulfide bonds has important implications for the topology of the A17L protein and supports a two-transmembrane model in which cysteines 101 and 121 are intraluminal and cysteine 178 is cytoplasmic. The structure of the A17L protein, however, was not dependent on these disulfide bonds, as a recombinant vaccinia virus with all three cysteine codons mutated to serines retained infectivity.

FIG. 1

Western blots of unreduced and reduced forms of the A17L protein. (A) BSC-1 cells in a six-well plate were infected with 10 PFU of vaccinia virus strain WR per cell. After 18 h, the cells were harvested in the absence or presence of 50 mM iodoacetamide. Washed cell pellets were resuspended in 30 μl of extraction buffer (1% NP-40, 150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl [pH 7.5], 1 mM phenylmethylsulfonyl fluoride) with or without 50 mM iodoacetamide. The samples were incubated for 10 min at room temperature and clarified by centrifugation, and 15 μl of the supernatant was treated with SDS with or without 2-ME and analyzed by SDS-PAGE and Western blotting with A17L-specific antibody. Sucrose gradient-purified vaccinia virions were resuspended in extraction buffer with or without iodoacetamide and analyzed as described above. Abbreviations: Iodo, iodoacetamide; Inf, lysate of infected cells; Un, lysate of uninfected cells; Virus, purified virions. The positions of marker proteins and their masses in kilodaltons are indicated on the left. A17L monomers and dimers are indicated by dots and wedges, respectively. (B) BSC-1 cells in six-well plates were transfected with 2 μg of plasmid in DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate; Boehringer Mannheim) and infected 4 h later with vA17LΔ5 in the absence of IPTG. After 48 h, the cells were harvested and the A17L protein was analyzed as for panel A. C101S, C121S, and C178S refer to plasmids containing codon 101, 121, or 178 mutated from cysteine to serine. Other abbreviations are as in panel A.

FIG. 2

Complementation with mutated A17L proteins. BSC-1 cells were transfected with a plasmid and infected with vA17LΔ5 in the absence of IPTG as described in the legend to Fig. 1B. After 48 h, the plates were stained with crystal violet. Plasmid abbreviations: pUC19sp, vector without the A17L ORF; A17L, unmutated A17L ORF; A17L-309A, stop codon at nucleotide 309; C101S, codon 101 changed from cysteine to serine; C121S, codon 121 changed from cysteine to serine; C178S, codon 178 changed from cysteine to serine; C121, 178S, codons 121 and 178 changed from cysteines to serines; C101, 121, 178S, all three cysteine codons changed to serines; TAG, equivalent number of codons between the second and third hydrophobic domains of the A17L ORF replaced with a hemagglutinin tag sequence. Two wells were transfected with a plasmid containing the unmutated A17L ORF.

FIG. 3

Virus yields. BSC-1 cells were infected with 1 PFU of vaccinia virus WR, vA17LΔ5, vA17L^C101,121S, or vA17L^{C101,121,178S} per cell. Infection with vA17LΔ5 was in the presence of IPTG. At 24, 48, and 72 h after infection, the medium was removed and clarified and the cells were harvested. C101, 121S and C101, 121, 178S are abbreviations of vA17L^C101,121S and vA17L^{C101,121,178S}, respectively. Virus titers of the cell lysates (A) and medium (B) were determined by plaque assay.

FIG. 4

Western blot analysis of A17L proteins of purified vA17L^C101,121S and vA17L^{C101,121,178S}. Infected BSC-1 cells were harvested after 48 h, and virions were purified by sedimentation through a sucrose cushion and CsCl gradient centrifugation. (A) Proteins were extracted in the presence or absence of NEM and treated with SDS in the presence or absence of 2-ME. (B) Purified virions were extracted with 1% Triton X-114 in phosphate-buffered saline for 10 min at 32°C without (lanes 1) or with (lanes 2) 100 mM dithiothreitol. The supernatants were recovered after centrifugation in a microcentrifuge. Dithiothreitol (100 mM) was added to the extracts without a reducing agent, and the samples were denatured by boiling in 2% SDS and analyzed by Western blotting.