FIG. 6.

Cys₇₁ is necessary and sufficient for intermolecular disulfide bond formation by A14, both in vitro and in vivo. (A) Synthesis of wt A14 (lane 1) and the C₇₁S A14 mutant (lane 2) by using IVTT. [³⁵S]methionine-labeled IVTT products were subjected to SDS-PAGE in the absence of reducing agents in order to assess the ability of the A14 proteins to undergo covalent dimerization; proteins were visualized by autoradiography. The unmodified (◂) and glycosylated (shaded circle) monomers of A14 were seen, as were several more slowly migrating forms that represent dimeric forms of wt A14 (stacked triangles). The positions of the 14,000-, 18,000-, and 29,000-M_r standards are shown at the left. (B) Immunoblot analysis of the wt and C₇₁S A14 proteins expressed in vivo. Transient-complementation analysis was performed by using the infection-transfection protocol described above. Lysates were resolved by nonreducing SDS-PAGE; A14 species were visualized by immunoblot analysis. Infections with vindA14 were performed in the presence (lane 1) or absence (lanes 2 to 5) of TET. The latter were transfected with vector alone (lane 3) or plasmids encoding wt A14 (lane 4) or the C₇₁S allele of A14 (lane 5). The A14 monomer ([◂]) and dimer (stacked triangles) are indicated; no dimer is seen in cells expressing the C₇₁S allele of A14 (lane 5). The positions of the 14,000-, 18,000-, and 29,000-M_r standards are shown at the left. (C) Transient-complementation assay using A14 C₇₁S. BSC40 monolayers were infected with vindA14 in the presence (+) or absence (−) of TET and were transfected with empty vector or plasmids expressing wt A14 or the C₇₁S allele. Twenty-four-hour viral yield was determined by plaque assay; the horizontal grey line represents the titer obtained upon transfection of empty vector.