Identification of a Vaccinia Virus Penetration Protein

Abstract

A vaccinia virus structural protein responsible for infection was identified by monoclonal antibodies (mAb). Two mAbs (2D5 and 8C2) neutralized the virus at a dilution of about 105. The 2D5 mAb reacted with VP29K under standard immunoblotting conditions and with a 23-kDa protein when virus was dissociated under nonreducing conditions. The 8C2 mAb reacted with the 23-kDa protein, but not with VP29K. Two-dimensional electrophoresis demonstrated that the 23-kDa protein was the nonreduced form of VP29K. Since they possess the same N-terminal amino acid sequence, the protein was renamed VP23-29K. The gene that encoded it was HindIII A17L ORF. The VP23-29K-dependent process of infection did not occur during the adsorption phase at 4°, and trypsin-treated virus could complete the process within 10 min at 37°. One half of the trypsin-treated intracellular mature virus (IMV) achieved the process within 20 min, but for normal IMV this time period was 2 hr. VP23-29K had function for the early step of penetration, and the functional site in the nonreduced 23-kDa form was masked to some extent in normal virus. The late cell fusion by the fusion positive (F+) D1 mutant proceeded in neutral pH. Cells infected with F- IHD-J strain virus did not fuse, but a short treatment with pH 5 medium developed cell fusion. Both of the cell fusions were inhibited by the 2D5 and 8C2 mAbs. Virion VP23-29K was suggested to be the fusion protein for the early penetration and the late cell fusion phases of vaccinia infection cycle.