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The mechanism by which the 14-kDa fusion protein of vaccinia virus (VV) is anchored in the envelope of intracellular naked virions (INV) is not understood. In this investigation, we demonstrate that the 14-kDa protein interacts with another virus protein with an apparent molecular mass of 21 kDa. Microsequence analysis of the N terminus of the 21-kDa protein revealed that this protein is encoded by the W A17L gene. The 21-kDa protein is processed from a 23-kDa precursor, by cleavage at amino acid position 16, at the consensus motif Ala-Gly-Ala, previously identified as a cleavage site for several W structural proteins. The 21-kDa protein contains two large internal hydrophobic domains characteristic of membrane proteins. Pulse-chase analysis showed that within 1 h after synthesis, the 14-kDa protein forms a stable complex with the 21-kDa protein. Formation of the complex was not inhibited by rifampin, indicating that the interaction between these two proteins occurs prior to virion morphogenesis. Immunoprecipitation analysis of disrupted virions showed the presence of the 21-kDa protein in the viral particle. Release of the 14-kDa-21-kDa protein complex from INV required treatment with the nonionic detergent Nonidet P-40 and a reducing agent. The protein complex consisted of 14-kDa trimers and of 21-kDa dimers. Since the 14-kDa fusion protein lacks a signal sequence and a large hydrophobic domain characteristic of membrane proteins, our findings suggest that the 21-kDa protein serves to anchor the 14-kDa protein to the envelope of INW.

Leishmania is a genus of parasitic protozoa capable of causing a spectrum of human diseases. The GP46/M-2 membrane glycoprotein has been demonstrated in a murine model system to elicit a protective immune response against infection with Leishmania amazonensis; in highly susceptible BALB/c mice, immunization leads to significant protection against infection. In the present study, for induction of long-term immunological effects, two recombinant vaccinia viruses, derived from the wild type and attenuated variant 48-7 and expressing the GP46/M-2 protein, were constructed; to ensure safety, we used the attenuated vaccinia virus mutant (48-7) as a live vector. Susceptible BALB/c mice immunized with either GP46/M-2-recombinant vaccinia virus were significantly protected against infection with L. amazonensis; 45 to 76% of the animals were completely protected (sterile) against a challenge inoculum of 103 infective organisms. The protectively immunized animals demonstrated T-and B-cell-dependent immunological responses; both lymphokine responses as well as antibody responses and long-term memory are indicative of T-celi activation. This first report of the use of a recombinant vaccinia virus to induce protection against a Leishmania infection indicates that recombinant vaccinia viruses should be of value in the design of a safe and effective vaccine against this parasitic disease.

An attenuated vaccinia virus mutant with specific genetic lesions has been used to develop a vehicle for safer live recombinant virus vaccines. The mutant virus 48-7 has an 8-MDa deletion starting 2.2 MDa from the left end of the viral genome and point mutations in the gene encoding the 14-kDa fusion protein that determines the plaque-size phenotype of the virus. Using the highly sensitive reporter gene luciferase, we have shown that this mutant can generate recombinant viruses that infect cultured cells and animals with normal vaccinia virus tropism. Insertion of the envelope and gag genes of human immunodeficiency virus type 1 into the attenuated vaccinia mutant resulted in their efficient expression and precursor processing in infected cultured cells. Infection of mice with human immunodeficiency virus-vaccinia recombinant viruses elicited human immunodeficiency virusspecific antibodies. Using mice pretreated with cyclophosphamide as a model for immunosuppression, the reduced virulence of the mutant recombinant virus was clearly evident. These findings demonstrate that the highly attenuated vaccinia virus mutant 48-7 can be used to generate effective and safer vaccines.

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