Gretchen Nelson
Johns Hopkins University - Cell, Molecular, Developmental Biology & Biophysics
NIH Advisor - Dr. Bernard Moss
Institute-Center - NIAID. 

Defense Date - May 19th at 2pm. 
Location - Mudd Hall / Room 100
                  3400 N. Charles St.
                  Baltimore, MD. 

CHARACTERIZATION OF VACCINIA VIRUS ENTRY-FUSION PROTEINS A28 AND H2.
The recently described vaccinia virus (VACV) entry/fusion complex (EFC) is comprised of at least eight polypeptides that are conserved in all poxviruses.  Neither the immunogenicity, the structure of the complex nor the roles of individual subunits are known. Here we begin to characterize two proteins of the EFC, A28 and H2. The first part of the work provides evidence for a direct interaction between the H2 and A28 subunits in the context of a virus infection as well as in uninfected cells transfected with expression plasmids. We focused on a 21 amino acid segment in H2 that is flanked by cysteine residues and contains LGYSGY, a motif similar to that found in the fusion peptides of other viruses. The effect of amino acid substitutions within the 21 amino acid segment was determined by an infectivity complementation assay using a conditional H2 null mutant of VACV. Three classes of mutations were found that: allowed wild-type complementation, resulted in a moderate decrease in complementation, or allowed lit le or no complementation. The latter class included glutamic acid substitutions of individual leucine and glycines and alanine substitution of both glycines within the LGYSGY motif. Loss of function correlated with decreased ability of the mutated H2 to interact with A28 in infected and uninfected cells. These data indicate that the LGYSGY motif is important for the interaction of H2 with A28 and suggest that it is buried within the EFC complex in the pre-fusion state.  In the second part of this work, A28 is investigated with regards to its immunogenicity.  We expressed and purified a recombinant 6X-HIS tagged A28 protein using the baculovirus system and Ni-NTA agarose.  Rabbit anti-serum to the protein was raised and shown to have neutralizing activity against VACV entry in a post-binding step. Using overlapping peptides we were able to show that at least one neutralizing epitope of A28 lies in the region between residues 73 and 92.
In addition passive immunization of mice with purified antibody from the rabbit serum gave protection against a VACV challenge.