Engineering a live attenuated vaccine: Mutational analysis of varicella-zoster virus (VZV) glycoproteins,gI andgE, in recombinant VZV strains

The predominant virion envelope glycoprotein, gE, noncovalently complexes with gI in VZV infected cells. To evaluate the functions of gE and gI, a series of deletions were made using VZV cosmids. The gE and gI ORFs were subcloned from one of four overlapping fragments of genomic DNA from the Oka strain that had been ligated into cosmid vectors. Mutations were made using restriction enzymes and/or PCR. The altered fragments were then ligated back into the cosmid. The mutated cosmid was transfected into melanoma cells with the three other cosmids using calcium phosphate. No virus was detected when both gI and gE were deleted while virus was obtained with positive controls. However, a virus producing smaller and slower growing plaques was generated upon deleting 60% of the N-terminus, 40% of the C-terminus, or the entire gI ORF. PCR was then used to rescue the change in phenotype by adding the gI ORF into a unique restriction site of the gI deleted cosmid. Cells infected with the gI mutant viruses form clustered, unorganized polykaryocytes versus organized, round polykaryocytes with nuclei surrounding a centralized Golgi in the parental and gI repaired viruses. In addition to a change in viral growth characteristics, gE was found to localize in patches on the surface of cells infected with the gI mutated viruses when studied by confocal microscopy. gE was found in a diffuse staining pattern in parental and gI repaired viruses. This study is the first investigation of VZV gI and gE mutants in whole virus and demonstrated that gI is dispensable for VZV replication and gE may be indispensable for VZV growth in vitro. Deletion of gI also alters plaque forming phenotype significantly, VZV growth in vitro, and distribution of gE on the cell surface. In addition, the N-terminus of gI is not sufficient to return the altered phenotype to that of the parental strain. The deletions in the above mentioned genes will result in a greater understanding of how this virus functions and this increase in knowledge will be essential in designing a live attenuated vaccine.