Characterization of the protein encoded by Varicella-Zoster Virus (VZV) open reading frame 25 (ORF25)

M.G. VIZOSO PINTO; R. HAASE; M. SOMMER; J.M. VILLEGAS; T. STELLENBERGER; A. BAIKER

Estoril

Workshop; The 33rd International Herpesvirus Workshop; 2008

Varicella-Zoster virus (VZV), a neurotropic alphaherpesvirus, causes chickenpox upon primary infection and shingles after reactivation from latency. VZV ORF25, the orthologue of UL33 in Herpes Simplex virus (HSV), is a 156 amino acid (aa) protein with a C-terminal region that is highly conserved among all herpesviruses. Little is known about ORF25 functions, but recent work in HSV has suggested a role in DNA packaging. We have performed a PCR mutagenesis strategy to construct nine different aa-to-alanine substitution mutants within the conserved C-terminal region of ORF25. All nine aa substitution mutants, a wildtype revertant mutant, a revertant mutant containing a synthetic ORF25 gene variant, and a complete deletion mutant of ORF25 were analyzed for their ability to reconstitute recombinant VZV by utilizing the cosmid system technology. So far, both wildtype revertant mutants and three ORF25 aa substitution mutants, namely: PP 82/83 AA, VV 123/124 AA, and TRRR 133-136 AAAA, were able to reconstitute infectious viruses. At least one ORF25 aa substitution mutant virus shows a phenotype of attenuated growth and smaller plaques. ORF25 seems to be essential for VZV replication, since no infectious virus could be reconstituted from the deletion mutant of ORF25. ORF25 comprises a cytoplasmic localization when expressed alone, but translocates to the nucleus in virally infected cells, as we could demonstrate by immunofluorescence microscopy with a polyclonal rabbit antiserum. To identify viral binding partners of ORF25, we have performed a comprehensive yeast-two-hybrid (Y2H) analysis of N- and C-terminal mutants of ORF25 against a library of VZV proteins. These Y2H analyses indicate that ORF25 interacts with itself at its C-terminus and with other viral (mainly tegument) proteins at its N-terminus. The strong interaction of ORF25 with the tegument proteins: ORF63, ORF47, and ORF66 (HSV: ICP22, UL13, and US3) could be reproduced in a LUMIER-based screening system in 293 cells.