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

VIZOSO PINTO, M. G.; SOMMER, M.; HAASE, R.; BAIKER, A.

Heidelberg, Alemania

Jornada; 18. Jahrestagung der Gesellschaft für Virologie; 2008

Sociedad alemana de Virología

The Varicella-Zoster virus (VZV) is a member of the neurotropic alphaherpesvirus subfamily,causing chickenpoxupon primary infection and shingles after reactivation from latency. VZVORF25, the orthologue of UL33 in HerpesSimplex virus (HSV), is a 156 aminoacid (aa) proteinwith a C-terminal region that is highly conserved amongall herpesviruses. ORF25 comprises anuclear localization in virally infected Melanoma cells, as demonstrated byimmunofluorescencemicroscopy with a polyclonal rabbit antiserum. A systematic yeast-two-hybrid (Y2H) screenofall known 71 VZV proteins revealed 31 putative interaction partners, indicating an important roleof this small proteinin viral replication (Science 311: 239-242).We have performed a PCR mutagenesis strategy to construct 9 differentaa-to-alanine substitutionmutants within the conserved C-terminal region of ORF25. All 9 aa substitution mutants,awildtype revertant mutant and a complete deletion mutant of ORF25 were analyzed for theirability to reconstituterecombinant VZV by utilizing the cosmid system technology within anORF25 depleted pOKA backbone. So far, thewildtype revertant mutant and 3 out of 9 ORF25 aasubstitution mutants, namely: PP 82/83 AA, VV 123/124 AA, andTRRR 133-136 AAAA, wereable to reconstitute infectious virus. No infectious virus could be reconstituted from the7remaining aa substitution mutants and the complete deletion mutant of ORF25, indicating thatORF25 is essentialfor viral replication of VZV. A phenotypical analysis of the ORF25 mutants isin progress and may contribute to ourunderstanding of ORF25 function(s).