FUNCTIONAL REGIONS OF THE HUMAN CYTOMEGALOVIRUS PROTEIN PUL97 INVOLVED IN NUCLEAR-LOCALIZATION AND PHOSPHORYLATION OF GANCICLOVIR AND PUL97ITSELF

In order to identify functional regions of the human cytomegalovirus p rotein pUL97 (i) different 5' fragments of the UL97 open reading frame (ORF) were fused to the coding region of the green fluorescent protei n and (ii) recombinant vaccinia viruses (rVV) were generated carrying two full-length and 11 mutated UL97 ORFs, The results indicated the pr esence of an N-terminal region within pUL97 which changed the intracel lular distribution of the fusion proteins. pUL97 was localized in the nucleus, but not in the nucleoli, and was detected in the nuclear matr ix fraction, Expression of all pUL97 mutants could be confirmed by Wes tern blot analysis. pUL97-associated ganciclovir (GCV) phosphorylation in rVV-infected cells, determined quantitatively by HPLC analysis, wa s abolished completely using individual UL97 deletion mutants. Phospho rylation of full-length and some of the mutated pUL97 was detected in cells infected with the rVVs, The UL97 constructs carrying point mutat ions from GCV-resistant HCMV isolates at positions 460M, 520H, 594V, a nd the 4 aa deletion 590AACR593, also resulted in decreased but not ab olished phosphorylation of GCV in the rVV system, whereas the phosphor ylation of pUL97 itself was not influenced. The rVV system is a suitab le method for quantitatively testing the functional relevance of pUL97 mutations.