We report the identification of a functional nuclear localization signal (NLS) in the human cytomegalovirus (HCMV) large tegument protein pUL48 that is required for nuclear localization in transfected cells and is essential for viral growth. The NLS was mapped to pUL48 amino acid residues 284 to 302. This sequence contains a bipartite NLS comprising two clusters of basic residues (bC1 and bC2) separated by 9 amino acids. Deletion or mutation of bC1 or mutation of bC2 abrogated the nuclear localization of full-length pUL48 in transiently expressing cells, thus strongly implying a bipartite character of the NLS. Nuclear localization could be restored by fusion of a functional NLS together with enhanced green fluorescent protein (EGFP) to the N terminus of these mutants. In HCMV-infected cells, pUL48 was found in both nuclear and cytoplasmic fractions, supporting a function of the NLS during virus infection. NLS mutant viruses, generated by markerless bacterial artificial chromosome mutagenesis, were not viable in cell culture, whereas coexpression of pUL48 complemented growth of these mutants. The fusion of a functional NLS to the N terminus of pUL48 in a nonviable NLS mutant virus partially rescued the growth defect. Furthermore, the replacement of the bipartite pUL48 NLS by the monopartite pUL36 NLS of herpes simplex virus 1 supported viral growth to some extent but still revealed a severe defect in focus formation and release of infectious virus particles. Together, these results show that nuclear targeting of pUL48 is mediated by a bipartite NLS whose function is essential for HCMV growth.

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Cholesterol oxidase (COase) is a bacterial enzyme catalyzing the first step in the biodegradation of cholesterol. COase is an important biotechnological tool for clinical diagnostics and production of steroid drugs and insecticides. It is also used for tracking intracellular cholesterol; however, its utility is limited by the lack of an efficient temporal control of its activity. To overcome this we have developed a regulatable fragment complementation system for COase cloned from sp. The enzyme was split into two moieties that were fused to FKBP (FK506-binding protein) and FRB (rapamycin-binding domain) pair and split GFP fragments. The addition of rapamycin reconstituted a fluorescent enzyme, termed split GFP-COase, the fluorescence level of which correlated with its oxidation activity. A rapid decrease of cellular cholesterol induced by intracellular expression of the split GFP-COase promoted the dissociation of a cholesterol biosensor D4H from the plasma membrane. The process was reversible as upon rapamycin removal, the split GFP-COase fluorescence was lost, and cellular cholesterol levels returned to normal. These data demonstrate that the split GFP-COase provides a novel tool to manipulate cholesterol in mammalian cells.