NUCLEAR TARGETING OF THE TEGUMENT PROTEIN PP65 (UL83) OF HUMAN CYTOMEGALOVIRUS - AN UNUSUAL BIPARTITE NUCLEAR-LOCALIZATION SIGNAL FUNCTIONSWITH OTHER PORTIONS OF THE PROTEIN TO MEDIATE ITS EFFICIENT NUCLEAR TRANSPORT

Large amounts of pp65 (UL83) of human cytomegalovirus are translocated to the cell nucleus during the first minutes after uptake of the tegu ment protein from infecting viral particles. Two stretches of basic am ino acids which resembled nuclear localization signals (NLS) of both t he simian virus 40 type and the bipartite type were found in the prima ry structure of pp65. Deletion of these sequences significantly impair ed nuclear localization of the truncated proteins after transient expr ession. The results indicated that both elements contributed to the nu clear localization of the protein. When fused to the bacterial beta-ga lactosidase, only one of the two basic elements was sufficient to medi ate nuclear translocation. This element consisted of two clusters of b asic amino acids (boxes C and D), which were separated by a short spac er sequence. In contrast to other bipartite NLS of animal cells, both basic boxes C and D functioned independently in nuclear transport, thu s resembling simian virus 40-type NLS. Yet, complete translocation of beta-galactosidase was only found in the bipartite configuration. When both boxes C and D were fused, thereby deleting the intervening seque nces, the nuclear transport of beta-galactosidase was reduced to level s seen with constructs in which only one of the boxes was present. App ropriate spacing, therefore, was important but not absolutely required . This was in contrast with results for other bipartite NLS, in which spacer deletions led to complete cytoplasmic retention. The presented results demonstrate that efficient nuclear transport of pp65 is mediat ed by one dominant NLS and additional targeting sequences. The major N LS of pp65 is an unusual signal sequence composed of two weak NLS whic h function together as one strong bipartite nuclear targeting signal.