The potential terminase subunit of human cytomegalovirus, pUL56, is translocated into the nucleus by its own nuclear localization signal and interacts with importin alpha

Human cytomegalovirus (HCMV) DNA-binding protein pUL56 is thought to be inv olved in the cleavage/packaging process of viral DNA and therefore needs to be transported into the nucleus. By using indirect immunofluorescence anal ysis, HCMV pUL56 (p130) was found to be localized predominantly in the nucl eus of infected cells. Solitary expression of wild-type as well as epitope- tagged pUL56 also resulted in nuclear distribution after transfection, sugg esting the presence of an endogenous nuclear localization signal (NLS), Del etion of a carboxy-terminal stretch of basic amino acids (aa 816-827) preve nted nuclear translocation, indicating that the sequence RRVRATRKRPRR of HC MV pUL56 mediates nuclear targetting, The signal character of the NLS seque nce was demonstrated by successful transfer of the NLS to a reporter protei n chimera. Furthermore, sequential substitutions of pairs of amino acids by alanine in the context of the reporter protein as well as substitutions wi thin the full-length pUL56 sequence indicated that residues at positions 7 and 8 of the NLS (R and K at positions 822 and 823 of pUL56) were essential for nuclear translocation, In order to identify the transport machinery in volved, the potential of pUL56 to bind importin alpha (hSRP1 alpha) was exa mined. Clear evidence of a direct interaction of a carboxy-terminal portion as well as the NLS of pUL56 with hSRP1 alpha was provided by in vitro bind ing assays. In view of these findings, it is suggested that nuclear translo cation of HCMV pUL56 is mediated by the importin-dependent pathway.