THE GENE-PRODUCT OF HUMAN CYTOMEGALOVIRUS OPEN READING FRAME UL56 BINDS THE PAC MOTIF AND HAS SPECIFIC NUCLEASE ACTIVITY

Using the cis-acting human cytomegalovirus (HCMV) packaging elements ( pac 1 and pac 2) as DNA probes, specific DNA-protein complexes were de tected by electrophoretic mobility shift assay (EMSA) in both HCMV-inf ected cell nuclear extracts and recombinant baculovirus-infected cell extracts containing the HCMV p130 (pUL56) protein, DNA-binding protein s, which were common in uninfected and infected cell extracts, were al so detected, Mutational analysis showed that only the AT-rich core seq uences in these cis-acting motifs, 5'-TAA AAA-3' (pac 1) and 5'-TTTTAT -3' (pac 2), were required for specific DNA-protein complex formation, The specificity of the DNA-protein complexes was confirmed by EMSA co mpetition. Furthermore, a specific endonuclease activity was found to be associated with lysates of baculovirus-infected cells expressing re combinant p130 (rp130). This nuclease activity was time dependent, rel ated to the amount of rp130 in the assay, and ATP independent. Nucleas e activity remained associated with rp130 after partial purification b y sucrose gradient centrifugation, suggesting that this activity is a property of HCMV p130. We propose a possible involvement of p130 in HC MV DNA packaging.