title: A "coiled-coil" motif is important for oligomerization and DNA binding properties of human cytomegalovirus protein UL77.
abstract: Human cytomegalovirus (HCMV) UL77 gene encodes the essential protein UL77, its function is characterized in the present study. Immunoprecipitation identified monomeric and oligomeric pUL77 in HCMV infected cells. Immunostaining of purified virions and subviral fractions showed that pUL77 is a structural protein associated with capsids. In silico analysis revealed the presence of a coiled-coil motif (CCM) at the N-terminus of pUL77. Chemical cross-linking of either wild-type pUL77 or CCM deletion mutant (pUL77ΔCCM) implicated that CCM is critical for oligomerization of pUL77. Furthermore, co-immunoprecipitations of infected and transfected cells demonstrated that pUL77 interacts with the capsid-associated DNA packaging motor components, pUL56 and pUL104, as well as the major capsid protein. The ability of pUL77 to bind dsDNA was shown by an in vitro assay. Binding to certain DNA was further confirmed by an assay using biotinylated 36-, 250-, 500-, 1000-meric dsDNA and 966-meric HCMV-specific dsDNA designed for this study. The binding efficiency (BE) was determined by image processing program defining values above 1.0 as positive. While the BE of the pUL56 binding to the 36-mer bio-pac1 containing a packaging signal was 10.0 ± 0.63, the one for pUL77 was only 0.2±0.03. In contrast to this observation the BE of pUL77 binding to bio-500 bp or bio-1000 bp was 2.2 ± 0.41 and 4.9 ± 0.71, respectively. By using pUL77ΔCCM it was demonstrated that this protein could not bind to dsDNA. These data indicated that pUL77 (i) could form homodimers, (ii) CCM of pUL77 is crucial for oligomerization and (iii) could bind to dsDNA in a sequence independent manner.
authors: Institute of Virology, Charité Universitätsmedizin Berlin, Berlin, Germany.
authors: Christina Sylvia
authors: CS
authors: Meissner
authors: Pánja
authors: P
authors: Köppen-Rung
authors: Alexandra
authors: A
authors: Dittmer
authors: Sara
authors: S
authors: Lapp
authors: Elke
authors: E
authors: Bogner
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020029:Base Pairing; D002213:Capsid / Q000378:metabolism; D019295:Computational Biology; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism; D042003:DNA Packaging; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space / Q000821:virology; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000378:metabolism
chemical_list: D004279:DNA, Viral; D014764:Viral Proteins