Identification of the interaction domain of the small terminase subunit pUL89 with the large subunit pUL56 of human cytomegalovirus.
The small terminase subunit pUL89 of human cytomegalovirus (HCMV) is thought to be required for cleavage of viral DNA into unit-length genomes in the cleavage/packaging process. Immunoprecipitations with a UL89-specific antibody demonstrated that pUL89 occurs predominantly as a monomer of approximate M(r) 75.000 together with a dimer of approximate 150.000. This was confirmed by gel permeation chromatography. In view of its putative function, pUL89 needs to be transported into the nucleus. By use of laser scanning confocal microscopy, pUL89 was found to be predominantly localized throughout the nucleus and in particular in viral replication centers of infected cells. By immunofluorescence, we demonstrated that both terminase subunits co-localized in viral replication centers. Furthermore, analysis with pUL89 GST-fusion protein mutants showed that amino acids 580-600 may represent the interaction domain with pUL56. To verify this result, a recombinant HCMV genome was constructed in which the UL89 open reading frame was disrupted. By transfection of the deletion BACmid alone, we showed that it has a lethal phenotype. Cotransfection assays demonstrated that, in contrast to pUL89 wild-type, a plasmid construct encoding a pUL89 variant without aa 580-590 as well as one encoding a variant without aa 590-600 could not complement the HCMV-pUL89 null genome, thus, suggesting that the 20 aa sequence GRDKALAVEQFISRFNSGYIK is sufficient for the interaction with pUL56 and in conclusion required for DNA packaging.
Thoma
Corina
C
Institute of Clinical and Molecular Virology, Schlossgarten 4, 91054 Erlangen, Germany.
Borst
Eva
E
Messerle
Martin
M
Rieger
Manuela
M
Hwang
Jae-Seon
JS
Bogner
Elke
E
D000818:Animals; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000737:chemistry*; D004279:DNA, Viral / Q000096:biosynthesis; D019281:Dimerization; D004706:Endodeoxyribonucleases / Q000737:chemistry*; D006801:Humans; D008297:Male; D018613:Microscopy, Confocal; D017434:Protein Structure, Tertiary; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry*; D015678:Viral Structural Proteins / Q000737:chemistry*
D004279:DNA, Viral; C090238:UL56 protein, cytomegalovirus; C436448:UL89 protein, Cytomegalovirus; D014764:Viral Proteins; D015678:Viral Structural Proteins; D004706:Endodeoxyribonucleases; C030894:terminase