Interactions between human cytomegalovirus helicase-primase proteins.
The human cytomegalovirus (HCMV) UL70, UL102, and UL105 genes are predicted to encode essential proteins that assemble the replicative helicase-primase complex based on sequence and genome position similarities to putative herpes simplex virus type 1 (HSV-1) counterparts. Consistent with this prediction, they are required for transient complementation of DNA synthesis. However, little is known about their physical interactions and biochemical activities, primarily because of their restricted expression in HCMV-infected cells. To look for assembly of the predicted complexes, we prepared rabbit polyclonal antisera and used Semliki Forest Virus (SFV) vectors to express untagged and glutathione-S-transferase (GST)-tagged UL70, UL102 and UL105 proteins. The UL70 and UL105 proteins co-purified with the GST-tagged UL102 protein from triply-infected baby hamster kidney cells (BHK-21), and pUL70, but not pUL105, co-purified with pGST-UL102 from dually infected BHK-21 cells. In immunoprecipitation experiments with untagged SFV-expressed proteins, pUL70 or pUL105 coprecipitated with pUL102, pUL102 or pUL70 co-precipitated with pUL105; and pUL102 or pUL105 coprecipitated with pUL70. Comparison of the GST-pull down and immunoprecipitation experiments suggested that the amino-terminal GST-tag interfered with certain pairwise interactions. These results support the prediction that the HCMV helicase-primase proteins assemble a three-protein heteromeric complex, and show that each protein contacts both partners.
McMahon
Timothy P
TP
Department of Biomedical Sciences, University at Albany School of Public Health, Albany, NY 12201-2002, USA.
Anders
David G
DG
D000914:Antibodies, Viral; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D004265:DNA Helicases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D016679:Genome, Viral; D006801:Humans; D014764:Viral Proteins / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D000914:Antibodies, Viral; C091942:UL102 protein, cytomegalovirus; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D019915:DNA Primase; D004265:DNA Helicases