Assembly of the Epstein-Barr virus BBLF4, BSLF1 and BBLF2/3 proteins and their interactive properties

Epstein-Barr virus (EBV) encodes putative helicase-primase proteins BBLF4, BSLF1 and BBLF2/3, which are essential for the lytic phase of viral DNA rep lication, The BSLF1, BBLF4 and BBLF2/3 proteins were expressed in B95-8 cel ls after induction of a virus productive cycle, possessing apparent molecul ar masses of 89 kDa, 90 kDa and 80 kDa, respectively. The anti-BSLF1 or ant i-BBLF2/3 protein-specific antibody, which recognizes its target protein in both Western blotting and immunoprecipitation analyses, immunoprecipitated all of the BSLF1, BBLF4 and BBLF2/3 proteins from the extract of the cells with a virus productive cycle, indicating that these viral proteins are as sembled together in vivo. To characterize their protein-protein interaction s in detail, recombinant baculoviruses capable of expressing each of these viral gene products in insect cells were constructed. The assembly of the t hree virus replication proteins was reproduced in insect cells co-infected with the three recombinant baculoviruses, indicating that complex formation does not require other EBV replication proteins. Furthermore, experiments performed by using the extracts from insect cells co-infected with each pai r of the recombinant viruses demonstrated that the BSLF1 protein could inte ract separately with both the BBLF4 and BBLF2/3 proteins and that the BBLF2 /3 protein also interacted with the BBLF4 protein. These observations stron gly suggest that within the BBLF4-BSLF1-BBLF2/3 complex each component inte racts directly with the other two, resulting in helicase-primase enzyme act ivity.