Genetic analysis of the Epstein-Barr virus-coded leader protein EBNA-LP asa co-activator of EBNA2 function

Co-operation between the Epstein-Barr virus (EBV)-coded leader protein EBNA -LP and the nuclear antigen EBNA2 appears to be critical for efficient viru s-induced B cell transformation. Here we report the genetic analysis of EBN A-LP function using two transient co-transfection assays of cooperativity, activation of latent membrane protein 1 (LMP1) expression from a resident E BV genome in Akata-BL cells and activation of an EBNA2-responsive reporter construct. Small deletions were introduced into each of five conserved regi ons (CRs) of EBNA-LP sequence present in type 1 and type 2 EBV strains and in several primate lymphocryptovirus EBNA-LP homologues. Deletions within a ll three CRs in the EBNA-LP W1W2 repeat domain completely abrogated functio n, through inhibition of nuclear localization in the cases of CR1 and CR2 b ut not of CR3; deletions within CR4 and CR5 in the Y1Y2 unique domain had r elatively little effect, yet loss of the whole Y2 sequence blocked activity . Alanine substitution of serine residues within potential phosphorylation sites identified two mutants of particular interest. Substitution of three such residues (S-34,S-36,S-63) within W1W2 not only abrogated EBNA-LP activ ity but was associated with a complete loss of EBNA2 detectability in co-tr ansfected cells, implying possible destabilization of the co-expressed EBNA 2 protein. More importantly the individual substitution of S., completely b locked EBNA-LP/EBNA2 co-operativity while retaining EBNA2 expression. We in fer critical roles for the CR3 domain and for the S-36 residue in EBNA-LP's co-operative function.