Genetic analysis of the Epstein-Barr virus-coded leader protein EBNA-LP as a 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 virus-induced B cell transformation. Here we report the genetic analysis of EBNA-LP function using two transient co-transfection assays of co-operativity, activation of latent membrane protein 1 (LMP1) expression from a resident EBV genome in Akata-BL cells and activation of an EBNA2-responsive reporter construct. Small deletions were introduced into each of five conserved regions (CRs) of EBNA-LP sequence present in type 1 and type 2 EBV strains and in several primate lymphocryptovirus EBNA-LP homologues. Deletions within all three CRs in the EBNA-LP W1W2 repeat domain completely abrogated function, through inhibition of nuclear localization in the cases of CR1 and CR2 but not of CR3; deletions within CR4 and CR5 in the Y1Y2 unique domain had relatively 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 (S34,36,63) within W1W2 not only abrogated EBNA-LP activity but was associated with a complete loss of EBNA2 detectability in co-transfected cells, implying possible destabilization of the co-expressed EBNA2 protein. More importantly the individual substitution of S36 completely blocked EBNA-LP/EBNA2 co-operativity while retaining EBNA2 expression. We infer critical roles for the CR3 domain and for the S36 residue in EBNA-LP's co-operative function.
McCann
Eamon M
EM
CRC Institute for Cancer Studies, The University of Birmingham, Edgbaston, Birmingham B15 2TA, UK1.
Kelly
Gemma L
GL
CRC Institute for Cancer Studies, The University of Birmingham, Edgbaston, Birmingham B15 2TA, UK1.
Rickinson
Alan B
AB
CRC Institute for Cancer Studies, The University of Birmingham, Edgbaston, Birmingham B15 2TA, UK1.
Bell
Andrew I
AI
CRC Institute for Cancer Studies, The University of Birmingham, Edgbaston, Birmingham B15 2TA, UK1.
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001402:B-Lymphocytes / Q000378:metabolism / Q000821:virology*; D002461:Cell Line, Transformed; D019309:Epstein-Barr Virus Nuclear Antigens / Q000502:physiology*; D017353:Gene Deletion; D005814:Genes, Viral; D004854:Herpesvirus 4, Human / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D012694:Serine / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*
C402641:EBNA-2 protein, Human herpesvirus 4; C402640:EBNA-LP protein, Human herpesvirus 4; D019309:Epstein-Barr Virus Nuclear Antigens; D014764:Viral Proteins; D012694:Serine; D000409:Alanine