Identification of latent membrane protein 2A (LMP2A) domains essential for the LMP2A dominant-negative effect on B-lymphocyte surface immunoglobulin signal transduction.
Epstein-Barr virus (EBV) recombinants which carry three different deletion mutations in the LMP2A cytoplasmic amino-terminal domain were constructed. The presence of each mutation, LMP2A delta 21-36, LMP2A delta 21-64, and LMP2A delta 21-85, in EBV-infected transformed lymphoblastoid cell lines was confirmed by PCR analysis and Southern blot hybridization. Confirmation of mutant LMP2A protein expression was by immunofluorescence and immunoblotting with a newly identified rat monoclonal antibody that recognizes each of the LMP2A deletion mutations. Lymphoblastoid cell lines infected with recombinant EBV DNAs containing the mutations were analyzed for loss of LMP2A's dominant-negative effect on surface immunoglobulin signal transduction by monitoring induction of tyrosine phosphorylation, calcium mobilization, and activation of lytic replication following surface immunoglobulin cross-linking. Domains of LMP2A important for induction of tyrosine phosphorylation, calcium mobilization, and activation of lytic replication were identified.
Fruehling
S
S
Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois, USA.
Lee
S K
SK
Herrold
R
R
Frech
B
B
Laux
G
G
Kremmer
E
E
Grässer
F A
FA
Longnecker
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D017931:DNA Primers; D004279:DNA, Viral; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009856:Oncogene Proteins, Viral / Q000502:physiology; D019000:Phosphotyrosine / Q000032:analysis; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D051381:Rats; D011947:Receptors, Antigen, B-Cell / Q000502:physiology*; D011995:Recombination, Genetic; D017384:Sequence Deletion; D015398:Signal Transduction; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D014779:Virus Replication
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004279:DNA, Viral; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D009856:Oncogene Proteins, Viral; D011947:Receptors, Antigen, B-Cell; D014763:Viral Matrix Proteins; D019000:Phosphotyrosine; D002118:Calcium