Mapping EBV Early Antigens in Human Cells after Microinjection of Subgenomic DNA Clones

Abstract

The early antigen complex of EBV include the diffuse (EAD) and restricted (EAR) antigens which are detectable by indirect immunofluorescence (IF) using heterologous human sera and monoclonal antibodies. In an effort to map these coding regions, human nasopharyngeal carcinoma cells (CNE) were microinjected with subgenomic restriction fragments of the EBV genome cloned into pBR322. Two to three days after microinjection of DNA, cells were fixed in acetone and stained by IF for antigen expression. The Bam HI M subgenomic fragment encodes EAD and cells injected with this fragment produce diffuse nuclear fluorescence with a monoclonal antibody, R3.1. The Bam HI A fragment at the right end of the EBV genome produces filamentous cytoplasmic fluorescence with a monoclonal antibody specific for the EAR antigen. The Eco RI D fragment of EBV DNA which overlaps the right side of Bam HI A and extends to the right terminus of the genome produces similar reactivity with EAR specific antibodies.