Early events associated with infection of Epstein-Barr virus infection of primary B-cells.
Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6-7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6-12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.
Sabyasachi
S
Halder
Masanao
M
Murakami
Subhash C
SC
Verma
Pankaj
P
Kumar
Fuming
F
Yi
Erle S
ES
Robertson
D000954:Antigens, Surface / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism* / Q000821:virology*; D002460:Cell Line; D002472:Cell Transformation, Viral; D022202:Chromosomes, Artificial, Bacterial; D017931:DNA Primers / Q000737:chemistry; D020031:Epstein-Barr Virus Infections / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D008213:Lymphocyte Activation; D008957:Models, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000954:Antigens, Surface; D017931:DNA Primers; D049452:Green Fluorescent Proteins