Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naive B cells, and facilitates recruitment of transcription factors to the viral genome

Abstract

. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/176099 doi: bioRxiv preprint first posted online Genetic analysis of EBNA-LP function host factors EBF1 and RBPJ to all latency promoters tested was severely delayed, 39 whereas these same factors were recruited efficiently to several host genes, some of 40 which exhibited increased EBNA2 recruitment. 41We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating 42 gene transcription, but rather facilitates the recruitment of several transcription factors to

Heterogeneity of the Epstein-Barr Virus (EBV) Major Internal Repeat Reveals Evolutionary Mechanisms of EBV and a Functional Defect in the Prototype EBV Strain B95-8

“…It is not formally possible to say whether the EBNA-LP produced in HB9 LCLs is truncated or not. However, we have found that deletion of the Y exons from the B95-8 BAC (in the context of heterogeneous IR1) results in very low protein levels ( 49 ), suggesting that any truncated EBNA-LP made may not be stable. Nevertheless, we have observed that the MVs in B95-8 do appear to have a detrimental impact on its transforming ability.…”

“…The y axis shows the cell count, and the x axis shows the log 10 CellTrace Violet fluorescence intensity. The left plot is for CD19 + (gated) live cells infected as mixed lymphocytes, and the right plot is for CD19-purified B cells infected and grown in the absence of other cells, based on data also published elsewhere ( 49 ).…”

“…Most strikingly, however, the more distant MV (G14617T) (highlighted in red in Table ST4) changes the last codon of the W1 exon of EBNA-LP from GAG (glutamate) to a TAG stop codon. As described elsewhere ( 49 ), we generated a B95-8 BAC (WT w ) in which the original IR1 was replaced with 6.6 copies of the consensus IR1 repeat unit, removing all MVs. LCLs established with WT w exhibit both higher and more consistent levels of EBNA-LP protein and a more consistent range of larger EBNA-LP isoforms ( Fig.…”

“…Peripheral blood leukocytes (PBLs) were isolated from blood by use of a Ficoll gradient, washed in RPMI medium supplemented with 1% fetal calf serum (FCS), and stored in RPMI-20% FCS-10% dimethyl sulfoxide (DMSO). Frozen PBLs from four donors were recovered, seeded at 2 × 10 6 cells per well in a 24-well plate, and infected with 2 × 10 5 Raji infectious units of either HB9 or WT w EBV ( 49 ). Medium was replaced on the following day and twice weekly thereafter.…”

“…Cyclosporine (100 ng/ml) was added to the medium for the first 2 weeks. Proliferation was assessed by staining cells—either CD19-purified B cells or peripheral blood leukocytes—with CellTrace Violet (Life Technologies) and analyzing them at 8 days postinfection by flow cytometry as described elsewhere ( 49 ).…”