Question

how you will verify whether Bcell markers (including CD19, CD20, and CD23) are down regulated with EBNA1 inhibitor treatment. What assays do you want to do do verify this? How will you look at this in the context of EBV gene expression and viral load.

Answer 1

EBNA1 is an EBV-encoded DNA-binding protein required for viral genome maintenance during latent infection.EBNA1 primarily resides in the nucleus of EBV-infected cells. The nuclear localization of EBNA1 is regulated through the interaction between a short EBNA1 sequence (NLS, nucleus localization sequence, a.a. 379-386) and two importins (nuclear import adaptor, α1 and α5). EBNA1 is essential for the immortalization of primary B-lymphocytes by EBV infection and its inhibition by siRNA depletion or by ectopic expression of dominant negative mutants induce apoptosis in EBV-infected B cells.

Marker Present over cell surface of B.cell such as CD19, CD20, and CD23 are down regulated with EBNA1 inhibitor treatment. this can be assayed using Immunofluorescence staining antibodies against the cell surface marker. Quantification of marker could be easier process to examine the down regulation in presence of EBNA1 inhibitor or in CONTROL (WITHOUT INHIBITOR)

Another technique could be used is the mRNA quantification of the B cell surface marker which can be potential indicator of the expression of cell surface marker in the presence and Absence of EBNA1 INHIBITOR.

To measure EBV loads, real-time polymerase chain reaction (PCR) is a standard and widely-used method The real-time PCR method measures the accumulation of amplified products with a laser scanning in a closed tube or 96-well plate format . Fluorogenic probes and SYBR green I dye are used as markers for accumulation of PCR products. This method is rapid, sensitive, reproducible, and is advantageous because the reaction is performed in closed tubes or wells, thereby reducing the risk of carry-over contamination