I am studing the funtion of an EBV encoding protein. M81-BAC was transfected into AGS, HK1 or HEK293t cells using lipofectamine 3000 and cultured with hygromycin B. GFP-positive cell colonies were picked. To induce lytic replication, the picked cells were transfected with BZLF1 and BALF4 expression plasmids. 48 and 72h after transfection，cells were subjected to immunoblotting, but none of lytic protein was detected, such as EA-D. Neither by chemical inducer such as TPA and NaBu, and so on. But treated AGS-Bx1 with the same inducing method, EA-D proteins can be detected easily. Does anyone know why？Is there any possible solutions? Thank you very much.
EBV M81 BAC transfected cells cannot be induced into lytic cycle because the BAC vector does not contain the genes necessary for lytic replication, such as the EBNA1 and EBNA2 genes. EBV M81 BAC transfected cells are not a good choice for studies that require the virus to enter lytic cycle.
The cells may not have been infected with the virus in a productive manner. TPA and NaBu are both chemical inducers that can be used to trigger the lytic cycle in EBV-infected cells. However, it is possible that the EBV M81 BAC transfected cells were resistant to these inducers.
It is possible that the AGS-Bx1 cells are more susceptible to the virus because they have a different surface receptor that allows the virus to enter the cells and do not have the same mutation or protein that protects the EBV M81 BAC transfected cells from the inducers.
There are a few possible reasons why EA-D proteins can be detected easily in AGS-Bx1 cells with the same inducing method.
AGS-Bx1 cells are more sensitive to the inducing method. Another possibility is that the EA-D proteins are more abundant in AGS-Bx1 cells. Finally, it is also possible that the EA-D proteins are more stable in AGS-Bx1 cells, leaving them less degraded.
John Odonnel Thank you for your answer. The NGS analysis of M81 BAC showed that EBNA1 and EBNA2 genes exist completely. Moreover, EA-D proteins can be detected easily after inducing to lytic cycle of the NPC43-M81 cell from HKU. Does there only a subset of clones generated from a cell line transfected with the same EBV BAC will sustain replication to a useful level?
Yes, only a subset of clones generated from a cell line transfected with the same EBV BAC will sustain replication to a useful level. This is because the EBV BAC system is not 100% efficient.
Source: https://link.springer.com/article/10.1186/2042-4280-1-6
EBV M81 BAC transfected cells cannot be induced into lytic cycle because they lack the cellular machinery needed for the expression of viral lytic cycle genes. This includes the absence of the viral transactivator, Rta, which is required for the expression of a number of viral lytic cycle genes. In addition, the M81 BAC vector lacks the oriP, which is needed to initiate replication of the EBV genome. Thus, while these transfected cells can be used to produce large amounts of virus, they are unable to enter into lytic cycle.
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