Up to 100-fold increase of apparent gene expression in the presence of Epstein-Barr virus oriP sequences and EBNA1: implications of the nuclear import of plasmids

Abstract

A 100-fold increase in luciferase activity was observed in 293 cells, stably expressing Epstein-Barr nuclear antigen 1 (EBNA1; 293-EBNA1 cells), that had been transiently transfected with plasmids carrying Epstein-Barr virus (EBV) oriP sequences. This increase was observed in comparison to reporter gene activity obtained after transfection with a plasmid carrying no oriP sequences. The luciferase gene on these plasmids was under the control of either the cytomegalovirus immediate-early 1 gene enhancer-promoter (CMV IE1) or the Rous sarcoma virus promoter. The increase of reporter gene activity was not due to plasmid replication, since a similar enhancement was observed in the presence of aphidicolin, an inhibitor of replicative DNA synthesis, or after deletion of the dyad symmetry (DS) element within oriP. Luciferase production was not increased in the presence of only the DS element. Microinjection of plasmids carrying the CMV IE1 promoter-driven luciferase gene with or without oriP sequences into the nuclei of 293-EBNA1 cells resulted in a 17-fold increase in luciferase activity. Cytoplasmic injection of these plasmids led to an enhancement of luciferase activity of up to 100-fold. This difference in the factor of activation after nuclear or cytoplasmic injection could be ascribed to increased transport of plasmids carrying oriP from the cytoplasm to the nucleus in the presence of EBNA1. These data suggest the possibility of substantially increasing the apparent expression of a gene under the control of a strong constitutive promoter in the presence of oriP sequences and EBNA1. This improvement in expression is due to intranuclear enhancement of gene expression. oriP-specific transport of plasmid DNA from the cytoplasm of 293-EBNA1 cells to the nucleus seems to contribute to the observed effect.

FIG. 1

(A) Schematic representation of the expression cassettes and oriP status of the tested plasmids. Plasmids were based on pCEP₄ (Invitrogen) deleted for the EBNA1-encoding sequence. The plasmids pTG11056, pTG11052, and pTG11182 carry the wild-type oriP sequences (positions 7334 to 9519 according to reference 3), indicated by oriP. The DS element (positions 8994 to 9134 according to reference 3) within oriP was deleted (pTG11155; ΔDS), or oriP was replaced by the DS element only (positions 9021 to 9133 according to reference 3) (pTG11157). The luciferase (luc) expression cassette contains the mouse HMG1 intron (intron) and the SV40 polyadenylation signal (pA) and was under the control of either the CMV IE1 promoter (CMV p) or the RSV promoter (RSV p; pTG11181 and pTG11182). pTG11052 carries an empty expression cassette. (B) Representation of the relative light units (RLU) obtained after quantification of luciferase activity in 293-EBNA1 cells (Invitrogen) transfected with the indicated plasmids: 3 × 10⁴ cells per well had been seeded on 48-well plates (Costar) and transfected with 30 ng of the indicated plasmid by using lipofectin (Gibco BRL) as described in the manufacturer’s protocol. Aphidicolin (Aph) treatment (1 μg/ml) of 293-EBNA1 cells before and during the transfection is indicated. Cells were harvested 16 h after transfection, and total cell protein was extracted (Promega lysis buffer). One-fifth of the material was used to quantify luciferase activity (Promega luciferase kit; Berthold Microlumat LB96P).

FIG. 2

(A) Representation of luciferase activity in 293-EBNA1 cells after microinjection. Plasmid pTG11056 (oriP+; 10 ng/μl) or pTG11077 (oriP−; 10 ng/μl) was injected into the nucleus (nuc) or cytoplasm (cyt) of 100 293-EBNA1 cells in three independent experiments. Cells were harvested 16 h after injection, and total protein extract was prepared. One-fifth of the material was used to quantify luciferase activity. Only nuclear injection was used in experiment 1. (B) Schematic representation of the ratio (oriP+/oriP−) in luciferase activity after nuclear or cytoplasmic injection of pTG11056 (oriP+) and pTG11077 (oriP−).

FIG. 3

Representation of luciferase activity in 293 cells after cotransfection and nuclear comicroinjection with EBNA1 expression plasmid pTG11149 together with pTG11056 (oriP+) or pTG11077 (oriP−). A total of 3 × 10⁴ 293 cells were seeded per well on 48-well plates. The next day, the cells were transfected with 30 ng of pTG11149 together with 30 ng of pTG11077 or pTG11056. Alternatively, a solution of 10 ng of pTG11149 per μl together with 10 ng of pTG11056 or pTG11077 per μl was injected into the nuclei of 100 293 cells. At 16 h after cotransfection or comicroinjection, the cells were harvested and total cell protein was prepared. One-fifth of the material was analyzed with respect to luciferase activity. The obtained RLU are indicated.