Dominant-negative inhibitors of EBNA-1 of Epstein-Barr virus

Abstract

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is required in trans to support replication of the EBV genome once per cell cycle via the latent origin of replication, oriP. EBNA-1 can also activate transcription on binding to the family of repeats of oriP to enhance some heterologous as well as native EBV promoters. We have made and screened derivatives of EBNA-1 for the ability to act as inhibitors of wild-type EBNA-1. These derivatives lack the linking or the retention functions of EBNA-1 and were analyzed for the residual ability to activate transcription and replication. We have identified derivatives of EBNA-1 that can inhibit up to 98% of wild-type EBNA-1's activities. We have also identified one derivative of EBNA-1 with only two of EBNA-1's three linking domains which can support transcription and replication inefficiently.

The plasmid replicon of EBV consists of multiplecis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element

“…We have now demonstrated, using two independent techniques, that oriP is recognized directly by the human DNA synthetic machinery, indicating why oriP, but not most other viral origins, is replicated once per cell cycle, and in synchrony with cellular chromosomes in proliferating cells. We note that the level of oriP DNA synthesized in the absence of EBNA-1 at 48 h post-transfection is within 2-fold of the levels of oriP DNA synthesized in cells that stably express EBNA-1 at 96 h post-transfection (Kirchmaier and Sugden, 1997), or in stably transfected cells , indicating that levels of DNA synthesis from oriP plasmids are independent of EBNA-1. Akin to chromosomal origins, oriP also contains multiple regions within it that independently facilitate DNA synthesis in cis.…”

“…A form of PCR shown to be quantitative (Kirchmaier and Sugden, 1997) was used to measure the number of newly synthesized, DpnI-resistant, oriP plasmids present 48 h after their transfection into C33A cells. The oriP plasmids were introduced along with an EBNA-1 expression plasmid, or a control expression plasmid.…”

“…Plasmid 1878, which does not contain oriP and which can be distinguished from 1920 by a length polymorphism in the neo R gene, was used as the backbone control plasmid. Plasmid 1878 was constructed by cloning the TK-neo cassette from 1381 (Kirchmaier and Sugden, 1997) into the same backbone as 1920. Plasmids 1920Plasmids , 1938 and 1878 were propagated in E.coli MC1061/P3 using conditions described previously (Aiyar et al, 1996).…”

“…Plasmids 1920Plasmids , 1938 and 1878 were propagated in E.coli MC1061/P3 using conditions described previously (Aiyar et al, 1996). Plasmid 1380, that is distinguished from 1878 and 1920 by a second length polymorphism in its neo R gene, was used as competitor during quantitative PCRs (Kirchmaier and Sugden, 1997). Plasmids RSVL (RSV LTR-luciferase) (de Wet et al, 1987) and 1033 (oriP-BamHI-C-luciferase) (Kirchmaier and Sugden, 1997) were used as transcription reporters.…”

Enhancement of hepatitis C virus replication by Epstein-Barr virus-encoded nuclear antigen 1

“…After transfection, cells (10 5 ) were suspended in RPMI-1640 medium containing 10% fetal calf serum, harvested after 72 h incubation and lysed. The assay was performed as described previously (Kirchmaier and Sugden, 1997). The luciferase light unit from each clone expressing EBNA1 or one of the EBNA1 derivatives was compared with that of mock-transfected cells and analyzed by Student's t-test with Bonferroni's correction.…”