An Imperfect Correlation between DNA Replication Activity of Epstein–Barr Virus Nuclear Antigen 1 (EBNA1) and Binding to the Nuclear Import Receptor, Rch1/importin α

Abstract

Epstein-Barr virus (EBV) replicates as a stable multicopy episome in latently infected mammalian cells. Latent cycle DNA replication requires only two viral elements, the cis-acting origin of plasmid replication (oriP) and the trans-acting origin binding protein (EBNA1). EBNA1 binds multiple recognition sites in oriP, but has not other enzymatic activities associated with replication functions. To identify human cellular proteins that mediate EBNA1 function, we designed a one-hybrid assay in yeast to select for proteins that bind to EBNA1 when bound to criP in vivo. A human cDNA encoding the Rch1/hSRP1 alpha/ importin alpha protein was isolated and shown to bind to full-length EBNA1, but not to an amino terminal deletion mutant of EBNA1 when bound to oriP in yeast. The interaction of EBNA1 with Rch1 was confirmed biochemically by coimmunoprecipitation from nuclear extracts and by direct binding of recombinant proteins in vitro. Internal deletion mutations in EBNA1 which compromised DNA replication activity were similarly reduced for binding to Rch1. Mutations with no effect on DNA replication activity were similarly unaffected for Rch1 binding. Rch1/importin alpha has been shown to bind to the nuclear localization sequence (NLS) of several proteins and stimulate nuclear import. A substitution mutation in the EBNA1 nuclear localization sequence reduced Rch1 binding, but had no effect on DNA replication function, indicating that Rch1 binding affinity does not correspond precisely with replication activity. Nevertheless, the identification of a stable interaction between Rch1 and EBNA1 at the origin of viral DNA replication raises the intriguing possibility that Rch1 contributes to the nuclear functions of EBNA1.

BSAP (Pax5)-Importin α1 (Rch1) Interaction Identifies a Nuclear Localization Sequence

“…Nonetheless, we detected interaction of BSAP in this assay only with importin ␣1. Other two-hybrid screens also revealed dominant, if not exclusive, importin ␣1 interactions with RAG1 (40), coilin (41), LEF (42), BRCA1 (43), and EBNA1 (44). (Both RAG1 and LEF were also found to bind to a second importin ␣, ␣5 (SRP1).)…”

Maintenance of Epstein-Barr virus (EBV) oriP-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1

“…In one study, deletion of EBNA-1 amino acids 325-376 had almost no effect on initial DNA replication but nearly eradicated episome persistence (31). The basic domains are also implicated in the looping between EBNA-1 bound to FR and DS, in linking among EBNA-1 molecules and in interactions with cellular proteins, one of which, EBP2, is associated with mitotic chromosomes (27)(28)(29)(30)(31)40).…”

“…Notably, the entire EBNA-1 C terminus, which includes the DNA-binding and dimerization domains, and NLS, cannot bind chromosomes. Although further dissection of the EBNA-1 basic domains could more precisely define the components critical for chromosome association and plasmid maintenance, these domains are also implicated in transcriptional activation, DNA looping and linking, and homo-and heterotypic protein-protein interactions (11,(26)(27)(28)(29)(30)(31). Because specific mutations would therefore likely have pleiotropic effects, we chose an alternative approach; to replace the EBNA-1 chromosome associating domains with cellular proteins that were similar to EBNA-1 in the pattern and salt sensitivity of their association with chromosomes.…”

The plasmid replicon of Epstein–Barr virus: Mechanistic insights into efficient, licensed, extrachromosomal replication in human cells

“…A nuclear localization signal (NLS) is encoded by AA379-386, which also associates with the cellular nuclear importation machinery (Kim, Maher et al 1997), (Fischer, Kremmer et al 1997). Sequences within the Arg-Gly rich regions of LR1 and LR2 may also function as NLSs due to their highly basic content.…”

“…These repeats have also been found to inhibit translation of EBNA1 in vitro and in vivo (Yin, Manoury et al 2003). However, the deletion of much of this domain has no apparent effect on functions of EBNA1 in cell culture, making the role that this domain plays difficult to elucidate.A nuclear localization signal (NLS) is encoded by AA379-386, which also associates with the cellular nuclear importation machinery (Kim, Maher et al 1997), (Fischer, Kremmer et al 1997). Sequences within the Arg-Gly rich regions of LR1 and LR2 may also function as NLSs due to their highly basic content.…”