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An imperfect correlation between DNA replication activity of Epstein-Barr virus nuclear antigen 1 (EBNA1) and binding to the nuclear import receptor, Rch1/importin alpha.
Virology. 1997 Dec 22; 239(2):340-51.V

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.

Authors+Show Affiliations

Roche Institute of Molecular Biology, Nutley, New Jersey, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

9434725

Citation

Kim, A L., et al. "An Imperfect Correlation Between DNA Replication Activity of Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) and Binding to the Nuclear Import Receptor, Rch1/importin Alpha." Virology, vol. 239, no. 2, 1997, pp. 340-51.
Kim AL, Maher M, Hayman JB, et al. An imperfect correlation between DNA replication activity of Epstein-Barr virus nuclear antigen 1 (EBNA1) and binding to the nuclear import receptor, Rch1/importin alpha. Virology. 1997;239(2):340-51.
Kim, A. L., Maher, M., Hayman, J. B., Ozer, J., Zerby, D., Yates, J. L., & Lieberman, P. M. (1997). An imperfect correlation between DNA replication activity of Epstein-Barr virus nuclear antigen 1 (EBNA1) and binding to the nuclear import receptor, Rch1/importin alpha. Virology, 239(2), 340-51.
Kim AL, et al. An Imperfect Correlation Between DNA Replication Activity of Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) and Binding to the Nuclear Import Receptor, Rch1/importin Alpha. Virology. 1997 Dec 22;239(2):340-51. PubMed PMID: 9434725.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - An imperfect correlation between DNA replication activity of Epstein-Barr virus nuclear antigen 1 (EBNA1) and binding to the nuclear import receptor, Rch1/importin alpha. AU - Kim,A L, AU - Maher,M, AU - Hayman,J B, AU - Ozer,J, AU - Zerby,D, AU - Yates,J L, AU - Lieberman,P M, PY - 1998/1/22/pubmed PY - 1998/1/22/medline PY - 1998/1/22/entrez SP - 340 EP - 51 JF - Virology JO - Virology VL - 239 IS - 2 N2 - 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. SN - 0042-6822 UR - https://www.unboundmedicine.com/medline/citation/9434725/An_imperfect_correlation_between_DNA_replication_activity_of_Epstein_Barr_virus_nuclear_antigen_1__EBNA1__and_binding_to_the_nuclear_import_receptor_Rch1/importin_alpha_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0042-6822(97)98874-7 DB - PRIME DP - Unbound Medicine ER -