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Journal of Virology, April 1999, p. 2974-2982, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Genetic Evidence that EBNA-1 Is Needed for
Efficient, Stable Latent Infection by Epstein-Barr Virus
May-Ann
Lee,
Margaret E.
Diamond, and
John L.
Yates*
Department of Genetics, Roswell Park Cancer
Institute, Buffalo, New York 14263
Received 5 October 1998/Accepted 7 January 1999
Replication and maintenance of the 170-kb circular chromosome of
Epstein-Barr virus (EBV) during latent infection are generally believed
to depend upon a single viral gene product, the nuclear protein EBNA-1.
EBNA-1 binds to two clusters of sites at oriP, an
1,800-bp sequence on the EBV genome which can support replication and
maintenance of artificial plasmids introduced into cell lines that
contain EBNA-1. To investigate the importance of EBNA-1 to latent
infection by EBV, we introduced a frameshift mutation into the EBNA-1
gene of EBV by recombination along with a flanking selectable marker.
EBV genomes carrying the frameshift mutation could be isolated readily
after superinfecting EBV-positive cell lines, but not if recombinant
virus was used to infect EBV-negative B-cell lines or to immortalize
peripheral blood B cells. EBV mutants lacking almost all of internal
repeat 3, which encode a repetitive glycine and alanine domain of
EBNA-1, were generated in the same way and found to immortalize B cells
normally. An EBNA-1-deficient mutant of EBV was isolated and found to
be incapable of establishing a latent infection of the cell line BL30
at a detectable frequency, indicating that the mutant was less than 1%
as efficient as an isogenic, EBNA-1-positive strain in this assay. The
data indicate that EBNA-1 is required for efficient and stable latent
infection by EBV under the conditions tested. Evidence from other
studies now indicates that autonomous maintenance of the EBV chromosome during latent infection does not depend on the replication initiation function of oriP. It is therefore likely that the viral
chromosome maintenance (segregation) function of oriP and
EBNA-1 is what is required.
*
Corresponding author. Mailing address: Department of
Genetics, Roswell Park Cancer Institute, Elm and Carlton St.,
Buffalo, NY 14263. Phone: (716) 845-8964. Fax: (716) 845-8449. E-mail: yates{at}sc3101.med.buffalo.edu.
Journal of Virology, April 1999, p. 2974-2982, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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