EBV epigenetically suppresses the B cell-to-plasma cell differentiation pathway while establishing long-term latency
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Published version
Author(s)
Type
Journal Article
Abstract
Mature human B cells infected by Epstein-Barr virus (EBV) become activated, grow, and
proliferate. If the cells are infected ex vivo, they are transformed into continuously proliferating
lymphoblastoid cell lines (LCLs) that carry EBV DNA as extra-chromosomal episomes,
express 9 latency-associated EBV proteins, and phenotypically resemble antigen-activated
B-blasts. In vivo similar B-blasts can differentiate to become memory B cells (MBC), in
which EBV persistence is established. Three related latency-associated viral proteins
EBNA3A, EBNA3B, and EBNA3C are transcription factors that regulate a multitude of cellular
genes. EBNA3B is not necessary to establish LCLs, but EBNA3A and EBNA3C are
required to sustain proliferation, in part, by repressing the expression of tumour suppressor
genes. Here we show, using EBV-recombinants in which both EBNA3A and EBNA3C can
be conditionally inactivated or using virus completely lacking the EBNA3 gene locus, that—
after a phase of rapid proliferation—infected primary B cells express elevated levels of factors
associated with plasma cell (PC) differentiation. These include the cyclin-dependent
kinase inhibitor (CDKI) p18INK4c, the master transcriptional regulator of PC differentiation B
lymphocyte-induced maturation protein-1 (BLIMP-1), and the cell surface antigens CD38
and CD138/Syndecan-1. Chromatin immunoprecipitation sequencing (ChIP-seq) and chromatin
immunoprecipitation quantitative PCR (ChIP-qPCR) indicate that in LCLs inhibition of
CDKN2C (p18INK4c) and PRDM1 (BLIMP-1) transcription results from direct binding of
EBNA3A and EBNA3C to regulatory elements at these loci, producing stable reprogramming.
Consistent with the binding of EBNA3A and/or EBNA3C leading to irreversible epigenetic
changes, cells become committed to a B-blast fate <12 days post-infection and are
unable to de-repress p18INK4c or BLIMP-1—in either newly infected cells or conditional
LCLs—by inactivating EBNA3A and EBNA3C. In vitro, about 20 days after infection with
EBV lacking functional EBNA3A and EBNA3C, cells develop a PC-like phenotype.
Together, these data suggest that EBNA3A and EBNA3C have evolved to prevent differentiation
to PCs after infection by EBV, thus favouring long-term latency in MBC and asymptomatic
persistence.
proliferate. If the cells are infected ex vivo, they are transformed into continuously proliferating
lymphoblastoid cell lines (LCLs) that carry EBV DNA as extra-chromosomal episomes,
express 9 latency-associated EBV proteins, and phenotypically resemble antigen-activated
B-blasts. In vivo similar B-blasts can differentiate to become memory B cells (MBC), in
which EBV persistence is established. Three related latency-associated viral proteins
EBNA3A, EBNA3B, and EBNA3C are transcription factors that regulate a multitude of cellular
genes. EBNA3B is not necessary to establish LCLs, but EBNA3A and EBNA3C are
required to sustain proliferation, in part, by repressing the expression of tumour suppressor
genes. Here we show, using EBV-recombinants in which both EBNA3A and EBNA3C can
be conditionally inactivated or using virus completely lacking the EBNA3 gene locus, that—
after a phase of rapid proliferation—infected primary B cells express elevated levels of factors
associated with plasma cell (PC) differentiation. These include the cyclin-dependent
kinase inhibitor (CDKI) p18INK4c, the master transcriptional regulator of PC differentiation B
lymphocyte-induced maturation protein-1 (BLIMP-1), and the cell surface antigens CD38
and CD138/Syndecan-1. Chromatin immunoprecipitation sequencing (ChIP-seq) and chromatin
immunoprecipitation quantitative PCR (ChIP-qPCR) indicate that in LCLs inhibition of
CDKN2C (p18INK4c) and PRDM1 (BLIMP-1) transcription results from direct binding of
EBNA3A and EBNA3C to regulatory elements at these loci, producing stable reprogramming.
Consistent with the binding of EBNA3A and/or EBNA3C leading to irreversible epigenetic
changes, cells become committed to a B-blast fate <12 days post-infection and are
unable to de-repress p18INK4c or BLIMP-1—in either newly infected cells or conditional
LCLs—by inactivating EBNA3A and EBNA3C. In vitro, about 20 days after infection with
EBV lacking functional EBNA3A and EBNA3C, cells develop a PC-like phenotype.
Together, these data suggest that EBNA3A and EBNA3C have evolved to prevent differentiation
to PCs after infection by EBV, thus favouring long-term latency in MBC and asymptomatic
persistence.
Date Issued
2017-08-03
Date Acceptance
2017-07-10
Citation
PLoS Biology, 2017, 15 (8), pp.1-30
ISSN
1544-9173
Publisher
Public Library of Science (PLoS)
Start Page
1
End Page
30
Journal / Book Title
PLoS Biology
Volume
15
Issue
8
Copyright Statement
© 2017 Styles et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
License URL
Sponsor
Medical Research Council (MRC)
Wellcome Trust
Identifier
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2001992
Grant Number
MR/L008432/1
099273/Z/12/Z
Subjects
Science & Technology
Life Sciences & Biomedicine
Biochemistry & Molecular Biology
Biology
Life Sciences & Biomedicine - Other Topics
EPSTEIN-BARR-VIRUS
CDK INHIBITOR P18(INK4C)
NUCLEAR-PROTEIN EBNA3C
TUMOR-SUPPRESSOR
LYTIC REACTIVATION
ESTROGEN-RECEPTOR
GERMINAL CENTER
HUMANIZED MICE
TRANSCRIPTION
GENERATION
Publication Status
Published
Article Number
e2001992
Date Publish Online
2017-08-03