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. 2019 Jan;4(1):78-88.
doi: 10.1038/s41564-018-0284-6. Epub 2018 Nov 12.

Epstein-Barr virus BORF2 inhibits cellular APOBEC3B to preserve viral genome integrity

Adam Z Cheng  1   2   3   4 , Jaime Yockteng-Melgar  5 , Matthew C Jarvis  1   2   3   4 , Natasha Malik-Soni  5 , Ivan Borozan  6 , Michael A Carpenter  1   2   3   4   7 , Jennifer L McCann  1   2   3   4 , Diako Ebrahimi  1   2   3   4 , Nadine M Shaban  1   2   3   4 , Edyta Marcon  8 , Jack Greenblatt  5   8 , William L Brown  1   2   3   4 , Lori Frappier  9 , Reuben S Harris  10   11   12   13   14
Affiliations

Epstein-Barr virus BORF2 inhibits cellular APOBEC3B to preserve viral genome integrity

Adam Z Cheng et al. Nat Microbiol. 2019 Jan.

Abstract

The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like (APOBEC) family of single-stranded DNA (ssDNA) cytosine deaminases provides innate immunity against virus and transposon replication1-4. A well-studied mechanism is APOBEC3G restriction of human immunodeficiency virus type 1, which is counteracted by a virus-encoded degradation mechanism1-4. Accordingly, most work has focused on retroviruses with obligate ssDNA replication intermediates and it is unclear whether large double-stranded DNA (dsDNA) viruses may be similarly susceptible to restriction. Here, we show that the large dsDNA herpesvirus Epstein-Barr virus (EBV), which is the causative agent of infectious mononucleosis and multiple cancers5, utilizes a two-pronged approach to counteract restriction by APOBEC3B. Proteomics studies and immunoprecipitation experiments showed that the ribonucleotide reductase large subunit of EBV, BORF26,7, binds APOBEC3B. Mutagenesis mapped the interaction to the APOBEC3B catalytic domain, and biochemical studies demonstrated that BORF2 stoichiometrically inhibits APOBEC3B DNA cytosine deaminase activity. BORF2 also caused a dramatic relocalization of nuclear APOBEC3B to perinuclear bodies. On lytic reactivation, BORF2-null viruses were susceptible to APOBEC3B-mediated deamination as evidenced by lower viral titres, lower infectivity and hypermutation. The Kaposi's sarcoma-associated herpesvirus homologue, ORF61, also bound APOBEC3B and mediated relocalization. These data support a model where the genomic integrity of human γ-herpesviruses is maintained by active neutralization of the antiviral enzyme APOBEC3B.

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Conflict of interest statement

Competing Interests RSH is a co-founder, shareholder, and consultant of ApoGen Biotechnologies Inc. The other authors declare no competing interests.

Figures

Figure 1 |
Figure 1 |. EBV BORF2 interacts with cellular A3B.
a, Total spectral counts from three independent affinity purification-mass spectrometry experiments using transfected BORF2-Flag as bait and empty Flag vector as negative control in 293T cells. b, Co-immunoprecipitation of endogenous A3B in 293T and AGS cells with BORF2-Flag, RRM1-Flag, or an empty vector control. c-f, Co-immunoprecipitation of indicated HA-tagged A3 constructs in 293T cells with BORF2-Flag. These data (a-f) are each representative of at least n = 3 biologically independent experiments.
Figure 2 |
Figure 2 |. EBV BORF2 inhibits A3B catalytic activity specifically.
a, Schematic of deaminase activity assay in which A3-mediated deamination of C-to-U in single-stranded DNA substrate, uracil excision by uracil DNA glycosylase (UDG), and abasic site cleavage by NaOH treatment yields a shorter product (6-FAM labeled for quantification by fluorescence scanning). b, Representative TBE-urea PAGE analysis of A3Bctd and A3H deaminase activity in the presence of increasing concentrations of EBV BORF2 (product percentage indicated below each lane). c, Quantification of the DNA deaminase activity data in panel (b) and two additional biologically independent experiments (normalized mean +/− SD with some error bars smaller than the symbols). These data (b-c) are representative of n = 3 biologically independent experiments.
Figure 3 |
Figure 3 |. BORF2 relocalizes A3B from the nuclear compartment to the endoplasmic reticulum.
a, Immunoblots of lysates from 293T cells transfected with equal amounts of BORF2-Flag and increasing amounts of A3B-HA (left) or the reciprocal set-up (right). b, Representative immunofluorescence microscopy images of latent (top panel) or lytic (bottom 3 panels) AGS-EBV stained with DAPI (blue) and antibodies against endogenous A3B (green) or BORF2 (red). A 10 μm scale is shown in the merged panel images. c, Quantification of A3B localization in AGS-EBV cells grown under latent (0 hrs) or lytic (8, 16, 24 hrs) conditions (n = 50 cells per condition). Parallel quantification of A3B localization in lytic BORF2-null AGS-EBV cells (24 hrs; n = 50 cells). d, Representative immunofluorescence microscopy images of U2OS expressing A3B-mCherry and BORF2-eGFP, and stained with an antibody against the endoplasmic reticulum protein, BiP/GRP-78 (purple; also see Supplementary Video 1). e, Representative immunofluorescence microscopy images of HeLa transiently expressing A3B-eGFP alone, BORF2-Flag alone, or both proteins together. f, Representative immunofluorescence microscopy images of endogenous A3B (green) and BORF2 (red) in a M81-transformed B cell that has spontaneously entered the lytic cycle. g, Representative immunofluorescence microscopy images of endogenous A3B (green) in AGS-EBV and ΔBORF2 derivative pools 24 hrs after lytic reactivation. Anti-BMRF1 (purple) marks sites of viral DNA replication in lytic cells and EdU (red) shows newly synthesized DNA. These data are representative of n = 2 (a,d,f,g) or n = 3 (b,c,e,f) biologically independent experiments.
Figure 4 |
Figure 4 |. BORF2 functions to preserve EBV genome integrity from A3B.
a, 3D-PCR differentiates between non-mutated and mutated DNA substrates by virtue of product accumulation at higher vs lower denaturation temperature (Tm) thresholds (e.g., A3B, orange enzyme, causes C/G-to-T/A mutations through uracil intermediates). b, Immunoblots of an AGS-EBV(Bx1g)ΔBORF2 clone engineered to express BORF2-Flag or vector control, shA3B or shControl, and UGI or vector control (see text for details). c, Representative agarose gel images showing the results of 3D-PCR experiments involving a 254 bp BRRF2 gene segment of AGS-EBV(Bx1g)ΔBORF2. The dashed red line shows the point at which non-mutated BRRF2 DNA fails to amplify under high denaturation conditions; visible PCR products below this Tm threshold represent lower temperature amplicons (i.e., mutated sequences). The right panels show the effect of complementing each condition with BORF2 expression. d, Pie charts showing the types of mutational events observed in Sanger sequences of cloned lower temperature amplicons from the 8 induced conditions shown in the bottom half of panel (c). Wild-type non-mutated sequences are depicted in green; base substitutions with number of mutations per sequence depicted in light green, yellow, orange, and red; deletions are depicted in black; other types of mutations (e.g., combination of base substitution and deletion) are depicted in navy. e, A summary of cytosine mutations detected in multiple EBV DNA regions under the indicated conditions following recovery by high-fidelity PCR (high temperature) and deep-sequencing. f, Titers of wild-type and ΔBORF2 viruses after lytic induction of AGS-EBV(Bx1g). Each symbol represents data from 4 independent cultures and the horizontal line shows the mean. g, Infectivity of wild-type and ΔBORF2 viruses produced by lytic replication in AGS-EBV(Bx1g). Each symbol represents the percent of GFP-positive Ramos reporter cells from n = 4 independent infections, and the horizontal line shows the mean. h, Infectivity of ΔBORF2 EBV produced by lytic replication in AGS-EBV(Bx1g) with endogenous A3B intact (shCtrl) or depleted (shA3B). Each symbol represents the percent of GFP-positive Ramos reporter cells from n = 3 independent infections, and the horizontal line shows the mean. These data are representative of n = 2 (f,g) or n = 3 (b,c,h) biologically independent experiments.
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Comment in

  • APOBEC restriction goes nuclear.
    Malim MH, Pollpeter D. Malim MH, et al. Nat Microbiol. 2019 Jan;4(1):6-7. doi: 10.1038/s41564-018-0323-3. Nat Microbiol. 2019. PMID: 30546096 No abstract available.

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