Analysis of Host Gene Expression Changes Reveals Distinct Roles for the Cytoplasmic Domain of the Epstein-Barr Virus Receptor/CD21 in B-Cell Maturation, Activation, and Initiation of Virus Infection

  1. Joyce D. Fingerothc,d*
  1. aDepartment of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA
  2. bDepartment of Medicine, Division of Interdisciplinary Medicine & Biotechnology-Genomics Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA
  3. cDivision of Infectious Diseases, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA
  4. dCommittee on Virology, Harvard Medical School, Boston, Boston, Massachusetts, USA
  1. R. M. Sandri-Goldin, Editor
  1. FIG 1
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    FIG 1

    Pre-B-cell lines Nalm6 and Laz221, which lack endogenous CR and variably express CR complex members, can stably express recombinant CR and CT and bind EBV. (A) Flow cytometric analysis (FC) of CD21, CD35, CD19/CD81/Leu13(CD225), and HLA-II (DR, DP, and DQ) expression on Nalm6 and Laz221 cells. x axis, log fluorescence intensity; y axis, cell number. An LCL is displayed as a staining control. (B) Expression of CR on two Nalm6 clones (1 and 2) compared with a lymphoma line (BL41) and a LCL that express endogenous CR at different levels. (C) Verification of CR and CT expression by immunoblot (left) and by PCR (right). (D) Demonstration that expression of associated proteins is unmodified in Nalm6CR although coreceptor, HLA-II expression is increased ∼3-fold (left shift of log mean fluorescence intensity). (E) Top four histograms, demonstration that EBV attaches to cells expressing CR and also CT by FC. CR and CT were directly detected with MAb HB-5 anti-CD21 (black line) compared with an isotype control (block). MAb 2L10 anti-EBVgp350/220 was used for detection of bound EBV (black line). No virus served as the negative control (block). An LCL is displayed as a positive control. Bottom histogram, EBV blockade, confirms that rabbit anti-CR blocks EBV attachment to Laz221CR and CT (dark gray block), whereas normal rabbit serum does not (light gray block). See Materials and Methods for details. (F) Demonstration that CT is not cleaved by phosphatidylinositol-specific phospholipase C (PIPLC) (not PI linked) by FC. Top, the GPI-linked proteins CD48 and CD59 are cleaved by PIPLC in HPB-ALL, a control T-cell line, resulting in decreased surface expression. Neither HLA-I nor CR (endogenous), which lack PI linkages, is altered. Bottom, concurrent analysis of Nalm6CT showed no evidence of CT cleavage. CD48 and CD59 are absent and HLA-I uncleaved on Nalm6CT.

  2. FIG 2
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    FIG 2

    EBV infection of CR-bearing pre-B-cell lines analyzed by RNA blot hybridization. (A) Top, expression of LMP1 and EBER in Nalm6CR and Laz221CR cells was detected 24 to 72 h after EBV incubation. Nalm6NEO cells served as a negative control. Expression of BHRF1, a highly expressed lytic RNA, was absent. Ak (Akata) cells served as the positive control. GAPDH or 28S RNA (arrow) provided loading controls. Bottom, preincubation of cells with OKB7, a MAb that blocks binding of EBV gp350/220 to CR, prevented infection of Nalm6CR, whereas UPC10, an isotype-matched control, did not. (B) EBNA-1 (top), EBNA-2 (middle), and LMP-2A (bottom) transcripts were also detected, as shown in Nalm6CR cells. (C) Cellular RNAs encoding RAG-1, TdT, and CD10 declined upon EBV infection, as previously observed (22). (D) Infection of CT-bearing cells induced LMP1 (Nalm6 and Laz221) and EBNA2 (Nalm6) expression with a time course similar to that of CR.

  3. FIG 3
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    FIG 3

    Unsupervised clustering and principal-component analysis of the gene expression data. (A) An unsupervised Euclidean distance-based cluster of arrays after normalizing the data demonstrates that B-cell lines that express CT, CR, or NEO form separate clusters that correlate better with one another than with cells expressing an alternate receptor form. (B) PCA analysis demonstrates that CT, CR, and NEO cells have distinct gene expression patterns. (C) Heat map of the differentially expressed genes by comparing NEO, CT, and CR at 1 min and 60 min demonstrates that NEO, CR, and CT cells have a set of unique differentially expressed genes. Rows represent genes, and columns represent samples from CT, CR, and NEO groups. Genes are clustered using row-normalized signals and mapped to the [−1,1] interval (shown in scales beneath each heat map). Red and green represent high and low expression values, respectively.

  4. FIG 4
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    FIG 4

    Differentially expressed genes in NEO, CR, and CT cells at 1 min (constitutive changes). (A) Venn diagrams of differentially expressed (downregulated and upregulated) genes in NEO, CT, and CR cells. (B) Heat map of selected top genes constitutively altered (1 min) after recombinant receptor expression. Left, NEO versus CT; middle, NEO versus CR; right, CT versus CR. Genes are clustered using row-normalized signals and mapped to the [−1,1] interval (shown in scales beneath each heat map). Red and blue represent high and low expression values, respectively.

  5. FIG 5
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    FIG 5

    Comparison of genes differentially altered in NEO, CT, and CR at 1 versus 60 min after incubation with EBV. (A) Venn diagrams of differentially expressed (downregulated and upregulated) genes in NEO, CT, and CR cells. (B) Heat map of selected top genes changing from 1 to 60 min within the cell lines NEO, CT, and CR. (C) Heat map depicting selected top genes changing at 60 min in NEO versus CT, NEO versus CR, and CT versus CR. Genes are clustered using row-normalized signals and mapped to the [−1,1] interval (shown in scales beneath each heat map). Red and blue represent high and low expression values, respectively.

  6. FIG 6
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    FIG 6

    Comparison of top signaling pathways affected in different receptor-bearing cell types. (A) CT; (B) CR. Sets of differentially expressed genes (LCB of >1.2) between cell lines were uploaded onto Ingenuity Pathway Analysis and corresponding signaling pathways predicted. Statistical significance was set at −log P = 2.

  7. FIG 7
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    FIG 7

    Comparison of predicted upstream regulators of NEO, CT, and CR transcripts at 60 min after EBV infection. Differentially expressed genes were processed through Ingenuity Pathway Analysis to predict the downstream regulators whose activation status was affected following EBV interaction. No regulators reached the significant activation z score of −2 or +2 in NEO (A), whereas several regulators were predicted to be activated (z-score of >2) in CT (B) and a significantly larger number of regulators was either activated or inhibited (z score of <−2) in CR (C).

  8. FIG 8
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    FIG 8

    RT-PCR validation of selected differentially expressed genes, including MAGE family, immediate early proto-oncogenes, histones, and novel candidates.

  9. FIG 9
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    FIG 9

    Schematic of CD21 complex formation on normal resting (primary) B cells compared with transfectant cell line models (CR and CT) used in this work.

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  1. Accepted manuscript posted online 5 March 2014, doi: 10.1128/JVI.03099-13 J. Virol. vol. 88 no. 10 5559-5577
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