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. 2013 May;87(10):5437-46.
doi: 10.1128/JVI.00281-13. Epub 2013 Mar 6.

A cluster of virus-encoded microRNAs accelerates acute systemic Epstein-Barr virus infection but does not significantly enhance virus-induced oncogenesis in vivo

Angela Wahl  1 , Sarah D Linnstaedt, Caitlin Esoda, John F Krisko, Francisco Martinez-Torres, Henri-Jacques Delecluse, Bryan R Cullen, J Victor Garcia
Affiliations

A cluster of virus-encoded microRNAs accelerates acute systemic Epstein-Barr virus infection but does not significantly enhance virus-induced oncogenesis in vivo

Angela Wahl et al. J Virol. 2013 May.

Abstract

Over 90% of the adult human population is chronically infected with the Epstein-Barr virus (EBV), an oncogenic herpesvirus. EBV primarily infects naive human B cells and persists latently in memory B cells. Most individuals experience an asymptomatic infection that is effectively controlled by the adaptive immune response. However, EBV-associated lymphomas can develop in immunocompromised individuals. These tumors typically express all nine EBV latent proteins (latency III). Latency III is also associated with the expression of three precursor microRNAs (miRNAs) located within the EBV BHRF1 gene locus. The role of these BHRF1 miRNAs was unclear until recent in vitro studies demonstrated that they cooperate to enhance virus-induced B cell transformation and decrease the antigenic load of virus-infected cells, indicating that the BHRF1 miRNA cluster may serve as a novel therapeutic target for the treatment of latency III EBV-associated malignancies. However, to date, it is not known if BHRF1 miRNAs enhance virus-induced oncogenesis and/or immune evasion of EBV in vivo. To understand the in vivo contribution of the BHRF1 miRNA cluster to EBV infection and EBV-associated tumorigenesis, we monitored EBV infection and assessed tumor formation in humanized mice exposed to wild-type virus and a viral mutant (Δ123) that lacks all three BHRF1 miRNAs. Our results demonstrate that while the BHRF1 miRNAs facilitate the development of acute systemic EBV infection, they do not enhance the overall oncogenic potential of EBV in vivo.

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Figures

Fig 1
Fig 1
Reconstitution of humanized mice with human hematopoietic cells. Reconstitution of humanized mice with human hematopoietic cells was monitored in the peripheral blood by flow cytometry. The mean percentages of human hematopoietic cells (CD45+), B cells (CD19+), and T cells (CD3+) in the peripheral blood of humanized mice prior to exposure to WT, REV or Δ123 EBV are shown. There was no significant difference in the percentages of human cells in the peripheral blood of mice exposed to WT, REV, or Δ123 EBV (two-tailed Mann-Whitney U test, significance defined as P ≤ 0.05).
Fig 2
Fig 2
Detection of viral DNA in the peripheral blood of humanized mice exposed to EBV. Spleens of humanized mice were injected with 3.6 × 105 GRU of WT (n = 7), REV (n = 3), or Δ123 (n = 11) EBV. Following splenic exposure, infection was monitored in peripheral blood with real-time PCR. A Kaplan-Meier plot illustrates the week postexposure when EBV DNA was first detected in the peripheral blood of mice exposed to WT/REV or Δ123 EBV. The rate of viral DNA detection in the peripheral blood of mice exposed to WT/REV or Δ123 EBV was compared with a log rank Mantel-Cox test (P = 0.0083).
Fig 3
Fig 3
Detection of cell-associated and cell-free EBV DNA in the peripheral blood of EBV-exposed humanized mice. The presence of EBV DNA in peripheral blood cells (cell-associated DNA) and plasma (cell-free DNA) of WT/REV (n = 10) and Δ123 (n = 11) virus-exposed mice was determined with real-time PCR. The corresponding cell-associated (A and C) and cell-free (B and D) peripheral blood viral loads for each mouse are indicated with the same symbol. A dashed line indicates the assay limit of detection (cell-associated EBV DNA, 375 DNA copies/ml; cell-free EBV DNA, 560 DNA copies/ml). Cell-associated EBV DNA was detected in the peripheral blood of 9/10 WT/REV EBV-exposed mice and 11/11 Δ123 EBV-exposed mice. Cell-free EBV DNA was detected in the peripheral blood of 9/10 WT/REV EBV-exposed mice and 9/11 Δ123 EBV-exposed mice. There was no significant difference in the peak cell-associated (E) or cell-free (F) EBV viral loads in the peripheral blood of WT/REV and Δ123 EBV-exposed mice with detectable viremia (two-tailed Mann-Whitney U test; cell-associated, P = 0.4964; cell-free, P = 0.6517).
Fig 4
Fig 4
Expansion, activation, and memory phenotype of CD8+ T cells in peripheral blood following EBV exposure. Flow cytometry was utilized to monitor the expansion, activation status, and memory phenotype of CD8+ T cells in the peripheral blood of mice pre- and postexposure to WT/REV or Δ123 EBV. (A) The mean (± standard error of the mean) percentage of CD3+ T cells that express CD8. (B) The mean (± standard error of the mean) percentage of activated (CD38+ HLA-DR+) CD8+ T cells. (C) The mean (± standard error of the mean) percentage of memory (CD45RA−) CD8+ T cells. A two-tailed Mann-Whitney U test was used to compare the percentages of human CD8+ T cells, activated CD8+ T cells, and memory CD8+ T cells in the peripheral blood of WT/REV and Δ123 EBV-exposed mice (*, P ≤ 0.05; **, P < 0.01).
Fig 5
Fig 5
Survival rate of EBV-infected humanized mice. A Kaplan-Meier plot depicts the survival rate of mice exposed to either WT/REV or Δ123 EBV. The rates of survival between WT/REV and Δ123 EBV-exposed mice were compared with a log rank Mantel-Cox test (P = 0.9171).
Fig 6
Fig 6
Tumor incidence in humanized mice exposed to WT/REV or Δ123 EBV. The presence and location of tumors in mice infected with WT/REV (n = 9) or Δ123 (n = 11) EBV were assessed at necropsy. (A) The number of WT/REV and Δ123 EBV-exposed mice with visible tumors at necropsy in the spleen, liver, kidney, and other organs. No significant (ns) difference in tumor incidence or tumor location was observed between WT/REV and Δ123 EBV-exposed mice at harvest (Fisher's exact test, significance defined as P ≤ 0.05). (B) Images of the spleen, liver, and kidney of WT/REV and Δ123 EBV-exposed mice. Tumors are indicated with arrows.
Fig 7
Fig 7
Expression of EBV latent proteins and BHRF1 miRNAs in tumors harvested from EBV-infected mice. We analyzed tumors harvested from four WT/REV and four Δ123 EBV-infected mice to assess latency status (A) and BHRF1 miRNA expression (B). The corresponding mouse number and location (S, spleen; L, liver; K, kidney) of each tumor are indicated. Latency status was determined by the relative expression level of EBV latent proteins LMP1 and EBNA2 and was defined as follows: latency I, LMP1− EBNA2−; latency II, LMP1+ EBNA2−; and latency III, LMP1+ EBNA2+. The relative expression level of each BHRF1 miRNA is also indicated (the typically less abundant of the two possible BHRF1-2 miRNAs is noted with an asterisk). LCLs were used as a positive control for latency III and BHRF1 miRNA expression.
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