A Gammaherpesvirus MicroRNA Targets EWSR1 (Ewing Sarcoma Breakpoint Region 1) In Vivo To Promote Latent Infection of Germinal Center B Cells

Abstract

Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), directly contribute to the genesis of multiple types of malignancies, including B cell lymphomas. In vivo, these viruses infect B cells and manipulate B cell biology to establish lifelong latent infection. To accomplish this, gammaherpesviruses employ an array of gene products, including microRNAs (miRNAs). Although numerous host mRNA targets of gammaherpesvirus miRNAs have been identified, the in vivo relevance of repression of these targets remains elusive due to species restriction. Murine gammaherpesvirus 68 (MHV68) provides a robust virus-host system to dissect the in vivo function of conserved gammaherpesvirus genetic elements. We identified here MHV68 mghv-miR-M1-7-5p as critical for in vivo infection and then validated host EWSR1 (Ewing sarcoma breakpoint region 1) as the predominant target for this miRNA. Using novel, target-specific shRNA-expressing viruses, we determined that EWSR1 repression in vivo was essential for germinal center B cell infection. These findings provide the first in vivo demonstration of the biological significance of repression of a specific host mRNA by a gammaherpesvirus miRNA.IMPORTANCE Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), directly contribute to the genesis of multiple types of malignancies. In vivo, these viruses infect B cells and manipulate B cell biology to establish lifelong infection. To accomplish this, gammaherpesviruses employ an array of gene products, including miRNAs, short noncoding RNAs that bind to and repress protein synthesis from specific target mRNAs. The in vivo relevance of repression of targets of gammaherpesvirus miRNAs remains highly elusive. Here, we identified a murine gammaherpesvirus miRNA as critical for in vivo infection and validated the host mRNA EWSR1 (Ewing sarcoma breakpoint region 1) as the predominant target for this miRNA. Using a novel technology, we demonstrated that repression of EWSR1 was essential for in vivo infection of the critical B cell reservoir. These findings provide the first in vivo demonstration of the significance of repression of a specific host mRNA by a gammaherpesvirus miRNA.

Keywords: EWSR1; MHV68; gammaherpesvirus; in vivo; miRNA.

FIG 1

MHV68 TMER5 is required for efficient establishment of splenic latency in vivo. (A) Schematic diagram of viruses carrying wild-type TMER5 (MHV68.WT) or mutated TMER5 carrying deletions of both pre-miRNA stem-loops (MHV68.ΔmiR7.12). (B) Virus titers during multistep lytic replication in vitro. NIH 3T12 fibroblasts were infected with MHV68.WT or MHV68.ΔmiR7.12 at an MOI of 0.05 and then overlaid with methylcellulose. At the indicated time points, cells and supernatants were harvested, and viral titers were determined by plaque assay. Values represent the means ± the standard errors of the mean (SEM). Each growth curve was performed in duplicate in each of two independent experiments. Significance was determined using a two-tailed, unpaired t test (*, P < 0.05). (C) Presence of viral genome in latently infected splenocytes harvested from in vivo samples. Three wild-type B6 mice per sample group per experiment were infected i.n. with 10⁴ PFU of MHV68.WT or MHV68.ΔmiR7.12. After 16 days, splenocytes were harvested, pooled, and then subjected to limiting-dilution nested PCR to detect the presence of viral genome. The frequencies of cells harboring viral genome were determined using a Poisson distribution, as indicated by the line at 63.2%. Values represent means ± the SEM of three independent experiments. Significance was determined using a two-tailed, unpaired t test (*, P < 0.05).

FIG 2

TMER5-encoded pre-miR-7 but not pre-miR-12 is required for efficient establishment of splenic latency in vivo. (A) Schematic diagram of viruses carrying wild-type TMER5 (MHV68.WT) or mutated TMER5 carrying deletions of either pre-miR-7 stem-loop (MHV68.ΔmiR7) or pre-miR-12 stem-loop (MHV68.ΔmiR12). (B) Virus titers during multistep lytic replication in vitro. NIH 3T12 fibroblasts were infected with MHV68.WT, MHV68.ΔmiR7, or MHV68.ΔmiR12 at an MOI of 0.05 and then overlaid with methylcellulose. At the indicated time points, cells and supernatants were harvested, and viral titers were determined by plaque assay. Values represent means ± the SEM. Each growth curve was performed in duplicate in each of two independent experiments. Significance was determined using a two-tailed, unpaired t test (*, P < 0.05). (C) Presence of viral genome in latently infected splenocytes harvested from in vivo samples. Three wild-type B6 mice per sample group per experiment were infected i.n. with 10⁴ PFU of MHV68.WT, MHV68.ΔmiR7, or MHV68.ΔmiR12. After 16 days, splenocytes were harvested, pooled, and then subjected to limiting-dilution nested PCR to detect the presence of viral genome. The frequencies of cells harboring viral genome were determined using a Poisson distribution, as indicated by the line at 63.2%. Values represent means ± the SEM of three independent experiments. Significance was determined using a two-tailed, unpaired t test (*, P < 0.05).

FIG 3

MHV68 mghv-miR-M1-7-5p targets and represses EWSR1 in vitro. (A) Level of endogenous mRNA targets in the presence of mghv-miR-M1-7-5p. NIH 3T12 cells were mock transfected or transfected with miR-7-5p mimic. The expression levels of host mRNA transcripts RGS16, EWSR1, ARHGEF18, and BIRC5 were determined by qRT-PCR 24 h posttransfection. Values represent means ± the SEM. Significance was determined by a two-tailed, unpaired t test (***, P < 0.001; **, P < 0.01). (B) Level of endogenous mRNA targets in the presence of mghv-miR-M1-7-3p. The expression levels of host mRNA transcripts TMEM38B, LARS2, RNF138, and ANAPC7 were determined by qRT-PCR, as described for miR-7-5p. *, P < 0.05. (C) Schematic representation of the miR-7-5p binding site within EWSR1 coding region. A vertical line between nucleotides indicates base pairing; a dashed line represents G:U wobble pairing. (D) Relative luciferase activities of transcripts carrying the EWSR1 miRNA target sequence and flanking region in the presence of miR-7-5p mimic. NIH 3T12 cells were transfected with firefly luciferase plasmid carrying the EWSR1-derived miR-7-5p binding site in the absence or presence of miR-7-5p. The firefly luciferase activity was normalized to renilla luciferase. *, P < 0.05. (E) Level of endogenous EWSR1 protein in the presence of miR-7-5p. NIH 3T12 cells were mock transfected or transfected with miR-7-5p mimic. After 48 h, EWSR1 protein expression was determined by Western blotting. β-Actin was included as a loading control. The values represent the densities of protein bands after normalization to β-actin.

FIG 4

EWSR1 is repressed in infected B cells in vivo. (A) Schematic of gating strategy used for flow sorting, including representative flow plot. Wild-type B6 mice were infected i.n. with 10⁴ PFU of MHV68-H2bYFP. At 16 days, splenocytes were harvested and subjected to flow cytometric sorting to isolate noninfected B cells (CD4^– CD8^– CD14^– CD19⁺ YFP^–) and infected B cells (CD4^– CD8^– CD14^– CD19⁺ YFP⁺). (B) Relative expression of endogenous mRNAs in noninfected versus infected B cells sorted from in vivo samples. The expression of miR-7-5p targets EWSR1 and BIRC5 was determined in each sample using qRT-PCR. The values represent the means ± the SEM of three independent experiments. Significance was determined by a two-tailed, unpaired t test (***, P < 0.001).

FIG 5

Generation and validation of recombinant viruses expressing anti-EWSR1 or scrambled shRNAs. (A) Mfold-predicted secondary structure of wild-type TMER5 and of mutated TMER5 sequences in which the pre-miR-7 and pre-miR-12 stem-loops have been replaced with anti-EWSR1 (EW-shR-3 and EW-shR-2) or scrambled sequence (SC-shR-3 and SC-shR-2) shRNAs. (B) Relative luciferase activity of transcripts carrying EWSR1 sequences at nt 1000 to 1539 in the coding region in the presence of TMER5-encoded scrambled (pUC57-SC.shR) or anti-EWSR1 (pUC57-EW.shR) shRNAs. NIH 3T12 cells were cotransfected with firefly luciferase plasmid carrying the EWSR1 sequence and plasmid carrying TMER5-encoded shRNAs. Firefly luciferase activity was normalized to renilla luciferase, and values are expressed relative to the activity in the presence of scrambled shRNAs. ***, P < 0.001. (C) Relative level of endogenous EWSR1 mRNA in the presence of TMER5-encoded scrambled (pUC57-SC.shR) or anti-EWSR1 (pUC57-EW.shR) shRNAs. NIH 3T12 cells were transfected with plasmids carrying TMER5-encoded shRNAs, and 24 h later endogenous EWSR1 mRNA levels were quantified using qRT-PCR. Values indicate the mRNA expression level relative to GAPDH (**, P < 0.01). (D) Level of endogenous EWSR1 protein in the presence of TMER5-encoded scrambled (pUC57-SC.shR) or anti-EWSR1 (pUC57-EW.shR) shRNAs. NIH 3T12 cells were transfected with plasmid carrying TMER5-encoded shRNAs or with scrambled (SC-siR-3) or anti-EWSR1 (EW-siR-3) siRNAs. After 48 h, EWSR1 protein expression was determined by Western blotting. β-Actin was included as a loading control. Values represent the density of EWSR1 protein bands after normalization to β-actin. (E) Schematic diagram of viruses carrying wild-type TMER5 (MHV68.WT) or TMER5 carrying anti-EWSR1 shRNAs (MHV68.EW.shR) or scrambled shRNAs (MHV68.SC.shR) in place of pre-miR-7 and pre-miR-12 stem-loops. (F) Relative levels of endogenous EWSR1 mRNA in cells infected with shRNA-expressing viruses. NIH 3T12 cells were infected with MHV68.EW.shR or MHV68.SC.shR at an MOI of 5. After 24 h, samples were harvested, and the EWSR1 mRNA levels were quantified by qRT-PCR. Values indicate the mRNA expression level relative to GAPDH and represent the means ± the SEM of three independent experiments (*, P < 0.05). (G) Level of endogenous EWSR1 protein in cells infected with shRNA-expressing viruses. NIH 3T12 cells were infected with MHV68.EW.shR or MHV68.SC.shR at an MOI of 5. After 48 h, samples were harvested, and the EWSR1 protein expression was determined by Western blotting. β-Actin was included as a loading control. Values represent the density of EWSR1 protein bands after normalization to β-actin. (H) Schematic of gating strategy used for flow sorting, including a representative flow plot. Wild-type B6 mice were infected i.n. with 10⁴ PFU of wild-type MHV68 (parental MHV68.ORF73βla), MHV68.EW.shR, or MHV68.SC.shR. At 16 days, the splenocytes were harvested, and B cells were then isolated by negative selection and subjected to flow cytometric sorting to isolate infected (CCF4-AM⁺ CD19⁺) B cells. (I) Relative expression of endogenous EWSR1 mRNA levels in infected B cells. The expression of EWSR1 transcript was determined in each sorted cell sample using qRT-PCR. The values represent the means ± the SEM of two independent experiments. Significance was determined by a two-tailed, unpaired t test (**, P < 0.01; *, P < 0.05).

FIG 6

In vivo repression of EWSR1 promotes splenic latency. (A) Splenomegaly in mice infected with shRNA-expressing viruses. Wild-type B6 mice were mock infected or infected i.n. with 10⁴ PFU of viruses carrying wild-type TMER5 (MHV68.WT) or TMER5 carrying anti-EWSR1 shRNAs (MHV68.EW.shR) or scrambled shRNAs (MHV68.SC.shR) in place of pre-miR-7 and pre-miR-12 stem-loops. At 16 days, the spleens were harvested and weighed. Values represent the means ± the SEM of four independent experiments (***, P < 0.001; **, P < 0.01; *, P < 0.05). (B) Presence of viral genome in latently infected splenocytes harvested from in vivo samples. Wild-type B6 mice (three per sample group per experiment) were infected i.n. with 10⁴ PFU of indicated viruses. After 16 days, splenocytes were harvested, pooled, and then subjected to limiting-dilution nested PCR to detect the presence of viral genome. The frequencies of cells harboring viral genome were determined, exactly as described for Fig. 1C. Values represent the means ± the SEM of three independent experiments. Significance was determined using a two-tailed, unpaired t test (**, P < 0.01; *, P < 0.05).

FIG 7

In vivo repression of EWSR1 promotes the infection of germinal center B cells. (A) Schematic of gating strategy used for multiparameter flow cytometric analysis of virus-positive B cell subsets, including representative flow plots. Wild-type B6 mice were infected i.n. with 10⁴ PFU of viruses carrying wild-type TMER5 (MHV68.WT) or TMER5 carrying anti-EWSR1 shRNAs (MHV68.EW.shR) or scrambled shRNAs (MHV68.SC.shR) in place of pre-miR-7 and pre-miR-12 stem-loops. After 16 days, the splenocytes were stained with antibodies directed against CD19, GL7, and IgM to identify naive, germinal center, and memory B cells and with β-lactamase substrate CCF4-AM to identify virus-infected cells. (B) Percent of virus-positive splenocytes. The percentage of total splenocytes staining positive for CCF4-AM cleavage is indicated for each infection group. (C) Percent of virus-positive CD19⁺ B cells displaying surface markers consistent with naive B cells (GL7^– IgM⁺). (D) Percent of virus-positive CD19⁺ B cells displaying surface markers consistent with germinal center B cells (GL7⁺). (E) Percent of virus-positive CD19⁺ B cells displaying surface markers consistent with memory B cells (GL7^– IgM^–). The values represent the means ± the SEM of five independent experiments. Significance was determined using a two-tailed, unpaired t test (**, P < 0.01; *, P < 0.05; ns, not significant).