Comprehensive profiling of functional Epstein-Barr virus miRNA expression in human cell lines

BACKGROUND: Epstein-Barr virus (EBV) establishes lifelong infections in its human host. The virus is associated with a broad range of malignancies of lymphoid and epithelial origin, including Burkitt's lymphoma, post-transplant lymphoproliferative disease, nasopharyngeal carcinoma and gastric carcinoma. During the latent phase of its life cycle, EBV expresses more than 40 mature miRNAs that are highly abundant in tumor cells and may contribute to oncogenesis. Although multiple studies have assessed the relative expression profiles of EBV miRNAs in tumor cells, data linking these expression levels to functional target knockdown are mostly lacking. Therefore we set out to systematically assess the EBV miRNA expression levels in EBV(+) tumor cell lines, and correlate this to their functional silencing capacity in these cells. RESULTS: We provide comprehensive EBV miRNA expression profiles of the EBV(+) cell lines C666-1 (nasopharyngeal carcinoma), SNU-719 (gastric carcinoma), Jijoye (Burkitt's lymphoma), and AKBM (Burkitt's lymphoma) and of EBV(-) cells ectopically expressing the BART miRNA cluster. By deep sequencing the small RNA population and conducting miRNA-reporter experiments to assay miRNA potency, we were able to compare the expression profiles of the EBV miRNAs with their functional silencing efficacy. We observe a strong correlation between miRNA expression levels and functional miRNA activity. There is large variation in expression levels between EBV miRNAs in a given cell line, whereas the relative expression profiles are well maintained between cell lines. Furthermore, we show that miRNA arm selection bias is less pronounced for gamma-herpesvirus miRNAs than for human miRNAs. CONCLUSION: We provide an in depth assessment of the expression levels and silencing activity of all EBV miRNAs in B- and epithelial cell lines of different latency stages. Our data show a good correlation between relative EBV miRNA expression levels and silencing capacity, and suggest preferential processing of particular EBV miRNAs irrespective of cell-type. In addition to encoding the largest number of precursor miRNAs of all human herpesviruses, EBV expresses many miRNAs precursors that yield two functional miRNA strands, rather than one guide strand and a non-functional passenger strand. This reduced strand bias may increase the size of the EBV miRNA targetome

Immune Evasion by Epstein-Barr Virus

Epstein-Bar virus (EBV) is widespread within the human population with over 90% of adults being infected. In response to primary EBV infection, the host mounts an antiviral immune response comprising both innate and adaptive effector functions. Although the immune system can control EBV infection to a large extent, the virus is not cleared. Instead, EBV establishes a latent infection in B lymphocytes characterized by limited viral gene expression. For the production of new viral progeny, EBV reactivates from these latently infected cells. During the productive phase of infection, a repertoire of over 80 EBV gene products is expressed, presenting a vast number of viral antigens to the primed immune system. In particular the EBV-specific CD4+ and CD8+ memory T lymphocytes can respond within hours, potentially destroying the virus-producing cells before viral replication is completed and viral particles have been released. Preceding the adaptive immune response, potent innate immune mechanisms provide a first line of defense during primary and recurrent infections. In spite of this broad range of antiviral immune effector mechanisms, EBV persists for life and continues to replicate. Studies performed over the past decades have revealed a wide array of viral gene products interfering with both innate and adaptive immunity. These include EBV-encoded proteins as well as small noncoding RNAs with immune-evasive properties. The current review presents an overview of the evasion strategies that are employed by EBV to facilitate immune escape during latency and productive infection. These evasion mechanisms may also compromise the elimination of EBV-transformed cells, and thus contribute to malignancies associated with EBV infection

CRISPR/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections

Herpesviruses infect the majority of the human population and can cause significant morbidity and mortality. Herpes simplex virus (HSV) type 1 causes cold sores and herpes simplex keratitis, whereas HSV-2 is responsible for genital herpes. Human cytomegalovirus (HCMV) is the most common viral cause of congenital defects and is responsible for serious disease in immuno-compromised individuals. Epstein-Barr virus (EBV) is associated with infectious mononucleosis and a broad range of malignancies, including Burkitt’s lymphoma, nasopharyngeal carcinoma, Hodgkin’s disease, and post-transplant lymphomas. Herpesviruses persist in their host for life by establishing a latent infection that is interrupted by periodic reactivation events during which replication occurs. Current antiviral drug treatments target the clinical manifestations of this productive stage, but they are ineffective at eliminating these viruses from the infected host. Here, we set out to combat both productive and latent herpesvirus infections by exploiting the CRISPR/Cas9 system to target viral genetic elements important for virus fitness. We show effective abrogation of HCMV and HSV-1 replication by targeting gRNAs to essential viral genes. Simultaneous targeting of HSV-1 with multiple gRNAs completely abolished the production of infectious particles from human cells. Using the same approach, EBV can be almost completely cleared from latently infected EBV-transformed human tumor cells. Our studies indicate that the CRISPR/Cas9 system can be effectively targeted to herpesvirus genomes as a potent prophylactic and therapeutic anti-viral strategy that may be used to impair viral replication and clear latent virus infection

Anti-HSV-1 CRISPR gRNAs are ineffective in targeting quiescent HSV-1 but do abrogate replication of reactivated HSV-1.

<p><b>a)</b> MRC5 cells exogenously expressing Cas9 were infected with quiescent HSV-1-eGFP. Anti-HSV-1 gRNAs were introduced into the MRC5-Cas9 cells via lentiviral transduction and cells were subsequently superinfected with HCMV to reactivate latent HSV-1. The percentage of cells with replicating HSV-1 were assessed 3 days post HCMV superinfection by flow cytometry. Two quiescent control (‘cells alone’) samples are presented: one not superinfected with HCMV to assess spontaneous HSV-1 reactivation levels, and one superinfected with HCMV to assess the reactivation potential and subsequent HSV-1 replication in these cells. Control vector corresponds to empty gRNA-vector. <b>b)</b> Relative amount of HSV-1 genomes in quiescent HSV-1-eGFP cells transduced with the indicated gRNAs as assessed by TaqMan qPCR. DNA input was normalized to RNAseP levels. The relative HSV-1 content was compared to quiescent MRC5 cells transduced with empty vector control lentivirus. <b>c)</b> Analysis of anti-HSV-1 genome editing by next generation sequencing. The percentage of WT sequences at the indicated target sites is presented upon introduction of the corresponding gRNA. For UL8 and UL52, two samples were analyzed. In control vector treated quiescent HSV-1 cells, no CRISPR/Cas9 editing was observed at these target sites (gRNA specific indels <0.1%). CRISPR/Cas9 genome editing was only observed for UL52 #2 and UL8 #2 where CRISPR/Cas9 editing was apparent in ±6 and ±1% of sequences. The nature of these mutations is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005701#ppat.1005701.s004" target="_blank">S4 Fig</a>.</p

Editing of the EBV genome in latently infected tumor cells using CRISPR/Cas9.

<p><b>a)</b> Latently infected gastric carcinoma SNU719 cells were transduced with lentiviral CRISPR/Cas vectors targeting the indicated EBV miRNA genes. The lines were subsequently selected with puromycin for 2 days and allowed to recover for 12 days. The activity of the targeted miRNAs was subsequently monitored by introduction of the indicated miRNA sensor vectors and assessment of the mCherry reporter expression after 4 days. Increased sensor expression indicates a loss of EBV miRNAs. The activities for the EBV miRNAs in the absence of gRNAs is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005701#ppat.1005701.s001" target="_blank">S1 Fig</a>. <b>b)</b> Sequencing of CRISPR-targeted EBV genomes indicates editing at the target sites. The EBV genomic locus of BART5 and BART16 were amplified by PCR, cloned in a DNA cloning vector, and subjected to Sanger DNA sequencing. The miRNA sequence is presented in yellow, the gRNA-target sites are displayed in bold, the PAM sequence as red, underlined text, and the cleavage site as a triangle. The number of times each variant has been sequenced is indicated.</p

Simultaneous targeting of HSV-1 with two gRNAs completely impairs virus replication.

<p><b>a)</b> Vero cells were transduced with the indicated single or double gRNAs and subsequently infected with HSV-1-eGFP at an MOI of 0.5. Cells were analyzed for eGFP-expression by flow cytometry at 1, 2, and 3 dpi to assess the percentage of virus-infected cells. <b>b)</b> Similar experiment as in a) but now performed in human MRC5 cells and at an MOI of 0.005, as MRC5 cells are more susceptible to HSV-1 infection. Cells were analyzed for eGFP-expression by flow cytometry at 1 dpi and 3 dpi to assess the percentage of successfully infected cells. <b>c)</b> Supernatants from b) were subjected to plaque assays to quantify the infectious HSV-1 titer produced by gRNA-expressing MRC5 cells that had been infected with HSV-1-eGFP three days earlier. Plaques were scored if visible by eye. <b>d)</b> Plaques from c) obtained after infection of cells with HSV-1 harvested from control or anti-<i>UL8</i> gRNA-expressing MRC5-cells were analyzed by light-microscopy. Whereas large plaques (‘P’) are observed in infected cells not carrying any gRNAs, virus harvested from UL8 gRNA- expressing cells induced microplaques. In double gRNA-treated cells, no signs of infection were observed. For all bar diagrams, measurements of triplicate experiments are presented + STD.</p

Anti-HCMV gRNAs efficiently abrogate HCMV replication in human cells.

<p><b>a)</b> Targeting essential HCMV genes with CRISPR/Cas9 impairs HCMV replication. MRC5 cells transduced with the indicated gRNAs were infected with HCMV-eGFP strain TB40/E at an MOI of 0.05 and subjected to flow cytometry at 2, 5, 8, and 11 dpi to assess the percentage of eGFP-positive infected cells. For each essential gene, four different gRNAs were monitored. Besides targeting human genes as controls, gRNAs targeting nonessential HCMV genes US6, US7, and US11 were included. <b>b)</b> Anti-HCMV gRNAs impair replication of both TB40/E and AD169 strains with the exception of anti-UL84 gRNAs. gRNA-expressing MRC5 cells were infected with eGFP-tagged TB40/E or AD169 and the percentage of eGFP-expressing cells was monitored at 2, 5, and 8 dpi. For bar diagrams in a) triplicate measurements + STD are presented. Bar diagrams in b) are from (at least) duplicate measurements.</p

Anti-HSV-1 gRNAs impair HSV-1 replication.

<p><b>a)</b> Vero cells were transduced with the indicated gRNAs and subsequently infected with HSV-1-eGFP at an MOI of 0.05. To assess the percentage of virus-infected cells, eGFP-expression was analyzed by flow cytometry at 2 dpi. Four gRNAs/gene targeting twelve essential HSV-1 genes and two non-essential genes (US3 and US8) were assessed. As controls, empty vector-transduced cells and gRNAs targeting the human genes <i>HLA-A</i> and <i>B2M</i> are presented. <b>b)</b> Prolonged inhibition of HSV-1 replication by gRNAs targeting essential genes UL8, UL29, and UL52. Select gRNA-expressing cells from a) were monitored for HSV-1-eGFP presence at 2 and 3 dpi. <b>c)</b> Indicated gRNA-expressing cells were infected with HSV-1-eGFP at MOI 0.5 or 0.05 and the amount of HSV-1 viral genomes present in the supernatant was assessed by qPCR at 4 dpi. The relative HSV-1 genome content was normalized to supernatant harvested from cells transduced with vector control. For all bar diagrams, measurements for triplicate experiments are presented + STD.</p