Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells’ transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we defined multiple properties of EVs and analyzed their capacity to deliver packaged miRNAs into target cells to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but EVs did not fuse detectably with cellular membranes to deliver their cargo. We engineered EVs to be fusogenic and document their capacity to deliver functional messenger RNAs. Engineered fusogenic EVs, however, did not detectably alter the functionality of cells exposed to miRNA-carrying EVs. These results suggest that EV-borne miRNAs do not act as effectors of cell-to-cell communication.

Cellular and viral microRNAs (miRNAs) are involved in many different processes of key importance and more than 10,000 miRNAs have been identified so far. In general, relatively little is known about their biological functions in mammalian cells because their phenotypic effects are often mild and many of their targets still await identification. The recent discovery that Epstein-Barr virus (EBV) and other herpesviruses produce their own, barely conserved sets of miRNAs suggests that these viruses usurp the host RNA silencing machinery to their advantage in contrast to the antiviral roles of RNA silencing in plants and insects. We have systematically introduced mutations in EBV's precursor miRNA transcripts to prevent their subsequent processing into mature viral miRNAs. Phenotypic analyses of these mutant derivatives of EBV revealed that the viral miRNAs of the BHRF1 locus inhibit apoptosis and favor cell cycle progression and proliferation during the early phase of infected human primary B cells. Our findings also indicate that EBV's miRNAs are not needed to control the exit from latency. The phenotypes of viral miRNAs uncovered by this genetic analysis indicate that they contribute to EBV-associated cellular transformation rather than regulate viral genes of EBV's lytic phase. Micro RNAs (miRNAs) are small, non-coding RNAs that bind to mRNA transcripts and abrogate their protein coding functions. Only a few of their mRNA targets are known, although miRNAs are found in all multicellular organisms and certain viruses. In particular, members of the herpesvirus family encode a surprisingly large number of miRNAs. Epstein-Barr virus (EBV) belongs to this virus family and is associated with several human malignancies including B-cell lymphomas. In vitro, this virus infects human primary B cells and transforms them into continuously proliferating lymphoblastoid cell lines (LCL), which is an amenable model covering key aspects of cellular transformation and lymphomagenesis. To assess the roles of EBV's miRNAs in this model, we generated EBV mutants that lack the capacity to encode viral miRNAs. Phenotypic analysis of human primary B cells infected with these mutant viruses revealed that miRNAs encoded in EBV's BHRF1 locus strongly promote B cell proliferation, regulate cell cycle functions, and prevent apoptosis early after infection. Our findings show that EBV has evolved discrete miRNAs to contribute to its well-known transforming capacity, which has not been appreciated previously.

Epstein–Barr virus (EBV) is a human tumor virus and a model of herpesviral latency. The virus efficiently infects resting human B lymphocytes and induces their continuous proliferation in vitro, which mimics certain aspects of EBV’s oncogenic potential in vivo. How lymphoblastoid cell lines (LCLs) evolve from the infected lymphocytes is uncertain. We conducted a systematic time-resolved longitudinal study of cellular functions and transcriptional profiles of newly infected naïve primary B lymphocytes. EBV reprograms the cells comprehensively and globally. Rapid and extensive transcriptional changes occur within 24 h and precede any metabolic and phenotypic changes. Within 72 h, the virus activates the cells, changes their phenotypes with respect to cell size, RNA, and protein content, and induces metabolic pathways to cope with the increased demand for energy, supporting an efficient cell cycle entry on day 3 postinfection. The transcriptional program that EBV initiates consists of 3 waves of clearly discernable clusters of cellular genes that peak on day 2, 3, or 4 and regulate RNA synthesis, metabolic pathways, and cell division, respectively. Upon onset of cell doublings on day 4, the cellular transcriptome appears to be completely reprogrammed to support the proliferating cells, but 3 additional clusters of EBV-regulated genes fine-tune cell signaling, migration, and immune response pathways, eventually. Our study reveals that more than 11,000 genes are regulated upon EBV infection as naïve B cells exit quiescence to enter a germinal center-like differentiation program, which culminates in immortalized, proliferating cells that partially resemble plasmablasts and early plasma cells.

Significance: Most humans are infected for their lifetime with Epstein–Barr virus (EBV), which can cause cancer and other EBV-associated diseases. Infected individuals develop strong immune responses to this virus, in particular cytotoxic CD8 ⁺ T cells, but viral infection is never cleared nor is EBV eliminated from the body. This suggests that certain viral molecules might prevent effective elimination of EBV-infected cells by CD8 ⁺ T cells. EBV is rich in genes coding for microRNAs, many with unknown function. We show that viral microRNAs interfere with recognition and killing of EBV-infected cells by CD8 ⁺ T cells. Multiple mechanisms and molecules are targeted by microRNAs to achieve this immune evasion. Therefore, targeting of viral microRNAs may improve antiviral immunity and therapy.

EBV, a member of the herpes virus family, is a paradigm for human tumor viruses and a model of viral latency amenable for study in vitro. It induces resting human B lymphocytes to proliferate indefinitely in vitro and initially establishes a strictly latent infection in these cells. BZLF1 , related to the cellular activating protein 1 (AP-1) family of transcription factors, is the viral master gene essential and sufficient to mediate the switch to induce the EBV lytic phase in latently infected B cells. Enigmatically, after infection BZLF1 is expressed very early in the majority of primary B cells, but its early expression fails to induce the EBV lytic phase. We show that the early expression of BZLF1 has a critical role in driving the proliferation of quiescent naïve and memory B cells but not of activated germinal center B cells. BZLF1’s initial failure to induce the EBV lytic phase relies on the viral DNA at first being unmethylated. We have found that the eventual and inevitable methylation of viral DNA is a prerequisite for productive infection in stably, latently infected B cells which then yield progeny virus lacking cytosine-phosphatidyl-guanosine (CpG) methylation. This progeny virus then can repeat EBV’s epigenetically regulated, biphasic life cycle. Our data indicate that the viral BZLF1 protein is crucial both to establish latency and to escape from it. Our data also indicate that EBV has evolved to appropriate its host’s mode of methylating DNA for its own epigenetic regulation.

Epstein-Barr virus (EBV) is a tumor virus that establishes lifelong infection in most of humanity, despite eliciting strong and stable virus-specific immune responses. EBV encodes at least 44 miRNAs, most of them with unknown function. Here, we show that multiple EBV miRNAs modulate immune recognition of recently infected primary B cells, EBV's natural target cells. EBV miRNAs collectively and specifically suppress release of proinflammatory cytokines such as IL-12, repress differentiation of naive CD4+ T cells to Th1 cells, interfere with peptide processing and presentation on HLA class II, and thus reduce activation of cytotoxic EBV-specific CD4+ effector T cells and killing of infected B cells. Our findings identify a previously unknown viral strategy of immune evasion. By rapidly expressing multiple miRNAs, which are themselves nonimmunogenic, EBV counteracts recognition by CD4+ T cells and establishes a program of reduced immunogenicity in recently infected B cells, allowing the virus to express viral proteins required for establishment of life-long infection.