Micro RNAs of Epstein-Barr Virus Promote Cell Cycle Progression and Prevent Apoptosis of Primary Human B Cells

Abstract

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.