Cells were then transfected with increasing amounts of BHRF1-3 miRNA sense oligos (Fig.4C, left and right, lanes 3-5) and analyzed for CXCL-11/I-TAC by RT-PCR (Fig.4C, left) and ELISA (Fig.4C, right). We observed a significant reduction of CXCL-11/I-TAC at both mRNA and protein levels when cells were transfected with BHRF1-3 miRNA.
The final known example of a cellular gene product downregulated by a viral miRNA is provided by CXCL-11, also called I-TAC, an interferon-inducible chemokine that acts as an attractant for T-cells. The mRNA encoding CXCL-11 is downregulated by EBV miR-BHRF1-3, which is expressed at high levels in many B cell tumors induced by EBV.
To confirm that EBV-miRNAs in exosomes led to gene silencing, we purified exosomes from EBV-negative BJAB cells. Although LCL and BJAB exosomes are similar macroscopically (Fig.3 A and G), in protein composition (Table S1), and in uptake by MoDC (Fig.3H), LCL exosomes are superior in repressing of CXCL11-3'UTR-mediated luciferase activity (Fig.3I). Collectively, these studies indicate that LCL exosomes internalized by MoDC led to silencing of ectopically expressed CXCL11 mRNA mediated through BHRF1-3.
From the targets identified to date (Table 1, Figure 1) it is apparent that viral miRNAs play an important role in immune evasion by inhibiting immune surveillance and extending the life of the infected host cell. Table 1 Immunomodulatory viral miRNAs.
Of particular interest is a host target, CXCL-11. As shown from miRNA inhibition studies in infected cells, EBV-miR-BHRF1-3 negatively regulates CXCL-11 (Pfeffer et al., 2004; Xia et al., 2008).
Each method has inherent advantages and limitations, but the combination of these approaches have been successful in identifying many miRNA targets (Table 1). Table 1 List of experimentally validated cellular targets of KSHV- and EBV- encoded miRNAs. The normal cellular functions of these proteins and the effect of their knockdown during viral infection are also listed. The targets that are underlined are repressed by both KSHV- and EBV-encoded miRNAs.
