MicroRNAs (miRNAs) are small (,22-nucleotide) RNAs that in lower organisms serve important regulatory roles in development and gene expression, typically by forming imperfect duplexes with target messenger RNAs. miRNAs have also been described in mammalian cells and in infections with Epstein–Barr virus (EBV), but the function of most of them is unknown. Although one EBV miRNA probably altered the processing of a viral mRNA, the regulatory significance of this event is uncertain, because other transcripts exist that can supply the targeted function. Here we report the identification of miRNAs encoded by simian virus 40 (SV40) and define their functional significance for viral infection. SVmiRNAs accumulate at late times in infection, are perfectly complementary to early viral mRNAs, and target those mRNAs for cleavage. This reduces the expression of viral T antigens but does not reduce the yield of infectious virus relative to that generated by a mutant lacking SVmiRNAs. However, wild-type SV40infected cells are less sensitive than the mutant to lysis by cytotoxic T cells, and trigger less cytokine production by such cells. Thus, viral evolution has taken advantage of the miRNA pathway to generate effectors that enhance the probability of successful infection. To search for virally encoded miRNAs, we developed a computer program to predict likely miRNA precursors (pre-miRNAs) and performed a screen in silico for these RNAs in SV40. After searching the entire viral genome in both orientations, we found only two candidates, one each in the early and late orientations (Supplementary Fig. 1). The early-polarity pre-miRNA was predicted at nucleotide positions 29–5,201. However, northern blotting of RNA from infected cells with a probe spanning this region detected no small RNAs of appropriate size (data not shown). The predicted latepolarity pre-miRNA maps to the untranslated region 3 0 to the polyadenylation cleavage site in the late pre-mRNA (Fig. 1a). An RNA in this position has not previously been reported; however, this region is just 5 0 to (and overlaps by 20 nucleotides (nt)) a ,62-nt transcript of unknown function called SAS (SV40-associated small RNA), initially discovered in 1980 (ref. 4). Several different oligonucleotides complementary to this region were used to probe SV40 total RNA (Fig. 1b). A probe that spans most of the computer-predicted LETTERS
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