End game: getting the most out of microRNAs.

V iruses are notorious for their ability to usurp cellular pathways for their own benefit, often by exploiting host factors in new and unusual ways. Hepatitis C virus (HCV), a causative agent of chronic liver disease, is no exception. As well as using host proteins and lipids, the virus is known to enlist an abundant liver-specific microRNA, miR-122, to aid in its replication (1). MicroRNAs are short (≈22-nt) sequences that typically bind, through complementary ≈6-nt “seed” sites, to the 3′ noncoding regions (NCRs) of certain cellular mRNAs. By recruiting the RNAinduced silencing factor complex (RISC), microRNA binding generally leads to translational suppression and/or degradation of the target transcript. HCV interactions with microRNAs, however, seem to defy all conventions. miR-122 binds the viral genome at not one but two sites. These tandem target sequences are located in the 5′ NCR rather than at the 3′ end, and recruitment of miR-122 does not repress translation but enhances viral replication through as-yet-unclear mechanisms. In PNAS, Machlin et al. (2) uncover a further unique feature of the miR-122– HCV association. Their work reveals that miR-122 binds the viral genome in a complex structure that requires not only the seed sequence but also functionally important associations beyond the seed site. The complex RNA interaction seems to be indispensable for HCV replication but is not required for the actions of miR-122 on cellular mRNAs. This model opens up new potential mechanisms for miR-122 in the HCV life cycle. In pursuit of understanding the role of miR-122 in HCV replication, Machlin et al. (2) hypothesize that specific regions of the microRNA required for viral growth can be uncovered. Dissecting the two miR-122 binding sites on the HCV RNA requires careful experimental design. By changing one critical nucleotide in site 1 or 2 of the viral genome, transfected miR-122 bearing the complementary change can be directed to a particular target, whereas the other site binds only the endogenous microRNA (3). Using this system, a series of miR-122 mutants is directed to each binding site, and the impact on viral replication is monitored by Northern blot analysis. Unexpectedly, miR-122 nucleotides 15 and 16, which are outside the canonical seed sequence (nucleotides 2–8), are shown to be critical for HCV replication when targeted to either genomic site. By engineering compensatory mutations, Machlin et al. (2) go on to demonstrate that these miR-122 nucleotides likely form direct base-pairing interactions with phylogenetically conserved HCV sequences (Fig. 1A). Most interestingly, the additional interactions of miR-122 upstream of site 1 result in binding to the extreme 5′ end of the HCV genome, converting a free terminus into an RNA duplex with 3′ overhang. Deletion or modification of the protruding region of miR-122 (nucleotides 20–23) severely impacts HCV replication. Remarkably, this 3′ tail and nucleotides 15–16 are not required for the conventional function of miR-122 as a microRNA or siRNA against a reporter transcript (Fig. 1B). These results suggest that the unusual tandem “bulge and tail” structure formed by miR-122 binding to the HCV genome might contribute to the unique outcomes of this interaction. In terms of microRNA biology, these findings present a few surprises. Although nucleotides 13–16 of other microRNAs have been shown to supplement recognition of the seed site sequences (4), binding to the extreme 5′ end of a target RNA is unprecedented. Furthermore, the requirement for microRNA nucleotides 20–23 is quite unusual. It is possible that these nucleotides are needed to stabilize the HCV-bound microRNA, perhaps by recruiting factors that protect from 3′–5′ exonucleases or other degradation schemes (Fig. 1C). Alternatively, this sequence may form a localization element that is important for HCV replication, such as the nuclear targeting sequence identified in the 3′ end of miR-29b (5). The entire miR-122 sequence is unusually highly conserved among mammals, and swapping of sequences between diverse microRNAs may help shed light on the role of the 3′ sequences in viral and cellular functions. More effort is needed to define the precise molecular complex at the 5′ end of the HCV genome and to understand the 5’-GCCA UGUGAGG