We show here that miR-122 stimulates HCV RNA translation at an early stage of association of the small ribosomal subunit with the viral RNA. Most experiments were performed with HCV reporter RNAs that do not allow HCV RNA replication in the transfected cells, and also a full-length HCV genome deficient in NS5B polymerase activity shows the same response to miR-122 stimulation.
The experiments with the mutations in the 5′- and 3′-UTR show that those sequences in the 5′-UTR are functional in translation stimulation, whereas that in the 3′-UTR does not significantly confer stimulation by miR-122. The incidental observation that the mutations in the variable region of the 3′-UTR affect translation efficiency confirms our previous finding that the variable region of the 3′-UTR is important for the overall translation regulation of HCV (Song et al, 2006
). The expression with the mutant HCV reporter RNAs and the mutated miR-122 showed that compensatory mutations in the miRNA can complement mutations in the miRNA target sites. This demonstrates a direct interaction of the miRNA with its HCV target RNA in translation stimulation. However, even the expression with the double mutant cannot reach the levels obtained with the wild-type HCV reporter in the presence of added wild-type miR-122, as the transfected Huh-7 cells already contain endogenous wild-type miR-122 that stimulates translation with the wild-type but not with the mutant reporter in excess of the ectopically added miR.
In a more mechanistic approach using gradient analysis of the translation initiation complexes formed, we show that miR-122 stimulates the association of the small ribosomal subunit with the HCV RNA. Both HCV RNA and miRNA are contained in the 48S translation initiation complexes identified by the stage-specific translation inhibitor GMP-PNP. The kinetic analysis shows that the stimulation takes place early in the initiation phase by accelerating the association of the small ribosomal subunit with the HCV RNA. At later stages, the formation of polysomes is found enhanced by miR-122 in cell-free extract as well as in living HeLa cells. All these experiments show clearly that miR-122 actually stimulates the translation of HCV RNA.
This finding may complement the result of an earlier study that showed that miR-122 causes the accumulation of HCV replicon RNA (Jopling et al, 2005
), and another study followed that experimental system to show that ectopic miR-122 increases the steady-state level of HCV RNA also in a non-hepatic cell line (Chang et al, 2008
). Although the effect of miR-122 on overall HCV replication was clearly shown by Jopling and coworkers, we suspected that an effect of miR-122 on HCV translation may have escaped detection in that study, as we know now that monocistronic reporter RNA systems are more appropriate to investigate translation than dicistronic RNAs that contain an additional picornavirus IRES (Song et al, 2006
; Jünemann et al, 2007
). As our present analysis was focused on the effect of miR-122 on HCV translation, we can only speculate whether or not later steps of the HCV replication cycle, for example, the synthesis of HCV progeny RNA genomes catalysed by the NS5B
polymerase, are also modulated by miR-122.
miR-122 may considerably contribute to the liver tropism of HCV on the level of translation regulation. The uptake of HCV into liver cells is obviously related to the metabolism of lipoprotein particles (reviewed in Andre et al, 2005
). Cellular surface receptors, such as CD81, scavenger receptor class B type I, claudin-1 and the low-density lipoprotein receptor, are involved in HCV entry into liver cells, whereas DC-SIGN and L-SIGN may help to internalize HCV into endothelial cells on its way to the liver (reviewed in Poumbourios and Drummer, 2007
). However, until now miR-122 was found to be expressed almost exclusively in liver cells and in the hepatoma cell line Huh-7 (Lagos-Quintana et al, 2002
; Sempere et al, 2004
; Chang et al, 2005
; Fu et al, 2005
). In this scenario, miR-122 can be speculated to provide the decisive increase in translation efficiency that results in more efficient replication of HCV in liver cells compared with other cells and by that finally makes HCV a hepatotropic virus.
The fact that those miR-122 target sequences in the HCV RNA that are functional in translation stimulation reside in the 5′-UTR is surprising only at first glance. Even if it could not be excluded that the coding sequence could also contain miRNA target sequences that are functional in translation regulation, they would most likely be rendered non-functional by traversing ribosomes during translation. In contrast, miRNA target sites that regulate translation of a given capped and polyadenylated ‘standard' mRNA could be expected to be located in the 3′-UTR to avoid interference of the bound miRNP complex with small ribosomal subunits scanning from the mRNA's 5′-end for an initiator AUG. Probably, this is the reason why until now nearly all miRNA-binding sites that function in translation repression have been found in the mRNA's 3′-UTR. However, an miRNA-binding site can be artificially relocated to the 5′-UTR without loss of function when the miRNP complex does not interfere with ribosomal scanning by the use of an IRES downstream of the miRNA-binding site (Lytle et al, 2007
). With respect to the location of the miRNA target site, a similar situation is found in HCV: both miR-122-binding sites that function in translation regulation are located upstream of the IRES.
Our results may also have consequences for the general understanding of miRNA function in translation regulation. So far, in the vast majority of cases the action of miRNAs on the 3′-UTRs of cellular mRNAs had been reported to result in the repression of translation or the destabilization of the mRNA (Pillai et al, 2007
). Only very recently, a report showed that an miRNA can stimulate translation by acting on the 3′-UTR (Vasudevan et al, 2007
), and translation of mRNAs coding for some ribosomal proteins was found to be slightly stimulated by miR-10a binding to the 5′-UTR (Orom et al, 2008
Why is HCV translation stimulated by the miRNA instead of being repressed? From the mechanistic point of view, we could just ask the reverse question: why not? From transcriptional regulation, we know enhancers and silencers of transcription; the difference in mechanics is just brought along by another kind of influence on the respective target protein complex, the activity of which is to be increased or decreased. And from the functional point of view, it is an advantage for a virus to take the stimulating turn of conformational changes, whereas the activity of cellular mRNAs stored for later use may usually be rather suppressed than stimulated by miRNAs.