Known to alter the expression of thousands of genes (36
), HIV-1 has been shown recently to alter the expression profile of cellular miRNAs. Comparing HIV-1-infected versus noninfected cells by microarray analysis, Triboulet et al.
) noticed that some miRNAs were up-regulated, whereas others were down-regulated by HIV-1. These findings are not compatible with a general shutdown or stimulation of the miRNA-guided RNA silencing machinery by the virus and rather argue for an alternative mechanism to explain how specific genes and miRNAs are modulated by HIV-1.
Initial studies aimed at investigating the existence of virus-derived miRNAs, through the use of small RNA cloning methods, were unable to provide experimental evidences in the case of HIV-1. In a study by Pfeffer et al.
), no miRNA of viral origin was found among 1540 small RNA sequences cloned from HeLa cells stably expressing CD4 and CXCR4, and infected with HIV-1, isolate Bru (LAV1). On the other hand, two independent groups have obtained positive signals from the use of northern blotting, compatible with small HIV-1 RNAs (15
). Here, we characterized and delineated miRNAs originating from the HIV-1 TAR element by making use of northern blotting, primer extension and RPA. As shown for SV40, this latter method may be suitable, better adapted and more sensitive for the detection of viral miRNAs (12
Why had HIV-1 TAR miRNAs eluded detection for so long? First, low expression levels of some viral miRNAs may render their isolation by cloning methods difficult among the more abundant cellular ribosomal RNAs, transfer RNAs and miRNAs. This is consistent with the facts that miRNA cloning frequency reflects miRNA abundance and that miRNAs cloned in a single copy may be more difficult to detect (37
). Second, viral and cellular proteins interacting with the TAR element within the 5′ LTR of HIV-1 mRNAs likely decrease its vulnerability to processing by RNases III. Third, HIV-1 TAR element may partially escape RNases III processing by adopting alternative conformations, such as one called TAR31, which is functionally similar but requires only 31 nt to form (38
). Fourth, the lack of a free 2-nt 3′ overhang at the base of TAR in HIV-1 mRNA transcript may hamper its processing, as demonstrated by the marked decrease in TAR RNA processing by Dicer upon extension of its 3′ overhang sequence from 2 to 10 nt. Finally, we cannot exclude the possibility that the TAR miRNAs are modified on the 2′ hydroxyl of the terminal ribose, which would significantly hamper the cloning efficiency of these modified small RNAs, as experienced in plants (39
). On the other hand, it is known that the HIV-1 promoter directs, in the absence of Tat, the synthesis of another class of short nonpolyadenylated transcripts with heterogeneous 3′ ends located around position +59 (40
). These transcripts may thus represent potential substrates for Dicer and argue for a specific sub-population of TAR as an alternative source of HIV-1 miRNAs.
When examining the TAR miRNA sequences elucidated by primer extension and RPA, we noticed a nt bias at the 5′ end of both miR-TAR-5p and miR-TAR-3p. The presence of a U at this position is in agreement with the preference of the RNases III Drosha and Dicer to cleave on the 5′ side of a U. Likely long enough to be recognized and processed by the RNase III Dicer, the TAR element may be too short for Drosha, whose pri-miRNA substrates typically contain a stem of ~3 helical turns (~33 bp) (41
). Moreover, TAR miRNAs do not result from cleavage ~11 bp away from the single-stranded junction at the base of TAR, as would be expected from the Drosha·DGCR8 complex (42
). Experimental data rather support the specific involvement of Dicer in HIV-1 TAR RNA processing. First, TAR RNA was efficiently cleaved by recombinant Dicer in vitro
. Similar findings were reported recently by Klase et al.
). Second, the size of the TAR RNA products (~23–24 nt) detected in vivo
correlates with that expected for RNAs issued from Dicer cleavage. Third, TAR RNA was processed into a miRNA duplex, a recognized Dicer signature. The most thermodynamically favorable pairing of the TAR miRNA duplex differs from the sequence structure located in the unprocessed TAR and exhibits two 3-nt 3′ overhangs (D.L. Ouellet, I. Plante and P. Provost, unpublished data). This opens the possibility of a pairing rearrangement within the duplex upon removal of the neighboring base pairs and sequences in the precursor molecule.
The extremities of some viral miRNA duplexes seem to diverge from the general 2-nt 3′ overhang rule established for endogenous miRNAs. Like the TAR miRNA duplex, miRNAs derived from SV40 (12
) and KSHV (11
) also exhibit atypical 3-nt 3′ overhangs. Computational positioning of mature KSHV miRNA sequences also revealed duplexes bearing 3′ overhangs as short as 1 nt (11
). The discrepancies between the viral and endogenous miRNAs may bear the signs of subtle processing irregularities, possibly related to the adaptive capacity of the RNA silencing components to recognize and process internal viral RNA structures like miRNA precursors. Initially reported in Zhang et al.
), Dicer excision of miRNAs from internal sites is not infrequent, as the long CAG and CUG repeats found within their natural context in transcripts from mutant genes involved in diseases such as myotonic dystrophy type 1, Huntington's disease and spinocerebellar ataxia type 1 are efficiently cleaved by Dicer (45
Mechanistically, endogenous substrate recognition by Dicer has been proposed to involve anchoring of the pre-miRNA 2-nt 3′ overhang in the pocket formed by its central PAZ domain (6
). Furthermore, the 2-nt overhang is measured by the alignment of the Dicer RNase III domains rather than by the distance between active residues on one peptide chain, whereas the length of the product (~21 nt) is determined by the distance between the PAZ domain and the active site (6
). Flanked by a single-stranded RNA sequence on its 3′, TAR RNA processing may be suboptimal, perhaps less accurate. In support to this possibility is a study by Vermeulen et al.
) reporting that shifts in Dicer cleavage sites result from dissimilar processing of substrates bearing overhangs of different lengths. In addition, folding of the TAR element itself may force Dicer dimers to adopt a slightly modified configuration. The 3-nt bulge, in particular, has been shown to introduce a bend of ~50° into the α helical RNA structure of TAR (48
), which otherwise exhibits a certain degree of flexibility. These structural constraints may explain the length of the TAR miRNAs, the nature of the overhangs generated and the relative degree of heterogeneity of the cleavage sites determined for miR-TAR-3p (please refer to the open arrowheads in A).
Transactivation by the viral protein Tat is severely compromised upon introduction of G/U mutations in the loop of the TAR element (49
). When studied in parallel with the wild-type TAR, however, the G/U loop TAR mutant was processed with similar efficiencies by Dicer in vitro
. These observations support the notion that the TAR structure that evolved to support transactivation by Tat remains susceptible to cleavage by Dicer, possibly reflecting the inability of TAR to escape from Dicer surveillance. Evocative of the rapid adaptive evolution of the antiviral pathway in Drosophila
), this ongoing interaction between HIV-1 and Dicer may be constantly shaping the antiviral functionalities of the host RNA silencing machinery.
When interpreting the results obtained from Dicer RNase assays in vitro
, the caveat has to be taken into account that recombinant human Dicer exhibits low processive activity in vitro
), a characteristic that has been attributed to the product remaining bound to the enzyme (44
). In more recent studies, Dicer has been shown to operate with TRBP within a pre-miRNA processing complex (51
) and to interact with Ago2 (53
), another component of miRNA-containing ribonucleoprotein (miRNP) complexes (54
). Although it has been reported to facilitate pre-miRNA processing in vitro
), the role of TRBP in modulating Dicer function remains largely undefined. It may be analogous to that played by DGCR8 within the Drosha·DGCR8 complex, in which DGCR8 may interact directly and specifically with pri-miRNAs, and proposed to function as a molecular anchor that determines the Drosha cleavage sites (41
). Alternatively, TRBP may contribute to releasing the miRNA product from Dicer and improving biosynthesis of miR-TAR-5p in vivo
Data obtained from Dicer knockdown and FMRP KO cells show that TAR miRNAs are recognized and channelled through the miRNA-guided RNA silencing pathway. Our TAR miRNA sensor activity assays revealed the miRNAs residing within the TAR element of HIV-1 are functional, as both miR-TAR-5p and miR-TAR-3p exerted downregulatory effects on gene expression, and that these effects were optimal in the presence of Dicer and FMRP. Together, our findings are compatible with TAR miRNAs being extracted from the HIV-1 TAR element by Dicer to be subsequently incorporated into active miRNP complexes in vivo, in which FMRP may act to facilitate their annealing to mRNAs bearing sites of perfect or imperfect complementarity. Whether Dicer mediates action of TAR miRNAs by acting also within miRNP effector complexes in vivo remains to be determined.
Although indicative of the presence of miRNAs derived from HIV-1 TAR element, the response of our sensor constructs in HIV-1-infected cells was relatively modest and less pronounced than that observed in cotransfected 293 cells. This observation may be related to the fact that cells infected with the virus express low physiological levels of TAR RNA, in contrast with vector-based, U6 promoter-driven overexpression of TAR RNA. In addition, expression in these cells of the viral TAR RNA binding Tat protein, which has been proposed as a suppressor of RNA silencing (16
), may have hampered Dicer processing of TAR RNA and contributed to attenuate the signal expected. Although a recent study reported that physiological levels of HIV-1 Tat neither inhibited the RNA silencing machinery of infected cells nor induced the accumulation of pre-miRNAs as would be predicted for an inhibitor of Dicer function (55
As assessed in mRNA target regulation, the silencing properties of miR-TAR-3p were slightly superior than those of miR-TAR-5p, supporting a relative functional asymmetry. This may be related to differences in the thermodynamic stability of the miRNA duplex extremities, as reported in Drosophila
where R2D2 has been shown to act as a protein sensor for siRNA thermodynamic asymmetry (56
). At that level, the human miRNA-guided RNA silencing machinery may differ from the fly system, as no evidence for a human homolog of R2D2 has been provided yet. Rather, the silencing potency of miR-TAR-3p in vivo
paralleled the preferential accumulation of mature miR-TAR-3p upon Dicer processing of TAR in vitro
. This latter step may thus represent a novel source of asymmetry between miRNAs comprised within a pre-miRNA. This observation is reminiscent to endogenous miRNA duplexes, where the level of mature miRNA detected is higher than that of its opposite miRNA* strand (3
). miR-TAR-3p and miR-TAR-5p may thus qualify as the miRNA and miRNA* of the duplex, respectively.
HIV-1 is known to perturb gene expression programming of infected cells, affecting thousands of genes (36
), which likely conceal key determinants of its pathogenesis. As a source of regulatory miRNAs, it is tempting to speculate about a contributory role of the TAR element. Our results indicate that HIV-1 mRNA transcripts are a source of regulatory miRNAs that are functionally competent in RNA silencing processes in vivo
. These findings can be reconciled with the recent findings that Dicer may act as natural anti-HIV-1 host defenses in human cells (14
). We can thus propose a scenario in which Dicer would interfere with the virus through processing of its structured RNAs, such as TAR, from which miRNA by-products could be released and influence expression of viral and/or host genes, with a potential impact on viral replication.
Biosynthesis and action of TAR miRNAs appear to be guided by the preferential release of miR-TAR-3p along asymmetrical processing events yielding miR-TAR-5p-loop as an intermediate RNA species. The lower efficiency of miR-TAR-5p in regulating gene expression may be inherent to this stepwise maturation process. Considering that miRNAs are usually expressed at low levels and that they act in synergy with other miRNAs, dismissal of a significant role for HIV-1 TAR miRNAs of low abundance would not be prudent. Rather, the possibility that TAR miRNA expression ultimately influences viral replication and/or the efficiency of host antiviral defenses is attractive and warrants further investigations. Our study provides the molecular basis required for elucidating the impact of TAR miRNAs on the gene expression programming of HIV-1-infected cells and for determining their role and importance in viral pathogenesis.