Report

A Cellular MicroRNA Mediates Antiviral Defense in Human Cells

See allHide authors and affiliations

Science  22 Apr 2005:
Vol. 308, Issue 5721, pp. 557-560
DOI: 10.1126/science.1108784

Abstract

In eukaryotes, 21- to 24-nucleotide-long RNAs engage in sequence-specific interactions that inhibit gene expression by RNA silencing. This process has regulatory roles involving microRNAs and, in plants and insects, it also forms the basis of a defense mechanism directed by small interfering RNAs that derive from replicative or integrated viral genomes. We show that a cellular microRNA effectively restricts the accumulation of the retrovirus primate foamy virus type 1 (PFV-1) in human cells. PFV-1 also encodes a protein, Tas, that suppresses microRNA-directed functions in mammalian cells and displays cross-kingdom antisilencing activities. Therefore, through fortuitous recognition of foreign nucleic acids, cellular microRNAs have direct antiviral effects in addition to their regulatory functions.

In plants and insects, viral double-stranded RNA is processed into small interfering RNAs (siRNAs) by the ribonuclease (RNase) III enzyme Dicer. These siRNAs are incorporated into the RNA-induced silencing complex to target the pathogen's genome for destruction (1, 2). Plant and insect viruses can counter this defense with silencing suppressor proteins, which often have adverse side effects on microRNA (miRNA) functions (3, 4). Although undisputed in plants and insects, a defensive role for RNA silencing in vertebrates has not been demonstrated. Virus-derived small RNAs have not been detected in infected vertebrate cells, with the exception of miRNAs produced by the Epstein-Barr virus, but the role of those molecules remains unclear (5). Moreover, some mammalian virus-encoded proteins that suppress RNA silencing have only been investigated in heterologous systems (6). Because RNA silencing suppresses mobilization of endogenous retroviruses in plants, yeast, worms, and flies (7), we reasoned that retrotransposition of mammalian exogenous viruses might also be subject to this process. Therefore, we studied the primate foamy virus type 1 (PFV-1), a complex retrovirus (akin to human immunodeficiency virus) that, in addition to the Gag, Pol, and Env proteins, produces two auxiliary factors, Bet and Tas, from the internal promoter (IP) (Fig. 1A) (8).

Fig. 1.

RNA silencing limits PFV-1 accumulation in mammalian cells. (A) Schematic of the PFV-1 genome. Bent arrows indicate the start of transcription between the 5′-proximal long-terminal repeat (LTR) and the IP. Viral sequences (F1 to F10) used for GFP transcriptional fusions are indicated. (B) mRNA accumulation from PFV-1 in 293T cells that do (+) or do not (–) stably express the P19 protein. Cells were harvested 48 hours after transfection. Northern analysis confirms P19 expression. rRNA, ethidium bromide staining of ribosomal RNA; NI, noninfected. (C) The GFP sensors F1 to F11 were transfected together with (+) or in the absence of (–) PFV-1. Their expression was assayed 48 hours later by Northern (first upper panel) and Western (fourth panel) analysis. (Second upper panel) PFV-1 RNA accumulation. (Bottom) Staining of total protein for loading control. Relative RNA or protein accumulation is shown at the bottom of each panel, with control levels arbitrarily set to 1.

PFV-1 accumulation was strongly enhanced in 293T cells expressing the P19 silencing suppressor (Fig. 1B). This suggested that a siRNA and/or miRNA pathway limits PFV-1 replication in human cells, because P19 specifically binds to and inactivates both types of small RNAs (4, 9, 10). Viral sequences spanning the 12-kb-long PFV-1 genome (Fig. 1A) were fused to the 3′ untranslated region (UTR) of a green fluorescent protein (GFP)–tagged reporter gene, and the resulting constructs (F1 to F11) were cotransfected with PFV-1 into baby hamster kidney (BHK) 21 cells. Any viral-derived siRNA would induce RNA silencing of the corresponding reporter fusions, diagnosed as reduced GFP mRNA accumulation. However, the mRNA levels from those constructs were similar in noninfected and infected cells (Fig. 1C). Use of a highly sensitive RNase protection assay likewise failed to provide evidence for viral-derived siRNAs (fig. S1).

The GFP levels from fusion F11 were disproportionably reduced compared to the accumulation of the F11 mRNA (Fig. 1C). They were also reduced compared to the GFP levels from constructs F2 and F10. Although a possible result of intrinsic protein instability, the effect was reminiscent of the translational inhibition directed by animal miRNAs (11). However, it was independent of the presence or absence of PFV-1 (Fig. 1C), suggesting that any miRNA involvement was likely cellular rather than viral. Using the DIANA-microT algorithm (12), we found a high probability hit (free energy of –21.0 kcal/mol) between the PFV-1 F11 sequence and the human miR-32 (Fig. 2A) (13). The predicted miR-32 target sequence was sufficient to promote translation inhibition of the GFP mRNA (Fig. 2B), unlike a derivative thereof that carried four mutations disrupting annealing of the small RNA. Moreover, translation inhibition by miR-32 was suppressed in P19-expressing cells (fig. S2).

Fig. 2.

miR-32 effectively limits PFV-1 replication. (A) Position of the computationally predicted miR-32 target relative to PFV-1 transcripts. (B) The miR-32 target sequence or a mutated form thereof (–) was fused to the 3′UTR of a GFP reporter gene (+). Constructs were transfected in HeLa cells and harvested 48 hours later. GFP and GFP mRNA accumulation were assessed by Western (top) and Northern (bottom) analysis. (C) HeLa cells were transfected with PFV-1 together with LNAs (10 nM) directed against miR-32 or miR-23. Total RNA was extracted 48 hours after transfection and subjected to Northern analysis. (D) PFV-1 was transfected in HeLa cells (transfection 1). Separate cells were transfected with a luciferase-based reporter (Luc) driven by the PFV-1 IP, which is activated by the transactivator Tas (transfection 2). Transfections 1 and 2 were mixed 24 hours later and further cocultured for 48 hours. Luciferase expression in cells from transfection 2, resulting from their infection by virions released from transfection 1, was then quantified. hpt, hours post-transfection. (E) The miR-32 target sequence within PFV-1Δ32 contains two synonymous mutations (arrows). Northern analysis of mutant and wild-type virus mRNAs was carried out 48 hours after transfection.

The miR-32 target is in open reading frame (ORF) 2, shared by the Bet and EnvBet proteins, and is also within the 3′UTR of all remaining PFV-1 mRNAs (Fig. 2A). To address the antiviral effect of miR-32, we used antisense locked nucleic acid (LNA) oligonucleotides (fig. S3), which yield highly stable hybrids (14). In HeLa and BHK-21 cells, the transfected anti-miR-32 LNA prevented translation inhibition by miR-32, whereas a control LNA with antisense sequence of the unrelated miR-23 did not (fig. S3). At LNA concentrations of 10 nM, accumulation of PFV-1 mRNAs was higher in the anti-miR-32–treated cells than in the anti-miR-23–treated cells (Fig. 2C). Use of a luciferase-based assay also indicated that the anti-miR-32, unlike the anti-miR-23, almost doubled progeny virus production (Fig. 2D).

Although these results are consistent with an antiviral effect of miR-32, we could not discard the possibility of an indirect action of anti-miR-32 LNA causing, for instance, ectopic expression of cellular miR-32 targets, which could in turn increase viral fitness. The miR-32 target sequence in PFV-1 was thus modified to contain two synonymous mutations that abolished the miR-32 pairing but preserved the Bet amino acid content (Fig. 2E). The mRNA levels from the miR-32–resistant virus (PFV-1Δ32) were three times as high as those from the unmodified virus, consistent with the anti-miR-32 results (Fig. 2, E and C). Therefore, miR-32 exerts a direct, sequence-specific effect against PFV-1.

Does PFV-1 encode a silencing suppressor to counter the antiviral effect of miR-32? The constitutive presence of miR-32 required that the putative suppressor be synthesized precociously, which is the case of the Tas and Bet proteins (Fig. 2A). As Bet is dispensable for productive replication, Tas appears the most likely candidate (15). miR-32–mediated translational inhibition was indeed suppressed in Tas-expressing BHK21 cells (Fig. 3A). This was not specific for the sequence or activity of miR-32, because Tas, like P19, also suppressed endonucleolytic cleavage of GFP sensors carrying a perfect miR-23 target (Fig. 3B and fig. S2). Probably as a consequence of its suppressor function, Tas promoted the nonspecific overaccumulation of all cellular miRNAs inspected, which we also observed 5 days after PFV-1 infection in BHK21 cells (Fig. 3C). miRNA overaccumulation is also seen with several plant viral suppressors that interfere with the miRNA pathway (3, 4).

Fig. 3.

Tas suppresses miRNA-directed silencing in mammalian cells. (A) The reporter constructs used in Fig. 2B were transected in control BHK21 cells (mock) or in cells stably expressing Tas. GFP expression was assayed by Western analysis (top) 48 hours after transfection. Tas expression was confirmed by Northern analysis (bottom). (B) A sequence with 100% complementarity to miR-23 (+) or a mutated derivative thereof (–) was inserted into the 3′UTR of the GFP reporter gene. Constructs were transfected in BHK21 cells (mock) or in cells stably expressing Tas (Tas), and the GFP mRNA was assayed by Northern analysis 48 hours later. (C) Northern analysis of cellular miRNAs from BHK21 cells expressing (+) or not expressing (–) Tas (left) and from noninfected (–) or PFV-1–infected (+) BHK21 cells (right). Total RNA was extracted 5 days after infection.

To validate the silencing suppression activity of Tas in a heterologous system, we used an Arabidopsis line expressing an RNA interference (RNAi) construct targeted against chalcone synthase (CHS), which is responsible for the brown seed-coat pigmentation (4). This line accumulates CHS siRNAs and, consequently, produces pale yellow seeds (Fig. 4A, left). Transgenic Tas expression restored anthocyanin synthesis (Fig. 4A, right) because of a strong decrease in CHS siRNA levels (Fig. 4B). Tas-expressing plants also exhibited developmental anomalies, including leaf elongation and serration (Fig. 4C), reminiscent of those elicited in Arabidopsis by viral suppressors interfering with miRNA functions (3, 4). As in mammalian cells, Tas enhanced miRNA accumulation (Fig. 4D), independently of their nature or mode of action, suggesting that it suppresses a fundamental step shared between the miRNA and siRNA pathways that is conserved from plants to mammals.

Fig. 4.

(A) Transgenic Tas suppresses CHS RNAi in Arabidopsis. (B) Northern analysis of CHS siRNAs in two independent Tas-expressing lines. Col0, nontransformed plants; CHS, the reference RNAi line. (C) Developmental defects and (D) miRNA accumulation in Tas-expressing Arabidopsis. miR156 and miR172 are evolutionarily conserved miRNAs that promote cleavage and translation inhibition, respectively. miR163 is a cleavage-promoting, Arabidopsis-specific miRNA.

These results indicate that RNA silencing limits the replication of a mammalian virus, PFV-1, and that a cellular miRNA contributes substantially to this response. As a counterdefense, PFV-1 produces Tas, a broadly effective silencing suppressor. Because all our experiments were conducted with Tas-expressing viruses, because of the essential role of the protein for replication (15), the strong effect of Tas on siRNA accumulation observed in Arabidopsis could account for our failure to detect siRNAs in mammalian cells (fig. S1). Therefore, we do not yet rule out their implication in the antiviral response reported here.

Our findings with miR-32 and PFV-1 were in fact anticipated in plants by Llave, who pointed out several near-perfect homologies between Arabidopsis small RNAs and viral genomes (16). The chances of a match between cellular miRNAs and foreign (i.e., viral) RNAs increase proportionally with the size of sampled sequences. The extent to which cellular miRNAs will be selected to target pathogen genomes upon their initial interaction with viruses may vary. Endogenous viruses might effectively coevolve with miRNAs for defensive or developmental purposes (17, 18), such that viral control might eventually constitute the sole function of some cellular miRNAs. Exogenous viruses with high mutation rates could, on the other hand, rapidly escape this miRNA interference through modification of the small RNA complementary regions (19).

Our results support the emerging notion that miRNAs might be broadly implicated in viral infection of mammalian cells, with either positive or negative effects on replication (5, 20). They also indicate that virtually any miRNA has fortuitous antiviral potential, independently of its cellular function. Moreover, because the repertoire of expressed miRNAs likely varies from one cell type to another (11), this phenomenon could well explain some of the differences in viral permissivity observed between specific tissues.

Supporting Online Material

www.sciencemag.org/cgi/content/full/308/5721/557/DC1

Materials and Methods

Figs. S1 to S4

References and Notes

References and Notes

View Abstract

Navigate This Article