Respiratory Syncytial Virus Utilizes a tRNA Fragment to Suppress Antiviral Responses Through a Novel Targeting Mechanism

Abstract

Target identification is highly instructive in defining the biological roles of microRNAs. However, little is known about other small noncoding RNAs; for example, tRNA-derived RNA Fragments (tRFs). Some tRFs exhibit a gene-silencing mechanism distinctly different from that of typical microRNAs. We recently demonstrated that a respiratory syncytial virus (RSV)-induced tRF, called tRF5-GluCTC, promotes RSV replication. RSV is the single most important cause of lower respiratory tract infection in children. By using biochemical screening and bioinformatics analyses, we have identified apolipoprotein E receptor 2 (APOER2) as a target of tRF5-GluCTC. The 3'-portion of tRF5-GluCTC recognizes a target site in the 3'-untranslated region of APOER2 and suppresses its expression. We have also discovered that APOER2 is an anti-RSV protein whose suppression by tRF5-GluCTC promotes RSV replication. Our report represents the first identification of a natural target of a tRF and illustrates how a virus utilizes a host tRF to control a host gene to favor its replication.

Identification of two novel functional tRNA-derived fragments induced in response to respiratory syncytial virus infection

“…The induction of tRFs is virus specific as the infection of hMPV, a closely related respiratory virus, does not result in such a significant change in tRF induction [10]. We scrutinized one of the most abundantly induced tRFs, namely tRF5-GluCTC, to establish the methods to study its gene regulatory mechanisms [10,13]. However, the functions of other inducible tRFs have not been explored and the experimental methods established from the tRF5-GluCTC study have not been validated.…”

“…Therefore, if they regulate gene expression, such regulation is likely to be at the level of post-transcription. Here, we first tested whether tRF5-GlyCCC and tRF5-LysCTT also have a gene regulatory capability [13]. If these tRFs have a gene trans-silencing function like tRF5-GluCTC, they would recognize the complementary sequence as a target site, therefore downregulating the expression of Pp harbouring a reverse complementary sequence of tRF5-GlyCCC or tRF5-LysCTT, namely the relative luciferase activity of 'Pp-anti_GlyCCC_WT' and 'Ppanti_LycCTT_WT', respectively [10,16].…”

“…To determine that the observed decrease of luciferase activity was tRF5-GlyCCCor tRF5-LysCTT-specific, we inhibited the individual tRF by transfecting cells with 20 nt antisense oligonucleotides whose sequences were complementary to tRF5-GlyCCC/ tRF5-LysCTT (see Figs 3(a) and 4(a) for their sequence), as previously described [10,13,17,18]. Compared to a nontargeting control oligoribonucleotide (designated as 'anticontrol'), transfection of either of these two tRFs efficiently restored the luciferase activity that was decreased by RSV infection (right two bars versus middle two bars in Figs 3(b) and 4(b)).…”

“…HEp-2 (Human epithelial type 2), A549 (human alveolar type II-like epithelial cells) and 293 (human embryonic kidney epithelial cells) cells were from ATCC, Manassas, VA, and maintained as described previously [13,39,40]. RSV A2 strain was grown in HEp-2 cells and purified by sucrosegradient as we previously described [10,13,40].…”

“…There is increasing evidence that some tRFs are not by-products of random degradation, but functional molecules [5,9,10,12]. We recently found that RSV specifically led to an abundant induction of tRFs [10,13]. We believe these tRFs are functional based on: (1) not all tRNAs being cleaved by RSV infection; (2) the cleavage is virus-specific, as a close member of RSV called human metapneumovirus does not induce tRFs; (3) the cleavage seems dependent on a specific RNase angiogenin (ANG); and (4) the cleavage happens right at the anticodon region of tRNAs.…”