Down-regulation of a host microRNA by a Herpesvirus saimiri noncoding RNA

Abstract

T cells transformed by Herpesvirus saimiri express seven viral U-rich noncoding RNAs of unknown function called HSURs. We noted that conserved sequences in HSURs 1 and 2 constitute potential binding sites for three host-cell microRNAs (miRNAs). Coimmunoprecipitation experiments confirmed that HSURs 1 and 2 interact with the predicted miRNAs in virally transformed T cells. The abundance of one of these miRNAs, miR-27, is dramatically lowered in transformed cells, with consequent effects on the expression of miR-27 target genes. Transient knockdown and ectopic expression of HSUR 1 demonstrate that it directs degradation of mature miR-27 in a sequence-specific and binding-dependent manner. This viral strategy illustrates use of a ncRNA to manipulate host-cell gene expression via the miRNA pathway.

Fig. 1

HSURs 1 and 2 bind host miRNAs in virally transformed T cells. (A) Sequences and predicted secondary structures of HSURs 1 and 2. Bold nucleotides are perfectly conserved in all available genome sequences from independent isolates of HVS A, B, and C strains and also in H. ateles (figs. S1 and S2). Complementarity between HSURs and miRNAs is represented by dots; miRNA seed regions are in yellow. (B) Coimmunoprecipitation of HSURs from extracts of virally transformed marmoset T cells with antibody to Flag (lane 3) or antibody to Ago2 (lane 5). I, input (5%); S, supernatant (5%); P, pellet (100%). (C) Coimmunoprecipitation of miRNAs from extracts of virally transformed marmoset T cell lines expressing (Wt, lanes 1 to 5) or lacking HSURs 1 and 2 (Mut, lanes 6 to 10) with Y12 antibody (αSm, lanes 4, 5 and 9, 10) or nonimmune serum (C, lanes 2, 3 and 7, 8). I, input (2%); S, supernatant (2%); P, pellet (100%). Northern blots in (B) and (C) were probed for HSURs, miRNAs, or U4atac, as an αSm immunoprecipitation control.

Fig. 2

The presence of HSURs 1 and 2 affects miR-27a abundance, decay, and target expression. (A) Relative levels of different mature miRNAs in virally transformed marmoset T cells expressing (Wt) or lacking (Mut) HSURs 1 and 2 were determined by means of quantitative real-time PCR. (B) Pulse-chase assay assessing the decay of radioactively labeled synthetic miR-27a and miR-16. (C) Western blot analysis of FOXO1 in marmoset T cells transformed by HVS expressing (Wt) or lacking (Mut) HSURs 1 and 2.

Fig. 3

HSUR 1 regulates the abundance of miR-27 in virally transformed T cells. (A) Northern blot analyses of miRNAs and HSURs after nucleofection with chimeric oligonucleotides anti-sense to GFP (lane 1), HSUR1 (lane 2), or HSUR 2 (lane 3). (B) Quantification of miRNAs from three independent experiments performed as in (A). (C) Western blot of FOXO1 in HVS-transformed marmoset T cells nucleofected as described in (A).

Fig. 4

HSUR 1 down-regulates mature miRNAs in a sequence-specific and binding-dependent manner. (A) Partial sequences of HSUR 1 (positions 40 to 62) and its mutants (in red) H1Mt and H1m20. Bold nucleotides are perfectly conserved (Fig. 1A). (B) Coimmunoprecipitation of miRNAs with αSm, as in Fig. 1C, from extracts of Jurkat T cells stably expressing HSURs 2 to 7 and either wild-type HSUR 1 (Wt, lanes 1 to 5), no HSUR 1 (ΔH1, lanes 6 to 10), mutant HSUR 1 H1Mt (lanes 11 to 15), or mutant HSUR 1 H1m20 (lanes 16 to 20). (C) miRNA levels in Jurkat T cells fluorescence-activated cell sorted for GFP after transient transfection with empty vector (GFP) or with plasmids expressing GFP and the following: HSUR 3 (GFP-H3), Wt HSUR 1 (GFP-H1), H1Mt (GFP-H1Mt), H1m20 (GFP-H1m20), or H1M1 (GFP-H1M1).