Functional analyses of mammalian virus 5'UTR-derived, small RNAs that regulate virus translation

Abstract

Enterovirus A71 (EV-A71¹) RNA contains an internal ribosomal entry site (IRES) to direct cap-independent translation. IRES-dependent translation requires the host's translation initiation factors and IRES-associated trans-acting factors (ITAFs). We previously showed that hnRNP A1, the mRNA stability factor HuR, and the RISC subunit Argonaute 2 (Ago2) are ITAFs that associate with stem loop II (SL-II) of the IRES and promote IRES-dependent translation. By contrast, the mRNA decay factor AUF1 is a negative-acting ITAF that also binds SL-II. Moreover, the small RNA-processing enzyme Dicer produces at least four virus-derived, small RNAs (vsRNAs 1-4) from the EV-A71 5'UTR in infected cells. One of these, vsRNA1, derived from SL-II, inhibits IRES activity via an unknown mechanism. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with SL-II. This presents a possible mechanism by which vsRNA1 could control association of ITAFs with the IRES and modulate viral translation. Here, we describe methods for functional analyses of vsRNA1-mediated regulation of IRES activity. These methods should be applicable to other virus-derived, small RNAs as well.

Keywords: Enterovirus A71; IRES trans-acting factor (ITAF); Internal ribosome entry site (IRES); Virus-derived small RNA.

Fig. 1.

The diagram depicts the bicistronic reporter plasmids used to synthesize RNA for transfections and dual-luciferase reporter assays. The expression of firefly luciferase is under the control of the EV-A71 5′UTR in the sense orientation (upper diagram). The EV-A71 5′UTR inserted in the antisense orientation (AS; lower diagram) serves as a negative control, bicistronic RNA.

Fig. 2.

Schematic diagrams/workflows of protein-IRES RNA interaction assays. (A) Streptavidin pull-down of protein-biotinylated RNA complexes. The biotinylated RNA in this example is the EV-A71 5′UTR. RNA not labeled with biotin serves as a negative control for the assay. The red box highlights SL-II from the IRES. For simplicity, proteins are shown only bound to SL-II. For the Western blots, the presence or absence of biotin in the RNAs are indicated by the plus and minus signs, respectively. The absence of a protein signal in the ‘minus’ lanes indicates that cellular proteins did not bind non-specifically to the paramagnetic particles, i.e., the detected proteins were purified via their association with RNA. (B) Immunoprecipitation of native RNP complexes from cell lysates. Native RNP complexes are incubated with an antibody directed against an RNA-binding protein of interest; non-immune antibody serves as a negative control. Dynabeads coupled to protein A permit magnetic purification of RNP–antibody–protein A-Dynabead complexes. Beads are washed and RNA is eluted and purified. Specific target RNAs associated with the protein of interest are detected by Northern blot or qRT-PCR.

Fig. 3.

Effects of vsRNA1 on the association of AUF1, Ago2, HuR, and hnRNP A1/A2 with SL-II. The SF268 lysate and biotin-labeled SL-II were prepared as described in Sections 2.3.1.1 and 2.2.2, respectively. The pull-down reactions were set up with 200 μg of cell lysate and 12.5 pmol of biotin-SL-II RNA as described in Section 2.3.1.1. To examine the effects of vsRNA1 on binding of AUF1, Ago2, HuR, and hnRNP A1/A2 to biotin-labeled SL-II RNA, the indicated pmoles of synthetic mimic vsRNA1 or synthetic scrambled RNA were added to reaction mixtures. Proteins eluted from the streptavidin pull-down were analyzed by Western blot using antibodies to the indicated proteins. This figure is reprinted from Ref. [12].