Structural elements in the internal ribosome entry site of Plautia stali intestine virus responsible for binding with ribosomes

Abstract

Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) at the intergenic region of the genome. The PSIV IRES initiates translation with glutamine rather than the universal methionine. To analyze the mechanism of IRES-mediated initiation, binding of IRES RNA to salt-washed ribosomes in the absence of translation factors was studied. Among the three pseudoknots (PKs I, II and III) within the IRES, PK III was the most important for ribosome binding. Chemical footprint analyses showed that the loop parts of the two stem-loop structures in Domain 2, which are highly conserved in related viruses, are protected by 40S but not by 60S ribosomes. Because PK III is close to the two loops, these structural elements were considered to be important for binding of the 40S subunit. Competitive binding analyses showed that the IRES RNA does not bind poly(U)-programmed ribosomes preincubated with tRNA(Phe) or its anticodon stem- loop (ASL) fragment. However, Domain 3-deleted IRES bound to programmed ribosomes preincubated with the ASL, suggesting that Domains 1 and 2 have roles in IRES binding to 40S subunits and that Domain 3 is located at the ribosome decoding site.

Figure 1

Effect of base pairing in IRES PKs on 80S ribosome binding. (A) Secondary structure model of PSIV IRES RNA. Asterisks and dots indicate base pairing in PKs and helical stems, respectively. Highly conserved nucleotides in CrP-like viruses are capitalized. Half-tones denote nucleotides mutated in these studies to produce the cognate IRES (see Materials and Methods). Nucleotides mutated in PKs, to disrupt base pairing and to restore base pairing by compensatory mutations, are in red. (B) Binding of ³²P-labeled IRES RNAs to 80S ribosomes. The fraction of IRES RNA bound is the ratio of [³²P]RNA retained on the filter to that of the input [³²P]RNA. (C) Effect of the compensatory mutations restoring base pairing in PK III on ribosome binding.

Figure 2

DMS and CMCT probing of the PSIV IRES. To detect modified bases, DMS- and CMCT-modified RNAs were analyzed by primer extension. (A) Modification with DMS (lanes 2 and 3) or CMCT (lanes 5 and 6) in the presence (lanes 2 and 5) or absence (lanes 3 and 6) of 40S ribosomes. Lanes 1 and 4 are the unmodified controls to detect non-specific termination. Bold bars mark IRES nucleotides protected from modification by 40S ribosomes. Nucleotide positions are shown at right. (B) Modification with DMS (lanes 2, 3, 5 and 6) or CMCT (lanes 8, 9, 11 and 12) in the presence of 60S (lanes 2 and 8) or 40S and 60S (lanes 5 and 11) ribosomes. Lanes 1, 4, 7 and 10 are the unmodified controls. The arrowhead marks A₆₀₁₄, which was only protected in the presence of both 40S and 60S ribosomes.

Figure 3

Hydroxyl radical probing of PSIV IRES RNA. The RNA was exposed to hydroxyl radicals in the presence (lane 2) or absence (lane 3) of 40S ribosomes. Lane 1 is the unmodified control, but 40S ribosomes were added to the sample before phenol–chloroform extraction to detect non- specific terminations caused by 18S rRNA as well as the sample in lane 3. Bold bars denote locations of protected residues and narrow bars denote sites of weak protection. Asterisks mark sites of non-specific terminations, as listed in the text. Nucleotide positions of the PSIV sequence are shown at left.

Figure 4

Summary of results of chemical probing and structural similarity analyses of the CrP-like virus IRESs. (A) Secondary structure model of the PSIV IRES showing the results of chemical probe studies. Filled circles and squares mark nucleotides modified by DMS and CMCT, respectively, and red symbols mark nucleotides protected from these modifications in the presence of 40S ribosomes. The sizes of these symbols denote band intensity in primer extension assays. The deep yellow shaded nucleotides mark sites protected from hydroxyl radicals in the presence of 40S ribosomes, and pale yellow shaded nucleotides mark sites with weak protection. Refined structures of some regions based on the results of chemical probing described in the text are shown in purple. (B) Manually aligned IGR sequences of CrP-like viruses. Accession numbers: PSIV, AB006531; HiPV (Himetobi P virus), AB017037; DCV (Drosophila C virus), AF014388; CrPV, AF218039; TrV (Triatoma virus), AF178440; BQCV (Black queen cell virus), AF183905; RhPV (Rhopalosiphum padi virus), AF022937; ALPV (Aphid lethal paralysis virus), AF536531; TSV (Taura syndrome virus), AF277675; ABPV (Acute bee paralysis virus), AF150629. Red, blue, green and pink letters denote nucleotides in stems in Domains 1, 2b, 2a and 3, respectively. Purple letters denote an additional putative stem in Domain 3 of TSV and ABPV. PK I, PK II, PK III, Domain 2a loop and Domain 2b loop nucleotides are in half-tone lines. Conserved short nucleotide sequences among these viruses are in boxes. Nucleotide positions in viral genomes are shown at right in parentheses.

Figure 5

Sedimentation profiles of Domain 2 RNA and ribosomal subunits. ³³P-labeled Domain 2 RNA was incubated with purified (A) 40S or (B) 60S ribosomes and centrifuged in sucrose gradients. Fractions were collected and the radioactivity in each fraction was measured. Black and white arrowheads indicate positions of 40S and 60S subunits, respectively, which were monitored by absorbance at 254 nm.

Figure 6

Filter binding analyses of complete IRES RNA (Domains 1–2–3) and truncated IRES RNA (Domains 1–2) binding to programmed 80S ribosomes. (A) Effects of poly(U) and/or tRNA^Phe programming on RNA binding. (B) Effects of poly(U) and/or 17mer tRNA^Phe ASL programming on RNA binding. ³²P-labeled IRES RNAs were incubated with 80S ribosomes that had been preincubated with poly(U), tRNA^Phe and/or ASL. Binding for samples containing 1 pmol RNA and 4 pmol ribosomes [without poly(U), tRNA^Phe or ASL] was taken as 100% binding. Results are the average of three independent experiments. Bars represent standard errors.