Functional Insights into the Adjacent Stem-Loop in Honey Bee Dicistroviruses That Promotes Internal Ribosome Entry Site-Mediated Translation and Viral Infection

Abstract

All viruses must successfully harness the host translational apparatus and divert it towards viral protein synthesis. Dicistroviruses use an unusual internal ribosome entry site (IRES) mechanism whereby the IRES adopts a three-pseudoknot structure that accesses the ribosome tRNA binding sites to directly recruit the ribosome and initiate translation from a non-AUG start site. A subset of dicistroviruses, including the honey bee Israeli acute paralysis virus (IAPV), encode an extra stem-loop (SLVI) 5' -adjacent to the IGR IRES. Previously, the function of this additional stem-loop is unknown. Here, we provide mechanistic and functional insights into the role of SLVI in IGR IRES translation and in virus infection. Biochemical analyses of a series of mutant IRESs demonstrated that SLVI does not function in ribosome recruitment but is required for proper ribosome positioning on the IRES to direct translation. Using a chimeric infectious clone derived from the related Cricket paralysis virus, we showed that the integrity of SLVI is important for optimal viral translation and viral yield. Based on structural models of ribosome-IGR IRES complexes, the SLVI is predicted to be in the vicinity of the ribosome E site. We propose that SLVI of IAPV IGR IRES functionally mimics interactions of an E-site tRNA with the ribosome to direct positioning of the tRNA-like domain of the IRES in the A site.IMPORTANCEViral internal ribosome entry sites are RNA elements and structures that allow some positive-sense monopartite RNA viruses to hijack the host ribosome to start viral protein synthesis. We demonstrate that a unique stem-loop structure is essential for optimal viral protein synthesis and for virus infection. Biochemical evidence shows that this viral stem-loop RNA structure impacts a fundamental property of the ribosome to start protein synthesis.

FIG 1

Secondary structure of Israeli acute paralysis virus (IAPV) and cognate stem-loop VI (SLVI) mutants. (A) The Dicistroviridae genome is comprised of a positive-sense, single-stranded RNA molecule that bears a 5′ genome-linked viral protein (VPg) and a 3′ poly(A) tail. The genome consists of two open reading frames (ORFs): the upstream cistron encodes viral nonstructural proteins, and the downstream cistron encodes viral capsid proteins. ORF1 and -2 expression is translationally and temporally regulated by the 5′ IRES and the IGR IRES, respectively. The IGR IRES adopts a triple-pseudoknot structure consisting of three pseudoknots (PKI, -II, and -III) which fold independently into two domains. PKII and -III together form the ribosome binding domain, while PKI forms the tRNA mimicry domain to establish the translational reading frame. SLVI located 5′ adjacent to the IGR IRES has been identified in a subset of dicistroviruses, including IAPV, ABPV, and KBV. (B) Structure of the related cricket paralysis virus IGR IRES bound to the 80S ribosome of Kluyveromyces lactis. The 40S and 60S ribosomal subunits are depicted in yellow and blue, respectively. SLVI of the honey bee dicistroviruses is predicted to be in the vicinity of the E site, near L1.1 of the IRES (shaded in gray). (Modified from reference under a Creative Commons CC BY 3.0 license [https://creativecommons.org/licenses/by/3.0/].) (C) 5′, 3′, and compensatory mutations of SLVI. The mutated nucleotides are depicted in white. For IAPV, the ORF1 stop codon is encompassed in the apical loop of SLVI (bold and red).

FIG 2

Dimethyl sulfate (DMS) structural probing analysis of SLVI mutants. (A) DMS modification profiles of wild-type and mutant IRESs in solution. Primer extension analysis was performed using PrHA40 to assess changes in the global conformation of the IGR IRES. The locations of the major structural elements are indicated to the left of the gel. (B) To identify structural changes within SLVI, primer extension was performed using PrHA190. Normalized DMS reactivities are shown as a function of the nucleotide position. Residues that exhibit high DMS reactivity (>0.5) are in red on the corresponding IRES secondary structures.

FIG 3

Binding affinities of wild-type and mutant IRESs. The dissociation constants of the wild-type and mutant IRESs were determined by filter binding. Radiolabeled IRES RNAs were incubated with increasing amounts of purified, salt-washed 40S and 60S ribosomal subunits and subsequently applied to a double membrane of nitrocellulose and nylon using a Bio-Dot filtration apparatus. The membranes were dried and subjected to autoradiography.

FIG 4

Disruption of SLVI formation impairs ribosome positioning on the IAPV IRES. (A) Wild-type (WT) or SLVI mutant IRES RNAs were incubated in the absence (−) or presence (+) of purified, salt-washed HeLa 80S ribosomes and analyzed by primer extension and denaturing polyacrylamide gel electrophoresis. The sequence of the wild-type IRES is shown on the left, with the position of the CCU triplet that occupies the A site as indicated (+1 indicates the first C). The positioning toeprint is observed at A6628, 14 nucleotides downstream. (B) Schematic of the IRES PKI domain, with the positioning toeprint as indicated. (C) Quantitation of the positioning toeprint observed for wild-type and mutant IRESs, calculated as a fraction of the total lane intensity and normalized to the wild-type IRES. Shown are the average toeprint intensities ± SDs from at least three independent experiments.

FIG 5

The chimeric IAPV/CrPV virus is infectious in Drosophila S2 cells. (A) Schematic of the chimeric virus derived from the full-length CrPV infectious clone by replacement of the CrPV IGR IRES with that of IAPV. −SLVI, chimera lacking SLVI; +SLVI^fused, SLVI fused in frame with ORF1 such that the stop codon (UAA) is encompassed in the apical loop; +SLVI, SLVI resides in the intercistronic region, downstream of the ORF1 stop codon. (B) In vitro-transcribed RNAs derived from the wild-type CrPV infectious clone (CrPV-3) or the chimera lacking SLVI (−SLVI) were incubated in Sf21 extract for 2 h at 30°C in the presence of [³⁵S]methionine-cysteine. A control without RNA was included to monitor background expression. Reactions were resolved by SDS-PAGE and visualized by autoradiography. A stop codon was introduced into ORF2 of each respective clone to demonstrate specificity of viral structural protein synthesis. The identities of the mature viral proteins, annotated based on predicted molecular mass, are indicated. (C) In vitro-transcribed RNAs from pCrPV-3, chimera −SLVI, or their corresponding ORF2 stop mutants were transfected into Drosophila S2 cells. At 48 h.p.t., cells were harvested and the expression of the VP2 viral capsid protein was monitored by Western blotting. (D) Viral titers were measured at 48 h after transfection of CrPV-3 and chimera −SLV RNAs, as described in Materials and Methods. Shown are the average values from three independent experiments, ± 1 SD. (E) In vitro translation profiles of chimeras +SLVI^fused and +SLVI in Sf21 extract. (F) The expression of the viral RNA-dependent RNA polymerase (RdRp) and capsid protein (VP2) was monitored by Western blotting at 48 h after transfection of chimera −SLVI, +SLVI^fused, and +SLVI RNAs. (G) Viral titers were measured at 48 h.p.t. for chimera −SLVI, +SLVI^fused, and +SLVI RNAs, as described in Materials and Methods. Shown are the average values from three independent experiments ± SDs.

FIG 6

Disruption of SLVI formation decreases viral yield. (A) In vitro-transcribed RNAs derived from chimera +SLVI or its cognate mutants were incubated in Sf21 extract for 2 h at 30°C in the presence of [³⁵S]methionine-cysteine. Reactions were resolved by SDS-PAGE and visualized by autoradiography. The respective mutations are as indicated in Fig. 1C. The ratio of ORF2 to ORF1 expression was determined by quantification of the bands indicated by asterisks. (B) Drosophila S2 cells were infected with chimera +SLV or its cognate mutants at an MOI of 10. [³⁵S]methionine-cysteine was added 30 min prior to the indicated time points (2, 4, and 6 h.p.i.) to metabolically label newly synthesized proteins. Labeled proteins were resolved by SDS-PAGE and subjected to phosphorimager analysis. The expression of viral RdRp and VP2 was monitored by Western blotting. (C) The accumulation of viral RNA was visualized by Northern blotting at 2, 4, and 6 h after infection with chimera +SLVI and mutant M2 (MOI = 10). (D) Viral titers were measured 6 h after infection with chimera +SLVI and mutant M2 at MOIs of 1 and 10, as described in Materials and Methods.