Complete genome sequences of two iflaviruses from the brown planthopper, Nilaparvata lugens.

The complete genome sequences of two new iflaviruses (genus Iflavirus, family Iflaviridae) were determined. These viral sequences were first identified in RNA-seq contig sequences of Nilaparvata lugens in two distinct colonies: Izumo and Kagoshima. The accuracy of the contig sequences of the two viruses was verified by restriction enzyme digestion of RT-PCR products from viruliferous insects. RT-PCR of RNA extracted from honeydews after viruliferous insect feeding detected the expected viral products, which suggested that viruses were excreted into the honeydews by the insects. Since we previously designated a similar iflavirus as "Nilaparvata lugens honeydew virus 1", the two new viruses have been tentatively named "Nilaparvata lugens honeydew virus 2" and "Nilaparvata lugens honeydew virus 3". The identity of the putative amino acid sequences of the capsid proteins of these viruses met the criterion for iflavirus species demarcation. Therefore, these two viruses are suggested to be members of distinct species in the genus Iflavirus.

The genome sequence and transmission of an iflavirus from the brown planthopper, Nilaparvata lugens.

A previously unknown iflavirus has been identified in a laboratory colony of the brown planthopper, Nilaparvata lugens. The iflavirus-like sequence was first identified in contig sequences obtained from transcriptome sequencing (RNA-seq) of the brown planthopper. The complete viral genome was resequenced using the Sanger method. The positive-strand RNA genome was 10,937 nucleotides excluding the 3' poly(A) tail, and contained a single large open reading frame encoding coat proteins in the 5' region and replicases in the 3' region. Conserved motifs for coat proteins, helicase, cysteine protease, and RNA-dependent RNA polymerase were identified in the deduced amino sequence, and the estimated molecular mass of the large polyprotein was 358.6kDa. RT-PCR detection of the viral genome indicated that viral shedding occurred through the honeydews of insects in the infected colony. To test transmission, the collected honeydews were used to feed insects in a non-viruliferous colony. After 7 days, the expected RT-PCR fragment was detected in the insects, indicating that the virus can be transmitted horizontally. This is the first iflavirus identified in planthoppers; thus, we propose the name of the virus as Nilaparvata lugens honeydew virus-1.

Structural basis for the binding of IRES RNAs to the head of the ribosomal 40S subunit.

Some viruses exploit internal initiation for their propagation in the host cell. This type of initiation is facilitated by structured elements (internal ribosome entry site, IRES) upstream of the initiator AUG and requires only a reduced number of canonical initiation factors. An important example are IRES of the virus family Dicistroviridae that bind to the inter-subunit side of the small ribosomal 40S subunit and lead to the formation of elongation-competent 80S ribosomes without the help of any initiation factor. Here, we present a comprehensive functional and structural analysis of eukaryotic-specific ribosomal protein rpS25 in the context of this type of initiation and propose a structural model explaining the essential involvement of rpS25 for hijacking the ribosome.

The genome sequence of pepper vein yellows virus (family Luteoviridae, genus Polerovirus).

The complete genome of pepper vein yellows virus (PeVYV) was sequenced using random amplification of RNA samples isolated from vector insects (Aphis gossypii) that had been given access to PeVYV-infected plants. The PeVYV genome consisted of 6244 nucleotides and had a genomic organization characteristic of members of the genus Polerovirus. PeVYV had highest amino acid sequence identities in ORF0 to ORF3 (75.9 - 91.9%) with tobacco vein distorting polerovirus, with which it was only 25.1% identical in ORF5. These sequence comparisons and previously studied biological properties indicate that PeVYV is a distinctly different virus and belongs to a new species of the genus Polerovirus.

Sugar transporter genes of the brown planthopper, Nilaparvata lugens: A facilitated glucose/fructose transporter.

The brown planthopper (BPH), Nilaparvata lugens, attacks rice plants and feeds on their phloem sap, which contains large amounts of sugars. The main sugar component of phloem sap is sucrose, a disaccharide composed of glucose and fructose. Sugars appear to be incorporated into the planthopper body by sugar transporters in the midgut. A total of 93 expressed sequence tags (ESTs) for putative sugar transporters were obtained from a BPH EST database, and 18 putative sugar transporter genes (Nlst1-18) were identified. The most abundantly expressed of these genes was Nlst1. This gene has previously been identified in the BPH as the glucose transporter gene NlHT1, which belongs to the major facilitator superfamily. Nlst1, 4, 6, 9, 12, 16, and 18 were highly expressed in the midgut, and Nlst2, 7, 8, 10, 15, 17, and 18 were highly expressed during the embryonic stages. Functional analyses were performed using Xenopus oocytes expressing NlST1 or 6. This showed that NlST6 is a facilitative glucose/fructose transporter that mediates sugar uptake from rice phloem sap in the BPH midgut in a manner similar to NlST1.

Identification of the 3C-protease-mediated 2A/2B and 2B/2C cleavage sites in the nonstructural polyprotein precursor of a Dicistrovirus lacking the NPGP motif.

Dicistroviruses have motifs for picornavirus 2C, 3C, and 3D proteins in their nonstructural polyprotein C-terminal region. The proteins from the nonstructural, N-terminal region of the polyprotein remain to be characterized. We have identified 3C-mediated cleavage sites in the N-terminal region of the nonstructural polyprotein of the dicistrovirus Plautia stali intestine virus (PSIV). The 2B/2C cleavage site mapped to amino acids (aa) 408-409 (QD). 2B/2C cleavage sites were suggested to be conserved in dicistroviruses. The most N-terminal PSIV cleavage site was aa 286-287 (QS). Including previous results, the polyprotein contains nine proteins arranged as follows: 2A, 2B, 2C, 3A, 3B1, 3B2, 3B3, 3C, and 3D.

Functional analysis of structural motifs in dicistroviruses.

The family Dicistroviridae is composed of positive-stranded RNA viruses which have monopartite genomes. These viruses carry genome-linked virus proteins (VPg) and poly (A) tails. The 5' untranslated region (UTR) is approximately 500 nucleotides and contains an internal ribosome entry site (IRES). These features resemble those of vertebrate picornaviruses, but dicistroviruses have other distinct characteristics. Picornaviruses have a single large open reading frame (ORF) encoding the capsid proteins at the 5'-end and the replicases at the 3'-end. In contrast, dicistroviruses have two nonoverlapping ORFs. The 5'-proximal ORF encodes the replicases and the 3'-proximal ORF encodes the capsid proteins. Usually, positive-stranded viruses which have capsid protein genes in the 3' part of the genome produce subgenomic RNA for synthesis of the capsid proteins, because abundant quantities of the capsid proteins are required for the viral replication cycle. In dicistroviruses, translation of the capsid proteins is controlled by an additional IRES. This IRES is located in the intergenic region (IGR) between the replicase and capsid coding regions, and mediates the initiation of translation for the capsid proteins. The IGR-IRES has a multiple stem-loop structure containing three pseudoknots. We describe the characteristics of dicistroviruses, including the RNA elements and viral proteins.

Cleavage sites of the "P3 region" in the nonstructural polyprotein precursor of a dicistrovirus.

Plautia stali intestine virus (PSIV) is a member of the family Dicistroviridae. Dicistroviruses, like picornaviruses, are thought to encode a 3C-like protease; however, the cleavage sites of dicistroviral nonstructural polyprotein precursors are unknown except for those in genome-linked virus protein (VPg) regions. We expressed part of the PSIV polyprotein in Escherichia coli and detected autoprocessed viral proteins. N-terminal sequence analysis of the autoprocessed proteins showed that Q/GWLSW and Q/NGVFD in the PSIV sequence correspond to 2C/3A and 3C/3D cleavage sites in picornaviruses. Alignments of deduced amino acid sequences of the "P3 region" suggest that these cleavage sites can be predicted.

Picornavirales, a proposed order of positive-sense single-stranded RNA viruses with a pseudo-T = 3 virion architecture.

Despite the apparent natural grouping of "picorna-like" viruses, the taxonomical significance of this putative "supergroup" was never addressed adequately. We recently proposed to the ICTV that an order should be created and named Picornavirales, to include viruses infecting eukaryotes that share similar properties: (i) a positive-sense RNA genome, usually with a 5'-bound VPg and 3'-polyadenylated, (ii) genome translation into autoproteolytically processed polyprotein(s), (iii) capsid proteins organized in a module containing three related jelly-roll domains which form small icosahedral, non-enveloped particles with a pseudo-T = 3 symmetry, and (iv) a three-domain module containing a superfamily III helicase, a (cysteine) proteinase with a chymotrypsin-like fold and an RNA-dependent RNA polymerase. According to the above criteria, the order Picornavirales includes the families Picornaviridae, Comoviridae, Dicistroviridae, Marnaviridae, Sequiviridae and the unassigned genera Cheravirus, Iflavirus and Sadwavirus. Other taxa of "picorna-like" viruses, e.g. Potyviridae, Caliciviridae, Hypoviridae, do not conform to several of the above criteria and are more remotely related: therefore they are not being proposed as members of the new order. Newly described viruses, not yet assigned to an existing taxon by ICTV, may belong to the proposed order.

Eukaryotic ribosomal protein RPS25 interacts with the conserved loop region in a dicistroviral intergenic internal ribosome entry site.

The intergenic region-internal ribosome entry site (IGR-IRES) of dicistroviruses binds to 40S ribosomal subunits in the absence of eukaryotic initiation factors (eIFs). Although the conserved loop sequences in dicistroviral IGR-IRES elements are protected from chemical modifications in the presence of the 40S subunit, molecular components in the 40S subunit, which interacts with the loop sequences in the IRES, have not been identified. Here, a chemical crosslinking study using 4-thiouridine-labeled IGR-IRES revealed interactions of the IGR-IRES with several 40S proteins but not with the 18S rRNA. The strongest crosslinking signal was identified for ribosomal protein S25 (rpS25). rpS25 is known to be a neighbor of rpS5, which has been shown to interact with a related IGR-IRES by cryo-electron microscopy. Crosslinking analysis with site-directed mutants showed that nucleotides UU(6089-6090), which are located in the loop region in conserved domain 2b in the IRES, appear to interact with rpS25. rpS25 is specific to eukaryotes, which explains why there is no recognition of the IGR-IRES by prokaryotic ribosomes. Although the idea that the IGR-IRES element may be a relict of a primitive translation system has been postulated, our experimental data suggest that the IRES has adapted to eukaryotic ribosomal proteins.

Binding mode of the first aminoacyl-tRNA in translation initiation mediated by Plautia stali intestine virus internal ribosome entry site.

Eukaryotic ribosomes directly bind to the intergenic region-internal ribosome entry site (IGR-IRES) of Plautia stali intestine virus (PSIV) and initiate translation without either initiation factors or initiator Met-tRNA. We have investigated the mode of binding of the first aminoacyl-tRNA in translation initiation mediated by the IGR-IRES. Binding ability of aminoacyl-tRNA to the first codon within the IGR-IRES/80 S ribosome complex was very low in the presence of eukaryotic elongation factor 1A (eEF1A) alone but markedly enhanced by the translocase eEF2. Moreover, eEF2-dependent GTPase activity of the IRES/80 S ribosome complex was 3-fold higher than that of the free 80 S ribosome. This activation was suppressed by addition of the antibiotics pactamycin and hygromycin B, which are inhibitors of translocation. The results suggest that translocation by the action of eEF2 is essential for stable tRNA binding to the first codon of the PSIV-IRES in the ribosome. Chemical probing analysis showed that IRES binding causes a conformational change in helix 18 of 18 S rRNA at the A site such that IRES destabilizes the conserved pseudoknot within the helix. This conformational change caused by the PSIV-IRES may be responsible for the activation of eEF2 action and stimulation of the first tRNA binding to the P site without initiation factors.

Characterization of the 5' internal ribosome entry site of Plautia stali intestine virus.

The RNA genome of Plautia stali intestine virus (PSIV; Cripavirus, Dicistroviridae) contains two open reading frames, the first of which is preceded by a 570 nt untranslated region (5' UTR). The 5' UTR was confirmed to be an internal ribosome entry site (IRES) using an insect cell lysate translation system: translation of a second cistron increased 14-fold in the presence of the 5' UTR and a cap analogue did not inhibit translation of the second cistron. Deletion analysis showed that 349 bases corresponding to nt 225-573 in the PSIV genome were necessary for internal initiation. The PSIV 5' IRES did not function in rabbit reticulocyte lysate or wheatgerm translation systems; however, the intergenic IRES for capsid translation of PSIV was functional in both systems, indicating that the 5' IRES and the intergenic IRES have distinct requirements for their activities. Chemical and enzymic analyses of the 5' IRES of PSIV indicate that its structure is distinct from that of Rhopalosiphum padi virus. Because 5' IRES elements in some dicistroviruses have been reported to be active in plant and mammalian cell-free translation systems, there appears to be variation among dicistroviruses in the mechanism of translation initiation mediated by 5' IRES elements.

Multiple coding sequences for the genome-linked virus protein (VPg) in dicistroviruses.

N-terminal Edman sequencing of the genome-linked viral protein (VPg) of Plautia stali intestine virus (PSIV, Dicistroviridae) detected heterologus residues. The VPg sequence determined was found to be triplicated in the nonstructural protein precursor. Multiple VPg-like sequences were also found in 10 of the 12 dicistroviruses with a maximum of six copies in Solenopsis invicta virus-1. We postulate that redundant VPg coding sequences facilitate multiplication of dicistroviruses, because fewer cycle of translation of the nonstructural protein precursor produces larger amounts of VPg proteins in parallel with the increased production of capsid proteins by the intergenic internal ribosome entry site mediated translation.

Characterization of a novel satellite virus and a strain of Himetobi P virus (Dicistroviridae) from the brown planthopper, Nilaparvata lugens.

Two distinct spherical virus-like particles were purified from the brown planthopper, Nilaparvata lugens. One was a geographical isolate of Himetobi P virus (Cripavirus, Dicistroviridae). The other was 30 nm in diameter and contained positive-stranded RNA. The RNA was 1647 nucleotides in length and encoded only its own capsid protein, indicating that this particle is a satellite virus. Transmission tests showed that the satellite was transmitted vertically; however, its helper virus was unknown. We named this satellite Nilaparvata lugens commensal X virus (NLCXV).

Structural variant of the intergenic internal ribosome entry site elements in dicistroviruses and computational search for their counterparts.

The intergenic region (IGR) located upstream of the capsid protein gene in dicistroviruses contains an internal ribosome entry site (IRES). Translation initiation mediated by the IRES does not require initiator methionine tRNA. Comparison of the IGRs among dicistroviruses suggested that Taura syndrome virus (TSV) and acute bee paralysis virus have an extra side stem loop in the predicted IRES. We examined whether the side stem is responsible for translation activity mediated by the IGR using constructs with compensatory mutations. In vitro translation analysis showed that TSV has an IGR-IRES that is structurally distinct from those previously described. Because IGR-IRES elements determine the translation initiation site by virtue of their own tertiary structure formation, the discovery of this initiation mechanism suggests the possibility that eukaryotic mRNAs might have more extensive coding regions than previously predicted. To test this hypothesis, we searched full-length cDNA databases and whole genome sequences of eukaryotes using the pattern matching program, Scan For Matches, with parameters that can extract sequences containing secondary structure elements resembling those of IGR-IRES. Our search yielded several sequences, but their predicted secondary structures were suggested to be unstable in comparison to those of dicistroviruses. These results suggest that RNAs structurally similar to dicistroviruses are not common. If some eukaryotic mRNAs are translated independently of an initiator methionine tRNA, their structures are likely to be significantly distinct from those of dicistroviruses.

Cell-free synthesis of polypeptides lacking an amino-terminal methionine by using a dicistroviral intergenic internal ribosome entry site.

An amino-terminal methionine corresponding to a recombinant AUG initiation codon sometimes affects the functions of proteins. To test the performance of translation mediated by a dicistroviral internal ribosome entry site (IRES), which initiates protein synthesis with elongator tRNAs, we optimized the conditions for cell-free translation. Although the IRES is 188 nucleotides long, a further 50 nucleotides of the upstream sequence stabilized translation efficiency. Optimal ion concentrations were affected by the sequences of the constructs. In a wheat-germ system, IRES-mediated translation produced 78 microg/ml of firefly luciferase from the AUG-deleted sequence, suggesting that dicistroviral IRESs will be able to yield polypeptides with a specific N-terminal amino acid other than methionine.

Conditional rather than absolute requirements of the capsid coding sequence for initiation of methionine-independent translation in Plautia stali intestine virus.

The positive-stranded RNA genome of Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) in an intergenic region (IGR). The IGR-IRES of PSIV initiates translation of the capsid protein by using CAA, the codon for glutamine. It was previously reported (J. Sasaki and N. Nakashima, J. Virol. 73:1219-1226, 1999) that IGR-IRES extended by several nucleotides into the capsid open reading frame (ORF). Despite the fact that the secondary structure model of the IGR-IRES is highly conserved, we were unable to find structural similarities in the 5' region of the capsid ORFs in related viruses. Therefore, we reevaluated the role of the capsid ORF in IGR-IRES-mediated translation in PSIV. Mutation of the CAA codon with various triplets did not inhibit IGR-IRES-mediated translation. N-terminal amino acid analyses of mutated products showed that the IGR-IRES could initiate translation by using various elongator tRNAs. By replacement of the capsid ORF with exogenous coding sequences having AUG deleted, translation products were produced in most cases, but capsid-exogenous fusion proteins were produced more efficiently than were the translation products. These data indicate that the 5' part of the capsid ORF is not an absolute requirement for the IGR-IRES-mediated translation. RNA structure probing analyses showed that the 5' part of the capsid ORF was a single strand, while that of exogenous reading frames was structured. Exogenous sequences also caused structural distortion in the 3' part of the IGR-IRES. We hypothesize that the single-stranded capsid ORF helps to form the tertiary structure of the IGR-IRES and facilitates precise positioning of ribosomes.

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

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.