Nobuhiko Nakashima

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.

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.

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.

COMPLETE NUCLEOTIDE SEQUENCE OF SPHEROIDIN GENE OF ANOMALA CUPREA ENTOMOPOXVIRUS

The complete nucleotide sequence of the spheroidin gene of Anomala cuprea entomopoxvirus (AcEPV) was determined. The sequence was compared with those of spheroidin genes of EPVs such as Melolontha melolontha EPV (MmEPV, Genus A), Choristoneura fumiferana EPV (CfEPV, Genus B) and Amsacta moorei EPV (AmEPV, Genus B). The gene harbored an open reading frame (ORF) of 2826 nt, with the same size as that of MmEPV belonging to the same genus, capable of coding for a polypeptide of 109.0 kDa. The predicted amino acid (aa) sequence showed a greater or moderate similarity to the corresponding sequence of the other EPVs, showing a 94, 40 and 41% aa identity with MmEPV, CfEPV and AmEPV, respectively; the identity was 89, 53 and 54% at the nucleotide level. The hydropathy plots also showed a greater similarity in organization to MmEPV and moderate similarity to the viruses of Genus B. In the polypeptide, 44 cysteine residues, which are likely to be involved in paracrystal formation and seven potential N-glycosylation sites were detected. The number of cysteine residues and N-glycosylation sites also depended on the difference in genera (A or B). Thus, the spheroidin gene of EPVs was well conserved within the same genus.