佐々木 潤

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PItransfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBPmediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs.

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for the replication of certain picornavirus genomes. We previously showed that nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB of Aichi virus (AiV), a picornavirus, interact with the Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3), which interacts with PI4KB. These five viral proteins, ACBD3, PI4KB, and the PI4KB product phosphatidylinositol 4-phosphate (PI4P) colocalize to the AiV RNA replication sites (J. Sasaki et al., EMBO J. 31:754-766, 2012). We here examined the roles of these viral and cellular molecules in the formation of AiV replication complexes. Immunofluorescence microscopy revealed that treatment of AiV polyprotein-expressing cells with a small interfering RNA targeting ACBD3 abolished colocalization of the viral 2B, 2C, and 3A proteins with PI4KB. A PI4KB-specific inhibitor also prevented their colocalization. Virus RNA replication increased the level of cellular PI4P without affecting that of PI4KB, and individual expression of 2B, 2BC, 2C, 3A, or 3AB stimulated PI4P generation. These results suggest that the viral protein/ACBD3/PI4KB complex plays an important role in forming the functional replication complex by enhancing PI4P synthesis. Of the viral proteins, 3A and 3AB were shown to stimulate the in vitro kinase activity of PI4KB through forming a 3A or 3AB/ACBD3/PI4KB complex, whereas the ACBD3-mediated PI4KB activation by 2B and 2C remains to be demonstrated. Copyright. © 2014, American Society for Microbiology.

Phosphatidylinositol 4-kinase III beta (PI4KB) is a host factor required for genome RNA replication of enteroviruses, small non-enveloped viruses belonging to the family Picornaviridae. Here, we demonstrated that PI4KB is also essential for genome replication of another picornavirus, Aichi virus (AiV), but is recruited to the genome replication sites by a different strategy from that utilized by enteroviruses. AiV non-structural proteins, 2B, 2BC, 2C, 3A, and 3AB, interacted with a Golgi protein, acylcoenzyme A binding domain containing 3 (ACBD3). Furthermore, we identified previously unknown interaction between ACBD3 and PI4KB, which provides a novel manner of Golgi recruitment of PI4KB. Knockdown of ACBD3 or PI4KB suppressed AiV RNA replication. The viral proteins, ACBD3, PI4KB, and phophatidylinositol-4-phosphate (PI4P) localized to the viral RNA replication sites. AiV replication and recruitment of PI4KB to the RNA replication sites were not affected by brefeldin A, in contrast to those in enterovirus infection. These results indicate that a viral protein/ACBD3/PI4KB complex is formed to synthesize PI4P at the AiV RNA replication sites and plays an essential role in viral RNA replication. The EMBO Journal (2012) 31, 754-766. doi: 10.1038/emboj.2011.429; Published online 29 November 2011

Picornavirus genomes are translated into a single large polyprotein, which is processed by virus-encoded proteases into individual functional proteins. 3C of all picornaviruses is a protease, and the leader (L) and 2A proteins of some picornaviruses are also involved in polyprotein processing. Aichi virus (AiV), which is associated with acute gastroenteritis in humans, is a member of the genus Kobuvirus of the family Picornaviridae. The AiV L and 2A proteins have already been shown to exhibit no protease activity. In this study, we investigated AiV polyprotein processing by 3C and 3CD using a cell-free translation system. 3C and 3CD were capable of processing the polyprotein in trans; 3C, however, cleaved the VP1/2A site inefficiently, while 3CD cleaved this site almost completely. Mammalian two-hybrid and coimmunoprecipitation assays showed an interaction between 2A and 3CD. Using a 3CD mutant and various 2A mutants of substrate proteins, we showed a clear correlation between the 2A-3CD interaction and the VP1/2A cleavage by 3CD. Thus, this study suggests that tight interaction of 3CD with the 2A region of a precursor protein is required for efficient cleavage at the VP1/2A site. (C) 2011 Elsevier B.V. All rights reserved.

We determined nucleotide sequences and inferred amino acid sequences of viral protein (VP) 4, VP6, VP7, and nonstructural protein 4 genes of a porcine rotavirus strain (SKA-1) from Japan. The strain was closely related to a novel group of human rotavirus strains (B219 and J19).

Aichi virus (AiV), which is associated with acute gastroenteritis in humans, is a member of the genus Kobuvirus of the family Picornaviridae. Picornavirus genome replication occurs in replication complexes that include viral nonstructural proteins, host proteins and viral RNA. In poliovirus, all nonstructural proteins are found in the replication complexes, suggesting the ability of the viral nonstructural proteins to interact with each other. In this study, we examined the interactions between the AiV nonstructural proteins using a mammalian two-hybrid system. The results showed that all of the tested proteins could interact with more than one protein. We observed homodimerization of five proteins, bidirectional heterodimerization of six protein pairs, and unidirectional heterodimerization of eighteen protein pairs. Among the interactions detected in this study, the 2A-2B, 2A-2BC, 2A-2C, 2BC-3CD, 2BC-3C, 2C-3C, 2C-3CD and 3AB-3C interactions have not been observed in the previous two-hybrid studies with other picornaviruses. The strongest interaction was observed between 2A and 3CD. AiV 2A has already been shown to be involved in genome replication. Domain mapping of the 2A and 3CD interaction in mammalian two-hybrid analysis revealed that the C-terminal quarter of 2A is not required for the interaction with 3CD. (c) 2009 Elsevier B.V. All rights reserved.

The Aichi virus 2A protein is not a protease, unlike many other picornavirus 2A proteins, and it is related to a cellular protein, H-rev107. Here, we examined the replication properties of two 2A mutants in Vero cells and a cell-free translation/replication system. In one mutant, amino acids 36 to 126 were replaced with an unrelated amino acid sequence. In the other mutant, the NC motif conserved in the H-rev107 family of proteins was changed to alanine residues. The two mutations abolished virus replication in cells. The mutations affected both negative-and positive-strand synthesis, the defect in positive-strand synthesis being more severe than that in negative-strand synthesis.

Secondary structural elements at the 5′ end of picornavirus genomic RNA function as cis-acting replication elements and are known to interact specifically with viral P3 proteins in several picornaviruses. In poliovirus, ribonucleoprotein complex formation at the 5′ end of the genome is required for negative-strand synthesis. We have previously shown that the 5′-end 115 nucleotides of the Aichi virus genome, which are predicted to fold into two stem-loops (SL-A and SL-C) and one pseudoknot (PK-B), act as a cis-acting replication element and that correct folding of these structures is required for negative-strand synthesis. In this study, we investigated the interaction between the 5′-terminal 120 nucleotides of the genome and the P3 proteins, 3AB, 3ABC, 3C, and 3CD, by gel shift assay and Northwestern analysis. The results showed that 3ABC and 3CD bound to the 5′-terminal region specifically. The binding of 3ABC was observed on both assays, while that of 3CD was detected only on Northwestern analysis. No binding of 3AB or 3C was observed. Binding assays using mutant RNAs demonstrated that disruption of the base pairings of the stem of SL-A and one of the two stem segments of PK-B (stem-B1) abolished the 3ABC binding. In addition, the specific nucleotide sequence of stem-B1 was responsible for the efficient 3ABC binding. These results suggest that the interaction of 3ABC with the 5′-terminal region of the genome is involved in negative-strand synthesis. On the other hand, the ability of 3CD to interact with the 5′-terminal region did not correlate with the RNA replication ability. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Recombinant rotavirus (RV) with eDNA-derived chimeric VP4 was generated using recently developed reverse genetics for RV. The rescued virus, KU//rVP4(SA11)-II(DS-1), contains SA11 (simian RV strain, G3P[2])-based VP4, in which a cross-reactive neutralization epitope (amino acids 381 to 401) on VF5* is replaced by the corresponding sequence of a different P-type DS-1 (human RV strain, G2P[4]). Serological analyses with a panel of anti-VP4- and -VP7-neutralizing monoclonal antibodies revealed that the rescued virus carries a novel antigenic mosaic of cross-reactive neutralization epitopes on its VP4 surface. This is the first report of the generation of a recombinant RV with artificial amino acid substitutions.

Osteopontin (OPN) knockout mice (OPN-KO mice) died of Plasinodium chabaudi chabaudi infection, although wild-type (WT) mice had self-limiting infections. OPN was detected in the WT mice at 2 days postinfection. OPN-KO mice produced significantly smaller amounts of interleukin-12 and gamma interferon than WT mice produced. These results suggested that OPN is involved in Th1-mediated immunity against malaria infection.

We describe here the successful establishment of a reverse genetics system for rotavirus (RV), a member of the Reoviridae family whose genome consists of 10-12 segmented dsRNA. The system is based on the recombinant vaccinia virus T7 RNA polymerase-driven procedure for supplying artificial viral mRNA in the cytoplasm. With the aid of helper virus (human RV strain KU) infection, intracellularly transcribed full-length VP4 mRNA of simian RV strain SA11 resulted in the rescue of the KU-based transfectant virus carrying the SA11 VP4 RNA segment derived from cDNA. In addition to the rescued transfectant virus with the authentic SA11 VP4 gene, three more infectious RV transfectants, into which silent mutation(s) were introduced to destroy both or one of the two restriction enzyme sites as gene markers in the SA11 VP4 genome, were also rescued with this method. The ability to artificially manipulate the RV genome will greatly increase the understanding of the replication and the pathogenicity of RV and will provide a tool for the design of attenuated vaccine vectors.

Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5′ end) formed at the 5′ end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5′-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5′-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5′-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5′-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5′ end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3′ end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5′ end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Aichi virus, a member of the family Picornaviridae, encodes a leader (L) protein of 170 amino acids (aa). The Aichi virus L protein exhibits no significant sequence homology to those of other picornaviruses. In this study, we investigated the function of the Aichi virus L protein in virus growth. In vitro translation and cleavage assays indicated that the L protein has no autocatalytic activity and is not involved in polyprotein cleavage. The L-VP0 junction was cleaved by 3C proteinase. Immunoblot analysis showed that the L protein is stably present in infected cells. Characterization of various L mutants derived from an infectious cDNA clone revealed that deletion of 93 as of the center part (aa 43 to 135), 50 as of the N-terminal part (aa 4 to 53), or 90 as of the C-terminal part (aa 74 to 163) abolished viral RNA replication. A mutant (Delta114-163) in which 50 as of the C-terminal part (aa 114 to 163) were deleted exhibited efficient RNA replication and translation abilities, but the virus yield was 4 log orders lower than that of the wild type. Sedimentation analysis of viral particles generated in mutant Delta114-163 RNA-transfected cells showed that the mutant has a severe defect in the formation of mature virions, but not in that of empty capsids. Thus, the data obtained in this study indicate that the Aichi virus L protein is involved in both viral RNA replication and encapsidation.

Aichi virus is a member of the family Picornaviridae. Computer-assisted secondary structure prediction suggested the formation of three stem-loop structures (SL-A, SL-B, and SL-C from the 5′ end) within the 5′-end 120 nucleotides of the genome. We have already shown that the most 5′-end stem-loop, SL-A, is critical for viral RNA replication. Here, using an infectious cDNA clone and a replicon harboring a luciferase gene, we revealed that formation of SL-B and SL-C on the positive strand is essential for viral RNA replication. In addition, the specific nucleotide sequence of the loop segment of SL-B was also shown to be critical for viral RNA replication. Mutations of the upper and lower stems of SL-C that do not disrupt the base-pairings hardly affected RNA replication, but decreased the yields of viable viruses significantly compared with for the wild-type. This suggests that SL-C plays a role at some step besides RNA replication during virus infection. © 2003 Elsevier Science (USA). All rights reserved.

We examined the utility of dried blood on filter paper for the source of cytokine messenger RNA (mRNA). Total RNA was isolated from the dried blood of mice infected with Plasmodium yoelii, and cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). As a reference, we extracted total RNA from peripheral blood collected at the same time as the preparation for dried blood. There was no difference in cytokine mRNA expression between the two sources; the dried blood on filter paper and the peripheral blood. Th1 cells, Th2 cells, and monocytes/ macrophagges derived cytokine mRNAs in the dried blood from infected mice were detected, and the increase of some of the cytokines mRNAs after infection was also observed. These results suggested that the dried blood on filter paper is satisfactory RNA source for immunological examination in field-based studies. (C) 2003 Elsevier Science B.V. All rights reserved.

Picornavirus positive-strand RNAs are selectively encapsidated despite the coexistence of viral negative-strand RNAs and cellular RNAs in infected cells. However, the precise mechanism of the RNA encapsidation process in picornaviruses remains unclear. Here we report the first identification of an RNA element critical for encapsidation in picornaviruses. The 5′ end of the genome of Aichi virus, a member of the family Picornaviridae, folds into three stem-loop structures (SL-A, SL-B, and SL-C, from the most 5′ end). In the previous study, we constructed a mutant, termed mut6, by exchanging the seven-nucleotide stretches of the middle part of the stem in SL-A with each other to maintain the base pairings of the stem. mut6 exhibited efficient RNA replication and translation but formed no plaques. The present study showed that in cells transfected with mut6 RNA, empty capsids were accumulated, but few virions containing RNA were formed. This means that mut6 has a severe defect in RNA encapsidation. Site-directed mutational analysis indicated that as the mutated region was narrowed, the encapsidation was improved. As a result, the mutation of the 7 bp of the middle part of the stem in SL-A was required for abolishing the plaque-forming ability. Thus, the 5′-end sequence of the Aichi virus genome was shown to play an important role in encapsidation.

Aichi virus is the type species of a new genus, Kobuvirus, of the family Picornaviridae. In this study, we constructed a full-length cDNA clone of Aichi virus whose in vitro transcripts were infectious to Vero cells. During construction of the infectious cDNA clone, a novel sequence of 32 nucleotides was identified at the 5′ end of the genome. Computer-assisted prediction of the secondary structure of the 5′ end of the genome, including the novel sequence, suggested the formation of a stable stem-loop structure consisting of 42 nucleotides. The function of this stem-loop in virus replication was investigated using various site-directed mutants derived from the infectious cDNA clone. Our data indicated that correct folding of the stem-loop at the 5′ end of the positive strand, but not at the 3′ end of the negative strand, is critical for viral RNA replication. The primary sequence in the lower part of the stem was also suggested to be crucial for RNA replication. In contrast, nucleotide changes in the loop segment did not so severely reduce the efficiency of virus replication. A double mutant, in which both nucleotide stretches of the middle part of the stem were replaced by their complementary nucleotides, had efficient RNA replication and translation abilities but was unable to produce viruses. These results indicate that the stem-loop at the 5′ end of the Aichi virus genome is an element involved in both viral RNA replication and production of infectious virus particles.

Protein synthesis is believed to be initiated with the amino acid methionine because the AUG translation initiation codon of mRNAs is recognized by the anticodon of initiator methionine transfer RNA. A group of positive-stranded RNA viruses of insects, however, lacks an AUG translation initiation codon for their capsid protein gene, which is located at the downstream part of the genome. The capsid protein of one of these viruses, Plautia stali intestine virus, is synthesized by internal ribosome entry site-mediated translation, Here we report that methionine is not the initiating amino acid in the translation of the capsid protein in this virus. Its translation is initiated with glutamine encoded by a CAA codon that is the first codon of the capsid-coding region. The nucleotide sequence immediately upstream of the capsid-coding region interacts with a loop segment in the stem-loop structure located 15-43 nt upstream of the 5' end of the capsid-coding region. The pseudoknot structure formed by this base pair interaction is essential for translation of the capsid protein. This mechanism for translation initiation differs from the conventional one in that the initiation step controlled by the initiator methionine transfer RNA is not necessary.

AUG-unrelated translation initiation was found in an insect picorna- like virus, Plautia stali intestine virus (PSIV). The positive-strand RNA genome of the virus contains two nonoverlapping open reading frames (ORFs). The capsid protein gene is located in the 3'-proximal ORF and lacks an AUG initiation codon. We examined the translation mechanism and the initiation codon of the capsid protein gene by using various dicistronic and monocistronic RNAs in vitro. The capsid protein gene was translated cap independently in the presence of the upstream cistron, indicating that the gene is translated by internal ribosome entry. Deletion analysis showed that the internal ribosome entry site (IRES) consisted of approximately 250 bases and that its 3' boundary extended slightly into the capsid-coding region. The initiation codon for the IRES-mediated translation was identified as the CUU codon, which is located just upstream of the 5' terminus of the capsid- coding region by site-directed mutagenesis. In vitro translation assays of monocistronic RNAs lacking the 5' part of the IRES showed that this CUU codon was not recognized by scanning ribosomes. This suggests that the PSIV IRES can effectively direct translation initiation without stable codon-anticodon pairing between the initiation codon and the initiator methionyl-tRNA.

We determined the complete genome sequence of Himetobi P virus (HiPV), an insect picorna-like virus, which was isolated from the small brown planthopper, Laodelphax striatellus. The genome of HiPV consists of 9,275 nucleotides excluding the poly (A) rail, and contains two large open reading frames (ORFs), which were separated by a 176-nucleotide noncoding region. The deduced amino acid sequence of the first ORF contains core motifs of picornaviral helicase, protease, and RNA-dependent RNA polymerase. The capsid protein-coding region was mapped onto the second ORF by determining the N-terminal amino acid sequences of the capsid proteins. Subgenomic RNA for the capsid protein gene was not detected in the infected tissue. The capsid protein precursor gene of HiPV lacks an AUG initiation codon at the expected position and the upstream sequence of the gene is predicted to form several stem-loop structures, suggesting that the precursor is produced by internal ribosome entry site (IRES) mediated-translation, as occurs in Plutia stall intestine virus (PSIV). These characteristics of the HiPV genome are similar to those of a new group of RNA viruses consisting of Drosophila C virus (DCV), Rhopalosiphum padi virus (RhPV), and PSIV.

The complete genome of an insect picorna-like virus, Plautia stali intestine virus (PSIV), was cloned and sequenced. The genome had 8797 nucleotides including two consecutive long open reading frames. The deduced amino acid sequence of the first open reading frame (nucleotides 571 to 6003) contained conserved sequence motifs for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase. The order of the three motifs in the genome was the same as those of mammalian picornaviruses. The coding regions of four capsid proteins (33, 30, 26, and 4.5 kDa) were mapped by determining their N-terminal sequences. Unlike mammalian picornaviruses, the genes for these proteins were in the 3' region of the PSIV genome. In vitro translation assay suggested that the capsid protein precursor of PSIV would be translated by internal initiation. The deduced amino acid sequence of the capsid proteins showed homology to those of the proteins encoded in the 3' part of the genomes of widely distributed insect picorna-like viruses, cricket paralysis virus, and Drosophila C virus. Some insect picorna-like viruses would have the same unique coding strategy as PSIV, (C) 1998 Academic Press.

A picorna-like virus was isolated from the brown-winged green bug, Plautia stali. The virus was named Plautia stall intestine virus (PSIV) based on the multiplication site of the virus in the infected insects. PSIV is a spherical particle with a diameter of 30 nm. Particles of PSIV were found to contain a 9.1-kb single-stranded RNA. Polyacrylamide gel electrophoresis of purified PSIV particles revealed three major (33, 30, 26 kDa), one medium (35 kDa), and one minor (4.5 kDa) structural proteins. The molar ratios of the proteins suggested that the 35-, 33-, 30-, 26-, and 4.5-kDa proteins corresponded to VP0, VP1, VP2, VP3, and VP4 of vertebrate picornaviruses. Immunological assays indicated that PSIV and Nezara viridula virus-1, which is a picorna-like virus of the green stinkbug in South Africa, were serologically distinct, PSIV was detected in the intestine with enzyme-linked immunosorbent assay but not in the salivary glands, fat bodies, Malpighian tubules, and reproductive organs of viruliferous P. stali. The virus was also detected on the surface of the eggs and in the feces of infected insects. These results suggest that excrement of infected insects are the primary inoculum of the virus in a colony of P. stali. The nonviruliferous adults of P. stali usually survive a few months in laboratory, while the average of adulthood lifetime in viruliferous P. stali was about 13 days. PSIV also infected two other stinkbugs, Nezara viridula and Halyomorpha halys. (C) 1998 Academic Press.