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Göran Akusjärvi - One of the best experts on this subject based on the ideXlab platform.
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Synthesis, Structure, and Function of Human Adenovirus Small Non-Coding RNAs.
Viruses, 2020Co-Authors: Tanel Punga, Mahmoud Darweesh, Göran AkusjärviAbstract:Human adenoviruses (HAdVs) are common pathogens causing a VAriety of respiratory, ocular and gastrointestinal diseases. To accomplish their efficient replication, HAdVs take an adVAntage of viral small non-coding RNAs (sncRNAs), which have multiple roles during the virus lifecycle. Three of the best-characterized HAdV sncRNAs; VA RNA, miVARNA and MLP-TSS-sRNA will be discussed in the present review. Even though VA RNA has been extensively characterized during the last 60 years, this multifunctional molecule continues to surprise us as more of its structural secrets unfold. Likely, the recent developments on miVARNA and MLP-TSS-sRNA synthesis and function highlight the importance of these sncRNA in virus replication. Collectively, we will summarize the old and new knowledge about these three viral sncRNAs with focus on their synthesis, structure and functions.
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Complementation of the human adenovirus type 5 VA RNAI defect by the VAccinia virus E3L protein and serotype-specific VA RNAIs
Virology, 2015Co-Authors: Raviteja Inturi, Göran Akusjärvi, Wael Kamel, Tanel PungaAbstract:Human adenoviruses (HAdVs) encode for multifunctional non-coding virus-associated (VA) RNAs, which function as powerful suppressors of the cellular interferon (IFN) and RNA interference (RNAi) systems. In this study we tested the ability of VArious plant and animal virus encoded RNAi and IFN suppressor proteins to functionally substitute for the HAdV-5 VA RNAI. Our results revealed that only the VAccinia virus (VACV) E3L protein was able to substitute for the HAdV-5 VA RNAI functions in virus-infected cells. Interestingly, the E3L protein rescues the translational defect but does not stimulate viral capsid mRNA accumulation observed with VA RNA. We further show that the E3L C-terminal region containing the dsRNA-binding domain is needed to enhance VA RNAI mutant virus replication. Additionally, we show that the HAdV-4 and HAdV-37 VA RNAI are more effective than the HAdV-5 VA RNAI in rescuing virus replication.
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Small RNA Sequence Analysis of Adenovirus VA RNA-Derived MiRNAs Reveals an Unexpected Serotype-Specific Difference in Structure and Abundance
PloS one, 2014Co-Authors: Wael Kamel, Bo Segerman, Tanel Punga, Göran AkusjärviAbstract:Human adenoviruses (HAds) encode for one or two highly abundant virus-associated RNAs, designated VA RNAI and VA RNAII, which fold into stable hairpin structures resembling miRNA precursors. Here we show that the terminal stem of the VA RNAs originating from Ad4, Ad5, Ad11 and Ad37, all undergo Dicer dependent processing into virus-specific miRNAs (so-called miVARNAs). We further show that the miVARNA duplex is subjected to a highly asymmetric RISC loading with the 3′-strand from all VA RNAs being the favored strand, except for the Ad37 VA RNAII, where the 5′-miVARNAII strand was preferentially assembled into RISC. Although the miVARNA seed sequences are not fully conserved between the HAds a bioinformatics prediction approach suggests that a large fraction of the VA RNAII-, but not the VA RNAI-derived miVARNAs still are able to target the same cellular genes. Using small RNA deep sequencing we demonstrate that the Dicer processing event in the terminal stem of the VA RNAs is not unique and generates 3′-miVARNAs with a slight VAriation of the position of the 5′ terminal nucleotide in the RISC loaded guide strand. Also, we show that all analyzed VA RNAs, except Ad37 VA RNAI and Ad5 VA RNAII, utilize an alteRNAtive upstream A start site in addition to the classical +1 G start site. Further, the 5′-miVARNAs with an A start appears to be preferentially incorporated into RISC. Although the majority of miVARNA research has been done using Ad5 as the model system our analysis demonstrates that the miVARNAs expressed in Ad11- and Ad37-infected cells are the most abundant miVARNAs associated with Ago2-containing RISC. Collectively, our results show an unexpected VAriability in Dicer processing of the VA RNAs and a serotype-specific loading of miVARNAs into Ago2-based RISC.
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The adenovirus VA RNA-derived miRNAs are not essential for lytic virus growth in tissue culture cells
Nucleic acids research, 2013Co-Authors: Wael Kamel, Bo Segerman, Tanel Punga, Daniel Öberg, Göran AkusjärviAbstract:At late times during a lytic infection human adenovirus type 5 produces ∼10(8) copies per cell of virus-associated RNA I (VA RNAI). This short highly structured RNA polymerase III transcript has previously been shown to be essential for lytic virus growth. A fraction of VA RNAI is processed by Dicer into small RNAs, so-called miVARNAIs, which are efficiently incorporated into the RNA-induced silencing complex. Here, we constructed recombinant adenoviruses with mutations in the seed sequence of both the 5'- and the 3'-strand of the miVARNAI duplex. The results showed that late viral protein synthesis, as well as new virus progeny formation, was essentially unaffected by the seed sequence mutations under lytic replicative conditions in HeLa or HEK293 cells. Collectively, our results suggest that either strand of the miVARNAI duplex does not have target mRNA interactions that are critical for the establishment of virus growth under lytic conditions. Further, by depletion of protein kinase R (PKR) in HEK293 cells, we show that the suppressive effect of VA RNAI on the interferon-induced PKR pathway is most critical for late gene expression.
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Old and new functions for the adenovirus virus-associated RNAs
Future Virology, 2013Co-Authors: Tanel Punga, Wael Kamel, Göran AkusjärviAbstract:Adenovirus type 5 encodes two short, highly structured noncoding RNAs, the virus-associated (VA) RNAI and VA RNAII. These RNAs are expressed in large amounts late during a lytic infection. Early studies established an important role for VA RNAI in maintaining efficient translation in late virus-infected cells by blocking actiVAtion of the key interferon-induced PKR protein kinase. More recent studies have demonstrated that the VA RNAs also target the RNAi/miRNA pathway. Collectively, aVAilable data suggest that the VA RNAs are multifunctional RNAs suppressing the activity of three dsRNA-sensing enzyme systems in human cells. Here, the known functions of the VA RNAs are summarized and the interplay between VA RNA expression and the activity of the interferon and RNAi pathways are discussed in more detail.
Michael B Mathews - One of the best experts on this subject based on the ideXlab platform.
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TERMINATION SEQUENCE REQUIREMENTS VARY AMONG GENES TRANSCRIBED BY RNA POLYMERASE III
Journal of Molecular Biology, 1999Co-Authors: Shobha Gunnery, Yuliang Ma, Michael B MathewsAbstract:Abstract RNA polymerase III (pol III) transcription generally terminates at a run of four or more thymidine (T) residues but some pol III genes contain runs of T residues that are not recognized as termination signals. Here, we investigate the terminal signal requirements that are operative in adenovirus virus-associated (VA) RNA genes. In the Xenopus 5 S RNA gene, efficient termination requires the T residues to be in a G + C-rich sequence context, but a run of five T residues in a G + C-rich context does not cause pol III termination when placed 30 nt downstream of the adenovirus-2 VA RNAIpromoter in a VA-Tat chimeric gene. The failure of pol III to recognize this putative termination signal is not due to the chimeric nature of the gene or to the proximity of the signal to the promoter, but to its sequence context. Termination at the VA RNA gene site requires a T-rich sequence and is inhibited by the proximity of G residues, but is insensitive to the presence of A residues. The T-rich sequence need not be uninterrupted, however. In the VA RNA gene of the avian adenovirus, CELO, the first of two tandem termination signals contains an interrupted run of T residues, TTATT, which functions as a terminator with high (although not complete) efficiency. These findings, together with a survey of sequences neighboring the terminal site of other pol III genes, lead to the conclusion that pol III termination signals are more complex than hitherto recognized, and that sequence context requirements differ between members of the class 1 and class 2 families of pol III genes.
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Surprising specificity of PKR binding to delta agent genomic RNA.
RNA (New York N.Y.), 1997Co-Authors: D A Circle, Paul A. Clarke, Olivia D. Neel, Hugh D. Robertson, Michael B MathewsAbstract:A direct, ribonuclease T1 protection assay was employed to study the binding of a delta agent genomic RNA transcript containing the conserved domain to the double-stranded RNA- (dsRNA-) dependent protein kinase of mammalian cells, PKR (also known as DAI, p1-elF2, or p68 kinase). In a control reaction, this assay identified a major portion of the same PKR binding site in VA RNA as deduced previously using a footprinting technique (Clarke PA, Mathews MB, 1995, RNA 1:1-20). Although delta agent RNA contains extensive secondary structure throughout the conserved region, we found a remarkable specificity in its PKR binding. The same region was protected by intact PKR and by a 184-amino acid fragment thereof containing the two RNA-binding motifs (dsRBMs) but lacking kinase activity. Two specific opposed, continuous segments of delta agent RNA (extending about 65-70 bases) were obtained reproducibly. Each is more than twice as long as those protected in VA RNA (about 25 bases), suggesting the involvement of PKR dimers in delta RNA binding. The PKR-protected region of delta agent RNA also contains a characteristic tertiary structural element that may be involved in binding specificity.
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Secondary and tertiary structure in the central domain of adenovirus type 2 VA RNA I.
RNA (New York N.Y.), 1996Co-Authors: Michael B MathewsAbstract:The small (160 nt) adenovirus RNA, VA RNAI, antagonizes the actiVAtion of the cellular protein kinase PKR (also known as DAI), a key regulator of gene expression. VA RNA consists of two stems separated by a complex region, the central domain, that is essential for its function. A notable feature of the central domain is a pair of tetranucleotides, GGGU and ACCC, which are mutually complementary and phylogenetically conserved. To investigate their role in the structure and function of VA RNA, we generated three sets of mutations designed to disrupt the putative stem and to restore it with different nucleotides. Substitutions in either of the tetranucleotides abrogated VA RNA function in two independent PKR-based assays, demonstrating the importance of these sequences in vivo. Compensating mutants restored function, indicating that the two tetranucleotides pair in the cell, but all of the compensating mutants were less active than wild-type VA RNA. The effects of the mutations on RNA structure were probed by nuclease sensitivity analysis. Pronounced changes in two loops in the central domain correlated closely with the formation and disruption of the stem, suggesting that the tetranucleotide stem defines a critical element in the structure of the central domain through tertiary interactions with the two loops. A model for the central domain is presented that accommodates these findings and also accounts for the known sites of PKR interaction.
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Structure, function, and evolution of adenovirus-associated RNA: a phylogenetic approach.
Journal of virology, 1996Co-Authors: Michael B MathewsAbstract:To explore the structure and function of a small regulatory RNA, we examined the virus-associated (VA) RNA species of all 47 known human adenovirus serotypes and of one simian virus, SA7. The VA RNA gene regions of 43 human adenoviruses were amplified and sequenced, and the structures of 10 representative VA RNAs were probed by nuclease sensitivity analysis. Most human viruses have two VA RNA species, VA RNA, and VA RNAII, but nine viruses (19%) have a single VA RNA gene. Sequence alignments classified the RNAs into eight families, corresponding broadly to the known virus groups, and three superfamilies. One superfamily contains the single VA RNAs of groups A and F and the VA RNAI species of group C; the second contains the VA RNAI species of groups B1, D, and E and the unclassified viruses (adenovirus types 42 to 47), as well as the single VA RNAs of group B2; and the third contains all VA RNAII species. Fourteen regions of homology occur throughout the molecule. The longest of these correspond to transcription signals; most of the others participate in RNA secondary structure. The previously identified tetranucleotide pair, GGGU:ACCC, is nearly inVAriant, diverging slightly (to GGGU:ACCU) only in the two group F viruses and forming a stem in the central domain that is critical for VA RNA structure and function. Secondary structure models which accommodate the nuclease sensitivity data and sequence VAriations within each family were generated. The major structural features-the terminal stem, apical stem-loop, and central domain-are conserved in all VA RNAs, but differences exist in the apical stem and central domains, especially of the VA RNAII species. Sequence analysis suggests that an ancestral VA RNA gene underwent duplication during the evolution of viruses containing two VA RNA genes. Although the VA RNAII gene seems to have been lost or inactiVAted by secondary deletion events in some viruses, the high degree of homology among the VA RNAII species implies that this RNA may play an undiscovered role in virus surviVAl. We speculate that the VA RNA genes originated from cellular sequences containing multiple tRNA genes.
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Functional characterization of the RNA-binding domain and motif of the double-stranded RNA-dependent protein kinase DAI (PKR).
Journal of molecular biology, 1995Co-Authors: Christian Schmedt, Simon R. Green, Lisa Manche, Deborah R. Taylor, Michael B MathewsAbstract:The double-stranded (ds) RNA-actiVAted protein kinase, DAI (also known as PKR), contains an RNA-binding domain comprising two tandem repeats of a motif, the dsRBM, which is shared with a number of other proteins that interact with structured RNAs. We have expressed the entire domain and the first copy of the motif in Escherichia coli and purified the two proteins, p20 and p10, to apparent homogeneity in order to study their interactions with RNA and with the intact kinase enzyme. Both p20 and p10 bound preferentially to structured RNA molecules. Competition assays showed that in both cases the order of affinity is dsRNA > VA RNA > tRNA, but the isolated motif bound much less tightly than the entire domain. Measurement of the dissociation constants for dsRNA by quantitative gel mobility shift analysis gave apparent Kd VAlues of 4 x 10(-9) M and 3.8 x 10(-7) M for p20 and p10, respectively. The binding of p20 molecules to dsRNA appeared to be cooperative. Multiple complexes were formed between the intact domain and dsRNA, saturating at a density of about one p20 molecule/11.25 base-pairs (or one turn) of duplex, whereas p10 achieved only about half of this packing density. The apparent Kd for the p20-VA RNA interaction was estimated as 3.5 x 10(-7) M and at least three complexes were detected, but no distinct complexes were visualized for the interaction between p10 and VA RNA. Both p20 and p10 inhibited autophosphorylation of intact DAI, probably by binding the dsRNA actiVAtor. Once actiVAted, DAI could phosphorylate both p10 and p20, suggesting that intermolecular phosphorylation can occur.
Hiroyuki Mizuguchi - One of the best experts on this subject based on the ideXlab platform.
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a microRNA derived from adenovirus virus associated RNAii promotes virus infection via posttranscriptional gene silencing
Journal of Virology, 2018Co-Authors: Keisaku Wakabayashi, Mitsuhiro Machitani, Masashi Tachibana, Fuminori Sakurai, Hiroyuki MizuguchiAbstract:ABSTRACT The adenovirus (Ad) serotype 5 genome encodes two noncoding small RNAs (virus-associated RNAs I and II [VA-RNAI and -II]), which are approximately 160-nucleotide (nt) RNAs transcribed by RNA polymerase III. It is well known that VA-RNAI supports Ad infection via the inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. Recent studies revealed that VA-RNAs are processed into VA-RNA-derived microRNAs (miRNAs) (miVARNAI and -II); however, we and another group recently demonstrated that miVARNAI does not promote Ad replication. On the other hand, the roles of VA-RNAII and miVARNAII in Ad replication have remained to be clarified. In this study, we demonstrated miVARNAII-mediated promotion of Ad replication. Transfection with chemically synthesized 3′-miVARNAII-138, one of the most abundant forms of miVARNAII, significantly enhanced Ad replication, while the other species of miVARNAII did not. We identified 8 putative target genes of 3′-miVARNAII-138 by microarray analysis and in silico analysis. Among the 8 candidates, knockdown of the cullin 4A (CUL4A) gene, which encodes a component of the ubiquitin ligase complex, most significantly enhanced Ad replication. CUL4A expression was significantly suppressed by 3′-miVARNAII-138 via posttranscriptional gene silencing, indicating that CUL4A is a target gene of 3′-miVARNAII-138 and miVARNAII functions as a viral miRNA promoting Ad infection. It has been reported that CUL4A is involved in degradation of c-Jun, which acts as a transcription factor in the Jun-N-terminal kinase (JNK) signaling cascade. Treatment with JNK inhibitors dramatically suppressed Ad replication, suggesting that miVARNAII-mediated downregulation of CUL4A enhanced JNK signaling and thereby promoted Ad infection. IMPORTANCE Several types of viruses encode viral miRNAs which regulate host and/or viral gene expression via posttranscriptional gene silencing, leading to efficient viral infection. Adenovirus (Ad) expresses miRNAs derived from VA-RNAs (miVARNAI and -II); however, recent studies have revealed that processing of VA-RNAI into miVARNAI inhibits Ad replication. Conversely, we demonstrate here that miVARNAII significantly promotes Ad replication and that miVARNAII-mediated suppression of CUL4A expression via posttranscriptional gene silencing induces accumulation of c-Jun, leading to promotion of Ad infection. These results exhibited the significance of VA-RNAII for supporting Ad infection through a mechanism complementary to that of VA-RNAI. These obserVAtions could provide important clues toward a new perspective on host-virus interaction. Moreover, Ad is widely used as a basic framework for viral vectors and oncolytic viruses. Our findings will help to regulate Ad infection and will promote the development of novel Ad vectors and oncolytic Ad.
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MicroRNA miR-27 Inhibits Adenovirus Infection by Suppressing the Expression of SNAP25 and TXN2.
Journal of Virology, 2017Co-Authors: Mitsuhiro Machitani, Keisaku Wakabayashi, Masashi Tachibana, Fuminori Sakurai, Kosuke Nakatani, Hiroyuki MizuguchiAbstract:: Recent studies have reported that host microRNAs (miRNAs) regulate infections by several types of viruses via VArious mechanisms and that inhibition of the miRNA processing factors enhances or prevents viral infection. However, it has not been clarified whether these effects of miRNAs extend to adenovirus (Ad) infection. Here we show that miR-27a and -b efficiently inhibit infection with an Ad via the downregulation of SNAP25 and TXN2, which are members of the SNARE proteins and the thioredoxin family, respectively. Approximately 80% reductions in Ad genomic copy number were found in cells transfected with miR-27a/b mimics, whereas there were approximately 2.5- to 5-fold larger copy numbers of the Ad genome following transfection with miR-27a/b inhibitors. Microarray gene expression analysis and in silico analysis demonstrated that SNAP25 and TXN2 are target genes of miR-27a/b. A reporter assay using plasmids containing the 3' untranslated regions of the SNAP25 and TXN2 genes showed that miR-27a/b directly suppressed SNAP25 and TXN2 expression through posttranscriptional gene silencing. Knockdown of SNAP25 led to a significant inhibition of Ad entry into cells. Knockdown of TXN2 induced cell cycle arrest at G1 phase, leading to a reduction in Ad replication. In addition, overexpression of Ad-encoded small noncoding RNAs (VA-RNAs) restored the miR-27a/b-mediated reduction in infection level with a VA-RNA-lacking Ad mutant due to the VA-RNA-mediated inhibition of miR-27a/b expression. These results indicate that miR-27a and -b suppress SNAP25 and TXN2 expression via posttranscriptional gene silencing, leading to efficient suppression of Ad infection.IMPORTANCE Adenovirus (Ad) is widely used as a platform for replication-incompetent Ad vectors (Adv) and replication-competent oncolytic Ad (OAd) in gene therapy and virotherapy. Regulation of Ad infection is highly important for efficient gene therapies using both Adv and OAd. In this study, we demonstrate that miR-27a and -b, which are widely expressed in host cells, suppress SNAP25 and TXN2 expression through posttranscriptional gene silencing. Suppression of SNAP25 and TXN2 expression leads to inhibition of Ad entry into cells and to cell cycle arrest, respectively, leading to efficient suppression of Ad infection. Our findings provide important clues to the improvement of gene therapies using both Adv and OAd.
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Enhanced Oncolytic Activities of the Telomerase-Specific Replication-Competent Adenovirus Expressing Short-Hairpin RNA against Dicer
Molecular cancer therapeutics, 2016Co-Authors: Mitsuhiro Machitani, Keisaku Wakabayashi, Masashi Tachibana, Fuminori Sakurai, Toshiyoshi Fujiwara, Hiroyuki MizuguchiAbstract:Oncolytic viruses have been receiving much attention as potential agents for cancer treatment. Among the VArious types of oncolytic viruses, the telomerase-specific replication-competent adenovirus (TRAD), which carries the tumor-specific promoter-driven E1 gene expression cassette, exhibits efficient antitumor effects. The development of a novel TRAD that shows higher replication efficiency and antitumor activity would be highly beneficial for safer and more efficient cancer therapy. We recently demonstrated that the endoribonuclease Dicer significantly inhibits the replication of wild-type adenovirus (Ad) via the processing of viral-associated (VA)-RNAs, which are Ad-encoded small noncoding RNAs, and that the knockdown of Dicer leads to enhanced VA-RNA expression and Ad replication after infection with wild-type Ad. Based on these findings, we herein developed a novel TRAD expressing short-hairpin RNA against Dicer (shDicer; TRAD-shDicer). After infection, TRAD-shDicer efficiently induced the knockdown of Dicer. TRAD-shDicer showed significantly higher replication efficiency and tumor cell lysis activity compared with the conventional TRAD in tumor cells. The Dicer expression levels and viabilities of normal cells were not altered by infection with TRAD-shDicer. These results indicate that TRAD-shDicer is a potent antitumor reagent by virtue of its enhanced oncolytic activity. Mol Cancer Ther; 16(1); 251–9. ©2016 AACR.
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Quantitative Analysis of Virus-associated RNAI Expression following Transduction with a Replication-incompetent Adenovirus Vector In Vitro and In Vivo
Journal of Molecular and Genetic Medicine, 2014Co-Authors: Keisaku Wakabayashi, Mitsuhiro Machitani, Masashi Tachibana, Fuminori Sakurai, Kahori Shimizu, Hiroyuki MizuguchiAbstract:The adenovirus (Ad) genome encodes one or two non-coding small RNAs called virus-associated (VA)-RNAs, that are transcribed by polymerase III and support Ad replication. As previously reported, a replication-incompetent Ad vector, which is widely used in not only gene therapy studies, including clinical trials, but also basic researches as a gene delivery vehicle, as well as wild-type Ad (WT-Ad) express VA-RNAs, and VA-RNAs actiVAte innate immunity, including the production of type I interferons. In addition, VA-RNAs perturb cellular microRNA (miRNA) expression profiles via competitive inhibition of key components involved in the miRNA maturation pathway. Although these characteristics of VA-RNAs might negatively affect the application of Ad vectors, VA-RNA expression profiles following transduction with an Ad vector have been not fully examined. In this study, we quantitatively analyzed the expression profiles of VA-RNAI, which is a major species of VA-RNAs, following transduction with Ad vectors in vitro and in vivo using real-time RT-PCR. The VA-RNAI expression levels in the cells transduced with a conventional Ad vector expressing luciferase (Ad-CAL2) at a multiplicity of infection (MOI) of 100 were approximately 2000- to 3000-fold lower than those infected with WT-Ad at the same MOI at 48 h after treatment. The expression levels of VA-RNAI in the mouse liver following administration with Ad-CAL2 were approximately 600-fold lower than those following administration with WT-Ad at 48 h post-administration. miRNA-mediated suppression of leaky expression of the Ad E4 genes resulted in about five-fold reduction in the VA-RNAI copy numbers in the liver following systemic administration in mice. These data provide informative clues for the development of novel safer Ad vectors.
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Improving adenovirus vector-mediated RNAi efficiency by lacking the expression of virus-associated RNAs.
Virus research, 2013Co-Authors: Mitsuhiro Machitani, Masashi Tachibana, Fuminori Sakurai, Kazufumi Katayama, Hayato Matsui, Takayuki Suzuki, Taketo Yamaguchi, Hiroyuki MizuguchiAbstract:Several studies have reported that short hairpin RNA (shRNA)-mediated RNA interference (RNAi) was competitively inhibited by the expression of adenovirus (Ad)-encoded small RNAs (VA-RNAs), which are expressed from a replication-incompetent Ad vector, as well as a wild-type Ad; however, it remained to be clarified whether an shRNA-expressing Ad vector-mediated knockdown was inhibited by VA-RNAs transcribed from the same Ad vector genome. In this study, we demonstrated that a lack of VA-RNA expression from the Ad vector leads to an increase in knockdown efficiencies of Ad vector-mediated RNAi. In the cells transduced with a first-generation Ad vector (FG-Ad) expressing shRNA (FG-Ad-shRNA), the copy numbers of shRNA and VA-RNAs incorporated into the RNA-induced silencing complex (RISC) was comparable. In contrast, higher amounts of shRNA were found in the RISC when the cells were transduced with an shRNA-expressing helper-dependent Ad (HD-Ad) vector, in which all viral genes, including VA-RNAs, were deleted (HD-Ad-shRNA), compared with FG-Ad-shRNA. HD-Ad vectors expressing shRNA against luciferase and p53 showed 7.4% and 37.3% increases in the knockdown efficiencies compared to the corresponding FG-Ad-shRNA, respectively, following in vitro transduction. Furthermore, higher levels of knockdown efficiencies were also found by the transduction with shRNA-expressing Ad vectors lacking VA-RNA expression (AdΔVR-shRNA) than by transduction with FG-Ad-shRNA. These results indicate that VA-RNAs expressed from an Ad vector inhibit knockdown by the shRNA-expressing Ad vector and that HD-Ad-shRNA and AdΔVR-shRNA are a powerful framework for shRNA-mediated knockdown.
Mitsuhiro Machitani - One of the best experts on this subject based on the ideXlab platform.
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a microRNA derived from adenovirus virus associated RNAii promotes virus infection via posttranscriptional gene silencing
Journal of Virology, 2018Co-Authors: Keisaku Wakabayashi, Mitsuhiro Machitani, Masashi Tachibana, Fuminori Sakurai, Hiroyuki MizuguchiAbstract:ABSTRACT The adenovirus (Ad) serotype 5 genome encodes two noncoding small RNAs (virus-associated RNAs I and II [VA-RNAI and -II]), which are approximately 160-nucleotide (nt) RNAs transcribed by RNA polymerase III. It is well known that VA-RNAI supports Ad infection via the inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. Recent studies revealed that VA-RNAs are processed into VA-RNA-derived microRNAs (miRNAs) (miVARNAI and -II); however, we and another group recently demonstrated that miVARNAI does not promote Ad replication. On the other hand, the roles of VA-RNAII and miVARNAII in Ad replication have remained to be clarified. In this study, we demonstrated miVARNAII-mediated promotion of Ad replication. Transfection with chemically synthesized 3′-miVARNAII-138, one of the most abundant forms of miVARNAII, significantly enhanced Ad replication, while the other species of miVARNAII did not. We identified 8 putative target genes of 3′-miVARNAII-138 by microarray analysis and in silico analysis. Among the 8 candidates, knockdown of the cullin 4A (CUL4A) gene, which encodes a component of the ubiquitin ligase complex, most significantly enhanced Ad replication. CUL4A expression was significantly suppressed by 3′-miVARNAII-138 via posttranscriptional gene silencing, indicating that CUL4A is a target gene of 3′-miVARNAII-138 and miVARNAII functions as a viral miRNA promoting Ad infection. It has been reported that CUL4A is involved in degradation of c-Jun, which acts as a transcription factor in the Jun-N-terminal kinase (JNK) signaling cascade. Treatment with JNK inhibitors dramatically suppressed Ad replication, suggesting that miVARNAII-mediated downregulation of CUL4A enhanced JNK signaling and thereby promoted Ad infection. IMPORTANCE Several types of viruses encode viral miRNAs which regulate host and/or viral gene expression via posttranscriptional gene silencing, leading to efficient viral infection. Adenovirus (Ad) expresses miRNAs derived from VA-RNAs (miVARNAI and -II); however, recent studies have revealed that processing of VA-RNAI into miVARNAI inhibits Ad replication. Conversely, we demonstrate here that miVARNAII significantly promotes Ad replication and that miVARNAII-mediated suppression of CUL4A expression via posttranscriptional gene silencing induces accumulation of c-Jun, leading to promotion of Ad infection. These results exhibited the significance of VA-RNAII for supporting Ad infection through a mechanism complementary to that of VA-RNAI. These obserVAtions could provide important clues toward a new perspective on host-virus interaction. Moreover, Ad is widely used as a basic framework for viral vectors and oncolytic viruses. Our findings will help to regulate Ad infection and will promote the development of novel Ad vectors and oncolytic Ad.
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MicroRNA miR-27 Inhibits Adenovirus Infection by Suppressing the Expression of SNAP25 and TXN2.
Journal of Virology, 2017Co-Authors: Mitsuhiro Machitani, Keisaku Wakabayashi, Masashi Tachibana, Fuminori Sakurai, Kosuke Nakatani, Hiroyuki MizuguchiAbstract:: Recent studies have reported that host microRNAs (miRNAs) regulate infections by several types of viruses via VArious mechanisms and that inhibition of the miRNA processing factors enhances or prevents viral infection. However, it has not been clarified whether these effects of miRNAs extend to adenovirus (Ad) infection. Here we show that miR-27a and -b efficiently inhibit infection with an Ad via the downregulation of SNAP25 and TXN2, which are members of the SNARE proteins and the thioredoxin family, respectively. Approximately 80% reductions in Ad genomic copy number were found in cells transfected with miR-27a/b mimics, whereas there were approximately 2.5- to 5-fold larger copy numbers of the Ad genome following transfection with miR-27a/b inhibitors. Microarray gene expression analysis and in silico analysis demonstrated that SNAP25 and TXN2 are target genes of miR-27a/b. A reporter assay using plasmids containing the 3' untranslated regions of the SNAP25 and TXN2 genes showed that miR-27a/b directly suppressed SNAP25 and TXN2 expression through posttranscriptional gene silencing. Knockdown of SNAP25 led to a significant inhibition of Ad entry into cells. Knockdown of TXN2 induced cell cycle arrest at G1 phase, leading to a reduction in Ad replication. In addition, overexpression of Ad-encoded small noncoding RNAs (VA-RNAs) restored the miR-27a/b-mediated reduction in infection level with a VA-RNA-lacking Ad mutant due to the VA-RNA-mediated inhibition of miR-27a/b expression. These results indicate that miR-27a and -b suppress SNAP25 and TXN2 expression via posttranscriptional gene silencing, leading to efficient suppression of Ad infection.IMPORTANCE Adenovirus (Ad) is widely used as a platform for replication-incompetent Ad vectors (Adv) and replication-competent oncolytic Ad (OAd) in gene therapy and virotherapy. Regulation of Ad infection is highly important for efficient gene therapies using both Adv and OAd. In this study, we demonstrate that miR-27a and -b, which are widely expressed in host cells, suppress SNAP25 and TXN2 expression through posttranscriptional gene silencing. Suppression of SNAP25 and TXN2 expression leads to inhibition of Ad entry into cells and to cell cycle arrest, respectively, leading to efficient suppression of Ad infection. Our findings provide important clues to the improvement of gene therapies using both Adv and OAd.
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Enhanced Oncolytic Activities of the Telomerase-Specific Replication-Competent Adenovirus Expressing Short-Hairpin RNA against Dicer
Molecular cancer therapeutics, 2016Co-Authors: Mitsuhiro Machitani, Keisaku Wakabayashi, Masashi Tachibana, Fuminori Sakurai, Toshiyoshi Fujiwara, Hiroyuki MizuguchiAbstract:Oncolytic viruses have been receiving much attention as potential agents for cancer treatment. Among the VArious types of oncolytic viruses, the telomerase-specific replication-competent adenovirus (TRAD), which carries the tumor-specific promoter-driven E1 gene expression cassette, exhibits efficient antitumor effects. The development of a novel TRAD that shows higher replication efficiency and antitumor activity would be highly beneficial for safer and more efficient cancer therapy. We recently demonstrated that the endoribonuclease Dicer significantly inhibits the replication of wild-type adenovirus (Ad) via the processing of viral-associated (VA)-RNAs, which are Ad-encoded small noncoding RNAs, and that the knockdown of Dicer leads to enhanced VA-RNA expression and Ad replication after infection with wild-type Ad. Based on these findings, we herein developed a novel TRAD expressing short-hairpin RNA against Dicer (shDicer; TRAD-shDicer). After infection, TRAD-shDicer efficiently induced the knockdown of Dicer. TRAD-shDicer showed significantly higher replication efficiency and tumor cell lysis activity compared with the conventional TRAD in tumor cells. The Dicer expression levels and viabilities of normal cells were not altered by infection with TRAD-shDicer. These results indicate that TRAD-shDicer is a potent antitumor reagent by virtue of its enhanced oncolytic activity. Mol Cancer Ther; 16(1); 251–9. ©2016 AACR.
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Quantitative Analysis of Virus-associated RNAI Expression following Transduction with a Replication-incompetent Adenovirus Vector In Vitro and In Vivo
Journal of Molecular and Genetic Medicine, 2014Co-Authors: Keisaku Wakabayashi, Mitsuhiro Machitani, Masashi Tachibana, Fuminori Sakurai, Kahori Shimizu, Hiroyuki MizuguchiAbstract:The adenovirus (Ad) genome encodes one or two non-coding small RNAs called virus-associated (VA)-RNAs, that are transcribed by polymerase III and support Ad replication. As previously reported, a replication-incompetent Ad vector, which is widely used in not only gene therapy studies, including clinical trials, but also basic researches as a gene delivery vehicle, as well as wild-type Ad (WT-Ad) express VA-RNAs, and VA-RNAs actiVAte innate immunity, including the production of type I interferons. In addition, VA-RNAs perturb cellular microRNA (miRNA) expression profiles via competitive inhibition of key components involved in the miRNA maturation pathway. Although these characteristics of VA-RNAs might negatively affect the application of Ad vectors, VA-RNA expression profiles following transduction with an Ad vector have been not fully examined. In this study, we quantitatively analyzed the expression profiles of VA-RNAI, which is a major species of VA-RNAs, following transduction with Ad vectors in vitro and in vivo using real-time RT-PCR. The VA-RNAI expression levels in the cells transduced with a conventional Ad vector expressing luciferase (Ad-CAL2) at a multiplicity of infection (MOI) of 100 were approximately 2000- to 3000-fold lower than those infected with WT-Ad at the same MOI at 48 h after treatment. The expression levels of VA-RNAI in the mouse liver following administration with Ad-CAL2 were approximately 600-fold lower than those following administration with WT-Ad at 48 h post-administration. miRNA-mediated suppression of leaky expression of the Ad E4 genes resulted in about five-fold reduction in the VA-RNAI copy numbers in the liver following systemic administration in mice. These data provide informative clues for the development of novel safer Ad vectors.
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Improving adenovirus vector-mediated RNAi efficiency by lacking the expression of virus-associated RNAs.
Virus research, 2013Co-Authors: Mitsuhiro Machitani, Masashi Tachibana, Fuminori Sakurai, Kazufumi Katayama, Hayato Matsui, Takayuki Suzuki, Taketo Yamaguchi, Hiroyuki MizuguchiAbstract:Several studies have reported that short hairpin RNA (shRNA)-mediated RNA interference (RNAi) was competitively inhibited by the expression of adenovirus (Ad)-encoded small RNAs (VA-RNAs), which are expressed from a replication-incompetent Ad vector, as well as a wild-type Ad; however, it remained to be clarified whether an shRNA-expressing Ad vector-mediated knockdown was inhibited by VA-RNAs transcribed from the same Ad vector genome. In this study, we demonstrated that a lack of VA-RNA expression from the Ad vector leads to an increase in knockdown efficiencies of Ad vector-mediated RNAi. In the cells transduced with a first-generation Ad vector (FG-Ad) expressing shRNA (FG-Ad-shRNA), the copy numbers of shRNA and VA-RNAs incorporated into the RNA-induced silencing complex (RISC) was comparable. In contrast, higher amounts of shRNA were found in the RISC when the cells were transduced with an shRNA-expressing helper-dependent Ad (HD-Ad) vector, in which all viral genes, including VA-RNAs, were deleted (HD-Ad-shRNA), compared with FG-Ad-shRNA. HD-Ad vectors expressing shRNA against luciferase and p53 showed 7.4% and 37.3% increases in the knockdown efficiencies compared to the corresponding FG-Ad-shRNA, respectively, following in vitro transduction. Furthermore, higher levels of knockdown efficiencies were also found by the transduction with shRNA-expressing Ad vectors lacking VA-RNA expression (AdΔVR-shRNA) than by transduction with FG-Ad-shRNA. These results indicate that VA-RNAs expressed from an Ad vector inhibit knockdown by the shRNA-expressing Ad vector and that HD-Ad-shRNA and AdΔVR-shRNA are a powerful framework for shRNA-mediated knockdown.
Fuminori Sakurai - One of the best experts on this subject based on the ideXlab platform.
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a microRNA derived from adenovirus virus associated RNAii promotes virus infection via posttranscriptional gene silencing
Journal of Virology, 2018Co-Authors: Keisaku Wakabayashi, Mitsuhiro Machitani, Masashi Tachibana, Fuminori Sakurai, Hiroyuki MizuguchiAbstract:ABSTRACT The adenovirus (Ad) serotype 5 genome encodes two noncoding small RNAs (virus-associated RNAs I and II [VA-RNAI and -II]), which are approximately 160-nucleotide (nt) RNAs transcribed by RNA polymerase III. It is well known that VA-RNAI supports Ad infection via the inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. Recent studies revealed that VA-RNAs are processed into VA-RNA-derived microRNAs (miRNAs) (miVARNAI and -II); however, we and another group recently demonstrated that miVARNAI does not promote Ad replication. On the other hand, the roles of VA-RNAII and miVARNAII in Ad replication have remained to be clarified. In this study, we demonstrated miVARNAII-mediated promotion of Ad replication. Transfection with chemically synthesized 3′-miVARNAII-138, one of the most abundant forms of miVARNAII, significantly enhanced Ad replication, while the other species of miVARNAII did not. We identified 8 putative target genes of 3′-miVARNAII-138 by microarray analysis and in silico analysis. Among the 8 candidates, knockdown of the cullin 4A (CUL4A) gene, which encodes a component of the ubiquitin ligase complex, most significantly enhanced Ad replication. CUL4A expression was significantly suppressed by 3′-miVARNAII-138 via posttranscriptional gene silencing, indicating that CUL4A is a target gene of 3′-miVARNAII-138 and miVARNAII functions as a viral miRNA promoting Ad infection. It has been reported that CUL4A is involved in degradation of c-Jun, which acts as a transcription factor in the Jun-N-terminal kinase (JNK) signaling cascade. Treatment with JNK inhibitors dramatically suppressed Ad replication, suggesting that miVARNAII-mediated downregulation of CUL4A enhanced JNK signaling and thereby promoted Ad infection. IMPORTANCE Several types of viruses encode viral miRNAs which regulate host and/or viral gene expression via posttranscriptional gene silencing, leading to efficient viral infection. Adenovirus (Ad) expresses miRNAs derived from VA-RNAs (miVARNAI and -II); however, recent studies have revealed that processing of VA-RNAI into miVARNAI inhibits Ad replication. Conversely, we demonstrate here that miVARNAII significantly promotes Ad replication and that miVARNAII-mediated suppression of CUL4A expression via posttranscriptional gene silencing induces accumulation of c-Jun, leading to promotion of Ad infection. These results exhibited the significance of VA-RNAII for supporting Ad infection through a mechanism complementary to that of VA-RNAI. These obserVAtions could provide important clues toward a new perspective on host-virus interaction. Moreover, Ad is widely used as a basic framework for viral vectors and oncolytic viruses. Our findings will help to regulate Ad infection and will promote the development of novel Ad vectors and oncolytic Ad.
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MicroRNA miR-27 Inhibits Adenovirus Infection by Suppressing the Expression of SNAP25 and TXN2.
Journal of Virology, 2017Co-Authors: Mitsuhiro Machitani, Keisaku Wakabayashi, Masashi Tachibana, Fuminori Sakurai, Kosuke Nakatani, Hiroyuki MizuguchiAbstract:: Recent studies have reported that host microRNAs (miRNAs) regulate infections by several types of viruses via VArious mechanisms and that inhibition of the miRNA processing factors enhances or prevents viral infection. However, it has not been clarified whether these effects of miRNAs extend to adenovirus (Ad) infection. Here we show that miR-27a and -b efficiently inhibit infection with an Ad via the downregulation of SNAP25 and TXN2, which are members of the SNARE proteins and the thioredoxin family, respectively. Approximately 80% reductions in Ad genomic copy number were found in cells transfected with miR-27a/b mimics, whereas there were approximately 2.5- to 5-fold larger copy numbers of the Ad genome following transfection with miR-27a/b inhibitors. Microarray gene expression analysis and in silico analysis demonstrated that SNAP25 and TXN2 are target genes of miR-27a/b. A reporter assay using plasmids containing the 3' untranslated regions of the SNAP25 and TXN2 genes showed that miR-27a/b directly suppressed SNAP25 and TXN2 expression through posttranscriptional gene silencing. Knockdown of SNAP25 led to a significant inhibition of Ad entry into cells. Knockdown of TXN2 induced cell cycle arrest at G1 phase, leading to a reduction in Ad replication. In addition, overexpression of Ad-encoded small noncoding RNAs (VA-RNAs) restored the miR-27a/b-mediated reduction in infection level with a VA-RNA-lacking Ad mutant due to the VA-RNA-mediated inhibition of miR-27a/b expression. These results indicate that miR-27a and -b suppress SNAP25 and TXN2 expression via posttranscriptional gene silencing, leading to efficient suppression of Ad infection.IMPORTANCE Adenovirus (Ad) is widely used as a platform for replication-incompetent Ad vectors (Adv) and replication-competent oncolytic Ad (OAd) in gene therapy and virotherapy. Regulation of Ad infection is highly important for efficient gene therapies using both Adv and OAd. In this study, we demonstrate that miR-27a and -b, which are widely expressed in host cells, suppress SNAP25 and TXN2 expression through posttranscriptional gene silencing. Suppression of SNAP25 and TXN2 expression leads to inhibition of Ad entry into cells and to cell cycle arrest, respectively, leading to efficient suppression of Ad infection. Our findings provide important clues to the improvement of gene therapies using both Adv and OAd.
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Enhanced Oncolytic Activities of the Telomerase-Specific Replication-Competent Adenovirus Expressing Short-Hairpin RNA against Dicer
Molecular cancer therapeutics, 2016Co-Authors: Mitsuhiro Machitani, Keisaku Wakabayashi, Masashi Tachibana, Fuminori Sakurai, Toshiyoshi Fujiwara, Hiroyuki MizuguchiAbstract:Oncolytic viruses have been receiving much attention as potential agents for cancer treatment. Among the VArious types of oncolytic viruses, the telomerase-specific replication-competent adenovirus (TRAD), which carries the tumor-specific promoter-driven E1 gene expression cassette, exhibits efficient antitumor effects. The development of a novel TRAD that shows higher replication efficiency and antitumor activity would be highly beneficial for safer and more efficient cancer therapy. We recently demonstrated that the endoribonuclease Dicer significantly inhibits the replication of wild-type adenovirus (Ad) via the processing of viral-associated (VA)-RNAs, which are Ad-encoded small noncoding RNAs, and that the knockdown of Dicer leads to enhanced VA-RNA expression and Ad replication after infection with wild-type Ad. Based on these findings, we herein developed a novel TRAD expressing short-hairpin RNA against Dicer (shDicer; TRAD-shDicer). After infection, TRAD-shDicer efficiently induced the knockdown of Dicer. TRAD-shDicer showed significantly higher replication efficiency and tumor cell lysis activity compared with the conventional TRAD in tumor cells. The Dicer expression levels and viabilities of normal cells were not altered by infection with TRAD-shDicer. These results indicate that TRAD-shDicer is a potent antitumor reagent by virtue of its enhanced oncolytic activity. Mol Cancer Ther; 16(1); 251–9. ©2016 AACR.
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Quantitative Analysis of Virus-associated RNAI Expression following Transduction with a Replication-incompetent Adenovirus Vector In Vitro and In Vivo
Journal of Molecular and Genetic Medicine, 2014Co-Authors: Keisaku Wakabayashi, Mitsuhiro Machitani, Masashi Tachibana, Fuminori Sakurai, Kahori Shimizu, Hiroyuki MizuguchiAbstract:The adenovirus (Ad) genome encodes one or two non-coding small RNAs called virus-associated (VA)-RNAs, that are transcribed by polymerase III and support Ad replication. As previously reported, a replication-incompetent Ad vector, which is widely used in not only gene therapy studies, including clinical trials, but also basic researches as a gene delivery vehicle, as well as wild-type Ad (WT-Ad) express VA-RNAs, and VA-RNAs actiVAte innate immunity, including the production of type I interferons. In addition, VA-RNAs perturb cellular microRNA (miRNA) expression profiles via competitive inhibition of key components involved in the miRNA maturation pathway. Although these characteristics of VA-RNAs might negatively affect the application of Ad vectors, VA-RNA expression profiles following transduction with an Ad vector have been not fully examined. In this study, we quantitatively analyzed the expression profiles of VA-RNAI, which is a major species of VA-RNAs, following transduction with Ad vectors in vitro and in vivo using real-time RT-PCR. The VA-RNAI expression levels in the cells transduced with a conventional Ad vector expressing luciferase (Ad-CAL2) at a multiplicity of infection (MOI) of 100 were approximately 2000- to 3000-fold lower than those infected with WT-Ad at the same MOI at 48 h after treatment. The expression levels of VA-RNAI in the mouse liver following administration with Ad-CAL2 were approximately 600-fold lower than those following administration with WT-Ad at 48 h post-administration. miRNA-mediated suppression of leaky expression of the Ad E4 genes resulted in about five-fold reduction in the VA-RNAI copy numbers in the liver following systemic administration in mice. These data provide informative clues for the development of novel safer Ad vectors.
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Improving adenovirus vector-mediated RNAi efficiency by lacking the expression of virus-associated RNAs.
Virus research, 2013Co-Authors: Mitsuhiro Machitani, Masashi Tachibana, Fuminori Sakurai, Kazufumi Katayama, Hayato Matsui, Takayuki Suzuki, Taketo Yamaguchi, Hiroyuki MizuguchiAbstract:Several studies have reported that short hairpin RNA (shRNA)-mediated RNA interference (RNAi) was competitively inhibited by the expression of adenovirus (Ad)-encoded small RNAs (VA-RNAs), which are expressed from a replication-incompetent Ad vector, as well as a wild-type Ad; however, it remained to be clarified whether an shRNA-expressing Ad vector-mediated knockdown was inhibited by VA-RNAs transcribed from the same Ad vector genome. In this study, we demonstrated that a lack of VA-RNA expression from the Ad vector leads to an increase in knockdown efficiencies of Ad vector-mediated RNAi. In the cells transduced with a first-generation Ad vector (FG-Ad) expressing shRNA (FG-Ad-shRNA), the copy numbers of shRNA and VA-RNAs incorporated into the RNA-induced silencing complex (RISC) was comparable. In contrast, higher amounts of shRNA were found in the RISC when the cells were transduced with an shRNA-expressing helper-dependent Ad (HD-Ad) vector, in which all viral genes, including VA-RNAs, were deleted (HD-Ad-shRNA), compared with FG-Ad-shRNA. HD-Ad vectors expressing shRNA against luciferase and p53 showed 7.4% and 37.3% increases in the knockdown efficiencies compared to the corresponding FG-Ad-shRNA, respectively, following in vitro transduction. Furthermore, higher levels of knockdown efficiencies were also found by the transduction with shRNA-expressing Ad vectors lacking VA-RNA expression (AdΔVR-shRNA) than by transduction with FG-Ad-shRNA. These results indicate that VA-RNAs expressed from an Ad vector inhibit knockdown by the shRNA-expressing Ad vector and that HD-Ad-shRNA and AdΔVR-shRNA are a powerful framework for shRNA-mediated knockdown.