Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5'UTR of Internal Ribosomal Entry Site

Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.

Artificial intelligence and machine learning could support drug development for hepatitis A virus internal ribosomal entry sites

Hepatitis A virus(HAV)infection is still an important health issue worldwide.Although several effective HAV vaccines are available,it is difficult to perform universal vaccination in certain countries.Therefore,it may be better to develop antivirals against HAV for the prevention of severe hepatitis A.We found that several drugs potentially inhibit HAV internal ribosomal entry site-dependent translation and HAV replication.Artificial intelligence and machine learning could also support screening of anti-HAV drugs,using drug repositioning and drug rescue approaches.

Phylogeny of Ptychostomum (Bryaceae,Musci) inferred from sequences of nuclear ribosomal DNA internal transcribed spacer (ITS) and chloroplast rps4

The phylogeny of Ptychostomum was first spacer （ITS） region of the nuclear ribosomal （nr） DNA DNA rps4 sequences. Maximum parsimony, maximum undertaken based on analysis of the internal transcribed and by combining data from nrDNA ITS and chloroplast likelihood, and Bayesian analyses all support the conclusion that the reinstated genus Ptychostomum is not monophyletic. Ptychostomum funkii （Schwagr.） J. R. Spence （≡ Bryum funkii Schwaigr.） is placed within a clade containing the type species of Bryum, B. argenteum Hedw. The remaining members of Ptychostomum investigated in the present study constitute another well-supported clade. The results are congruent with previous molecular analyses. On the basis of phylogenetic evidence, we agree with transferring B. amblyodon Mull. Hal. （≡ B. inclinatum （Brid.） Turton≡ Bryum archangelicum Bruch ＆ Schimp.）, Bryum lonchocaulon Mull. Hal., Bryum pallescens Schleich. ex Schwaigr., and Bryum pallens Sw. to Ptychostomum.

Post-transcriptional regulation of vascular endothelial growth factor: Implications for tumor angiogenesis

Vascular endothelial growth factor （VEGF） is a potent secreted mitogen critical for physiologic and tumor angiogenesis. Regulation of VEGF occurs at several levels, including transcription, mRNA stabilization, translation, and differential cellular localization of various isoforms. Recent advances in our understanding of post-transcriptional regulation of VEGF include identification of the stabilizing mRNA binding protein, HuR, and the discovery of internal ribosomal entry sites in the 5＇UTR of the VEGF mRNA. Monoclonal anti-VEGF antibody was recently approved for use in humans, but suffers from the need for high systemic doses. RNA interference （RNAi） technology is being used in vitro and in animal models with promising results. Here, we review the literature on post-transcriptional regulation of VEGF and describe recent progress in targeting these mechanisms for therapeutic benefit.

CONSTRUCTION OF THE DICISTRONIC ADENOVIRUS VECTOR EXPRESSING BIOACTIVE HUMAN INTERLEUKIN-12

The full-length cDNA encoding the subunits p40 and p35 of human interleukin12(hIL12) were cloned separately by RTPCR, linked together by internal ribosomal entry site (IRES) of encephalomyocarditis virus which initiates capindependent translation to form a dicistronic gene fragment. The dicistronic fragment was placed between the cytomegalovirus (CMV) promoter and SV40 polyA signal to form a dicistronic expression cassette. Subsequently, the dicistronic expression cassette was inserted into E1 region of Ad5 genome in cosmid vector pAx1cw of E1substitution type. By homologous recombination with EcoT22Idigested Ad5 DNATPC in 293 cells, the replicationdeficient recombinant adenoviruses of hIL12 were generated efficiently. After infected with hIL12 recombinant adenoviruses in vitro, 293 cells, human hepatocellular carcinoma cells HepG2, and primary human skin fibroblasts expressed and secreted hIL12 at comparable levels (30～60ng/ 106cells/24hr), which could stimulate the proliferation and IFNγ production of human lymphoblasts. These suggest that the dicistronic adenovirus vector of hIL12 could effectively mediate the expression of bioactive hIL12 and might be used in cancer gene therapy.

RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are required to produce the 56-kDa isoform

We have previously reported that the human ACAT1 gene produces a chimeric mRNA through the interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG1397-1399 and GGC1274-1276 as translation initiation codons to produce normal 50-kDa ACAT1 and a novel enzymatically active 56-kDa isoform, respectively, with the latter being authentically present in human cells, including human monocyte- derived macrophages. In this work, we report that RNA secondary structures located in the vicinity of the GGC1274-1276 codon are required for production of the 56-kDa isoform. The effects of the three predicted stem-loops （nt 1255-1268, 1286-1342 and 1355-1384） were tested individually by transfecting expression plasmids into cells that contained the wild-type, deleted or mutant stem-loop sequences linked to a partial ACAT1 AUG open reading frame （ORF） or to the ORFs of other genes. The expression patterns were monitored by western blot analyses. We found that the upstream stem-loop1255-1268 from chromosome 7 and downstream stem-loop1286-1342 from chromosome 1 were needed for production of the 56-kDa isoform, whereas the last stem-loop135s-1384 from chromosome 1 was dispensable. The results of experi- ments using both monocistronic and bicistronic vectors with a stable hairpin showed that translation initiation from the GGC1274-1276 codon was mediated by an internal ribosome entry site （IRES）. Further experiments revealed that translation initiation from the GGC1274-1276 codon requires the upstream AU-constituted RNA secondary structure and the downstream GC-rich structure. This mechanistic work provides further support for the biological significance of the chimeric nature of the human ACAT1 transcript.

Positional effect of mutations in 5'UTR of hepatitis C virus 4a on patients' response to therapy

AIM： To investigate the effects of mutations in domain Ⅲ of the hepatitis C virus （HCV） internal ribosome entry sequences （IRES） on the response of chronic HCV genotype 4a patients to interferon therapy.METHODS： HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance. IRES domain Ⅲ was cloned and 15 clones for each patient were sequenced. The obtained sequences were aligned with genotype 4a prototype using the ClustaIW program and mutations scored. Prediction of stem-loop secondary structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing.RESULTS： Analysis of RNA secondary structure revealed that insertions in domain Ⅲ altered WatsonCrick base pairing of stems and reduced molecular stability of RNA, which may ultimately reduce binding affinity to ribosomal proteins. Insertion mutations in domain - were statistically more prevalent in sustained viral response patients （SVR, n = 14） as compared to breakthrough （BT, n = 5） patients.CONCLUSION： The influence of mutations within domain Ⅲ on the response of HCV patients to combination therapy depends primarily on the position, but not the frequency, of these mutations within IRES domain Ⅲ.

Establishment of chronic hepatitis C virus infection:Translational evasion of oxidative defence

Hepatitis C virus(HCV)causes a clinically important disease affecting 3%of the world population.HCV is a single-stranded,positive-sense RNA virus belonging to the genus Hepacivirus within the Flaviviridae family.The virus establishes a chronic infection in the face of an active host oxidative defence,thus adaptation to oxidative stress is key to virus survival.Being a small RNA virus with a limited genomic capacity,we speculate that HCV deploys a different strategy to evade host oxidative defence.Instead of counteracting oxidative stress,it utilizes oxidative stress to facilitate its own survival.Translation is the first step in the replication of a plus strand RNA virus so it would make sense if the virus can exploit the host oxidative defence in facilitating this very first step.This is particularly true when HCV utilizes an internal ribosome entry site element in translation,which is distinctive from that of cap-dependent translation of the vast majority of cellular genes,thus allowing selective translation of genes under conditions when global protein synthesis is compromised.Indeed,we were the first to show that HCV translation was stimulated by an important prooxidant-hydrogen peroxide in hepatocytes,suggesting that HCV is able to adapt to and utilize the host antiviral response to facilitate its own translation thus allowing the virus to thrive under oxidative stress condition to establish chronicity.Understanding how HCV translation is regulated under oxidative stress condition will advance our knowledge on how HCV establishes chronicity.As chronicity is the initiator step in disease progression this will eventually lead to a better understanding of pathogenicity,which is particularly relevant to the development of anti-virals and improved treatments of HCV patients using anti-oxidants.

Tricistronic hepatitis C virus subgenomic replicon expressing double transgenes

AIM: To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons.

Immune Responses to a Dicistronic Plasmid Expressing HBsAg of Hepatitis B Virus and Interferon-γ

DNA vaccines encoding a viral protein have been shown to induce antiviral immune responses and provide pro-tection against subsequent viral challenge. The present article deals with the efficacy of a DNA vaccine greatly im-proved by the simultaneous expression of HBsAg and interferon-T gene. We constructed a dual expression vector pHIN encoding the HBsAg of Hepatitis B virus and murine IFN-T which are connected with Internal Ribosome Entry Site（IRES）. Mice immunized with this dual expression DNA vaccine exhibited the enhancement of cellular immune response and increased the production of anti-HBV surface antibody, compared with the mice of single gene expres-sion control. Taken together, these results demonstrate that the application of a cytokine gene in a DNA vaccine for-mulation as an adjuvant can improve its immunigenicity.

Construction of the recombinant vector carrying herpes simplex virus thymidine kinase and cytokine genes expressed in cell line Tca8113

Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell line Tca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E.coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFP-N 3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N 3, and then transferred into E.coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFP-N 3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60% of the total amount. Conclusion: pFGFP-tk-IRES-IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be beneficial for gene therapy in cell line Tca8113.

Structure-function relationship in viral RNA genomes: The case of hepatitis C virus

The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structural units in the genomic RNA, termed functional RNA domains. These elements interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. The genomic RNA of the hepatitis C virus(HCV) is a good model for investigating about conserved structural units. It contains functional domains, defined by highly conserved structural RNA motifs, mostly located in the 5'-untranslatable regions(5'UTRs) and 3'UTR, but also occupying long stretches of the coding sequence. Viral translation initiation is mediated by an internal ribosome entry site located at the 5' terminus of the viral genome and regulated by distal functional RNA domains placed at the 3' end. Subsequent RNA replication strongly depends on the 3'UTR folding and is also influenced by the 5' end of the HCV RNA. Further increase in the genome copy number unleashes the formation of homodimers by direct interaction of two genomic RNA molecules, which are finally packed and released to the extracellular medium. All these processes, as well as transitions between them, are controlled by structural RNA elements that establish a complex, direct and long-distance RNARNA interaction network. This review summarizes current knowledge about functional RNA domains within the HCV RNA genome and provides an overview of the control exerted by direct, long-range RNA-RNA contacts for the execution of the viral cycle.

IRESfinder:Identifying RNA internal ribosome entry site in eukaryotic cell using framed k-mer features

In eukaryotic cells, initiation of protein translation is to recruit the ribosome to a specific mRNA, which is generally dependent on the 5＇ cap structure. However, protein translation can also be initiated in a cap-independent manner by using a cis-regulatory element termed the internal ribosome entry site （IRES）. The first experimentally validated IRES was reported in the poliovirus （Pelletier and Sonenberg, 1988）. Then eukaryotic cellular mRNAs were also validated to contain IRES elements.

外来入侵植物刺萼龙葵(Solanum rostratum Dunal)根际土壤真菌多样性及其次生代谢产物的化感作用研究

【目的】分析外来入侵植物刺萼龙葵根际土壤真菌群落多样性,了解其根际可培养真菌次生代谢产物的植物生长调节活性,并从中筛选具有开发为植物生长调节剂及生物除草剂潜力的菌株。【方法】采集刺萼龙葵根际土壤,提取总DNA,利用高通量测序技术分析土壤真菌群落多样性;同时,利用纯培养手段获得可培养真菌菌株,以乙酸乙酯萃取其发酵产物,并以单子叶植物早熟禾和双子叶植物反枝苋为受试植物,检测其次生代谢产物的促生/抑生活性。【结果】子囊菌门真菌广泛存在于刺萼龙葵根际土壤中,且占绝对优势。Alpha多样性分析发现,刺萼龙葵根际土壤中真菌群落有着非常高的多样性和丰富性。此外,菌株SR02、SR05和SR13对受试植物具有较强的生长抑制作用。【结论】刺萼龙葵根际土壤真菌优势类群明显,尖孢镰刀菌和链格孢在真菌群落中占优势地位;从根际土壤中发现多个具有显著生长促进或抑制作用的菌株,其在刺萼龙葵根际土壤中所起到的生理生态学意义,以及其次生代谢产物是否具有开发为植物生长调节剂及生物农药的潜力,值得进行深入的研究。

Phylogeny and Biogeography of Thuja L. （Cupressaceae）, an Eastern Asian and North American Disjunct Genus

: In order to develop better insights into biogeographic patterns of eastern Asian and North American disjunct plant genera, sequences of nuclear ribosomal DNA internal transcribed spacer (nr DNA ITS) region were used to estimate interspecific relationships of Thuja L. (Cupressaceae) and infer its biogeography based on the phylogeny. According to the phylogenetic analysis, two clades were recognized. The first clade included Thuja plicata D. Don (western North America) and T. koraiensis Nakai (northeastern Asia), and the second one contained T. occidentalis(Gord.) Carr. (Japan). The ancestral area of Thuja was inferred to be eastern Asia, and two dispersal events were responsible for the modern distribution of Thuja in North America. Both the North Atlantic land bridge and Bering land bridge were possible routes for the migration of ancestral populations to North America.

The unbearable lightness of sequenced-based identification

Using the basic GenBank local alignment search tool program(BLAST)to identify fungi collected in a recently protected beech forest at Montricher(Switzerland),the number of ITS sequences associated to the wrong taxon name appears to be around 30%,even higher than previously estimated.Such results rely on the in-depth re-examination of BLAST results for the most interesting species that were collected,viz.first records for Switzerland,rare or patrimonial species and problematic species(when BLAST top scores were equally high for different species),all belonging to Agaricomycotina.This paper dissects for the first time a number of sequence-based identifications,thereby showing in every detail—particularly to the user community of taxonomic information—why sequence-based identification in the context of a fungal inventory can easily go wrong.Our first conclusion is that in-depth examination of BLAST results is too time consuming to be considered as a routine approach for future inventories:we spent two months on verification of approx.20 identifications.Apart from the fact that poor taxon coverage in public depositories remains the principal impediment for successful species identification,it can be deplored that even very recent fungal sequence deposits in GenBank involve an uncomfortably high number of misidentifications or errors with associated metadata.While checking the original publications associated with top score sequences for the few examples that were here re-examined,a positive consequence is that we uncovered over 80 type sequences that were not annotated as types in GenBank.Advantages and pitfalls of sequence-based identification are discussed,particularly in the light of undertaking fungal inventories.Recommendations are made to avoid or reduce some of the major problems with sequence-based identification.Nevertheless,the prospects for a more reliable sequence-based identification of fungi remain quite dim,unless authors are ready to check and update the metadata associated with previously deposited sequences in their publications.