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.

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.

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.

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.

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.

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.

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.