Internal ribosome entry site ofRhopalosiphum padivirus is functional in mammalian cells and has cryptic promoter activity in baculovirus-infected Sf21 cells1
Abstract:Aim: To substantiate the in vitro translational studies of a cross-kingdom, internal ribosome entry site (IRES), the 5´untranslated region of the Rhopalosiphum padi virus (RhPV), can function in mammalian cells and act as a shuttle IRES between insect cells and mammalian cells. Methods: Cytomegalovirus (CMV) promoter-based bicistronic mammalian cell expression vectors, either in plasmids or baculovirus vectors, were generated. Plasmid transient transfection and baculovirus transduction assays were performed to… Show more
“…Figure 4Efficiency of baculovirus-incorporated promoters in C6/36 cells. ( a ) Schematic representation of transfer vectors used to generate the recombinant baculoviruses expressing EGFP driven by several baculovirus, mammalian viral, and mosquito host promoters from the following genes: pag1 , a HzNV-1 viral early expressing gene 23 ; p10 , baculovirus late gene 46 ; cmv , cytomegalovirus 47 ; sv40 , simian virus 40 24 ; cir , chimeric internal ribosome entry site (IRES) of RhPV virus and EV71 virus 48,49 ; b1 , cecropin b1 gene 50 ; pub , polyubiquitin gene 42 and a4 , defensin a4 gene 51 . ( b ) Fluorescence images.…”
Efficient gene delivery technologies play an essential role in the gene functional analyses that are necessary for basic and applied researches. Mosquitoes are ubiquitous insects, responsible for transmitting many deadly arboviruses causing millions of human deaths every year. The lack of efficient and flexible gene delivery strategies in mosquitoes are among the major hurdles for the study of mosquito biology and mosquito-pathogen interactions. We found that Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type baculovirus species, can efficiently transduce mosquito cells without viral propagation, allowing high level gene expression upon inducement by suitable promoters without obvious negative effects on cell propagation and viability. AcMNPV transduces into several mosquito cell types, efficiently than in commonly used mammalian cell lines and classical plasmid DNA transfection approaches. We demonstrated the application of this system by expressing influenza virus neuraminidase (NA) into mosquito hosts. Moreover, AcMNPV can transduce both larvae and adults of essentially all blood-sucking mosquito genera, resulting in bright fluorescence in insect bodies with little or no tissue barriers. Our experiments establish baculovirus as a convenient and powerful gene delivery vector in vitro and in vivo that will greatly benefit research into mosquito gene regulation, development and the study of mosquito-borne viruses.
“…Figure 4Efficiency of baculovirus-incorporated promoters in C6/36 cells. ( a ) Schematic representation of transfer vectors used to generate the recombinant baculoviruses expressing EGFP driven by several baculovirus, mammalian viral, and mosquito host promoters from the following genes: pag1 , a HzNV-1 viral early expressing gene 23 ; p10 , baculovirus late gene 46 ; cmv , cytomegalovirus 47 ; sv40 , simian virus 40 24 ; cir , chimeric internal ribosome entry site (IRES) of RhPV virus and EV71 virus 48,49 ; b1 , cecropin b1 gene 50 ; pub , polyubiquitin gene 42 and a4 , defensin a4 gene 51 . ( b ) Fluorescence images.…”
Efficient gene delivery technologies play an essential role in the gene functional analyses that are necessary for basic and applied researches. Mosquitoes are ubiquitous insects, responsible for transmitting many deadly arboviruses causing millions of human deaths every year. The lack of efficient and flexible gene delivery strategies in mosquitoes are among the major hurdles for the study of mosquito biology and mosquito-pathogen interactions. We found that Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type baculovirus species, can efficiently transduce mosquito cells without viral propagation, allowing high level gene expression upon inducement by suitable promoters without obvious negative effects on cell propagation and viability. AcMNPV transduces into several mosquito cell types, efficiently than in commonly used mammalian cell lines and classical plasmid DNA transfection approaches. We demonstrated the application of this system by expressing influenza virus neuraminidase (NA) into mosquito hosts. Moreover, AcMNPV can transduce both larvae and adults of essentially all blood-sucking mosquito genera, resulting in bright fluorescence in insect bodies with little or no tissue barriers. Our experiments establish baculovirus as a convenient and powerful gene delivery vector in vitro and in vivo that will greatly benefit research into mosquito gene regulation, development and the study of mosquito-borne viruses.
“…TAAG sequences are relatively rare in the AcMNPV genome and are found primarily in late or very late promoter regions [ 20 ]. However, the cDNA of the RhPV 5′UTR IRES possesses six TAAG motifs and can act as a cryptic promoter in baculovirus-infected Sf21 cells [ 19 ]. In order to characterize which of the six TAAG motifs mediated the promoter activity, truncated RhPV 5′UTR sequences were produced by making stepwise deletions from either ends; then, these truncated sequences were subject to functional promoter assays.…”
Section: Resultsmentioning
confidence: 99%
“…The M300/429 fragment that spans the RP110 promoter region also exhibited IRES activity in all of the tested translation systems [ 24 ]. Our previous studies also demonstrated that the 579 nts RhPV 5′UTR function as an IRES, but not as a promoter, in mammalian cells [ 19 ]. Thus, it would be interesting to explore whether the 110 nt RP110 promoter or any other constructs listed in Figure 1 A can function as an IRES element in mammalian cells, given that no promoter activity was found in vCMV-DRhir(L1-2)E, vCMV-DRhir(L5-6)E or vCMV-DRhir(L6)E infected Sf21 cells ( Figure 2 ).…”
Section: Resultsmentioning
confidence: 99%
“…Due to its cross-kingdom activity, the RhPV 5′UTR IRES has the potential to be used in eukaryotic cell expression vectors. Interesting, we have shown that the cDNA of the RhPV 5′UTR IRES possesses six TAAG motifs and can act as a cryptic promoter activity in baculovirus-infected Sf21 cells [ 19 ]. In this report, we demonstrate that a small fragment in the RhPV 5′UTR IRES cDNA, corresponding to nt 309–418, not only retains significant IRES activity in CHO cells, but also possesses baculovirus-dependent promoter activity in Sf21 insect cells.…”
The 579-nucleotide 5′ untranslated region (5′UTR) of the Rhopalosiphum padi virus (RhPV) possesses a cross-kingdom internal ribosome entry site (IRES) activity that functions in insect, mammalian, and plant-derived in vitro translation systems, and six TAAG motifs within the DNA fragment encoding the RhPV 5′UTR were previously found to confer the RhPV 5′UTR with late promoter activity in baculovirus. In the present study, various truncated RhPV 5′UTR sequences were produced, and among them, a fragment of 110 bp ranging from nucleotides 309 to 418 was identified to be the shortest fragment responsible for the late promoter activity in baculovirus infected Sf21 cells. This 110 bp fragment contains a TAAG tandem repeat that retains more than 60% of the late promoter activity of the full length RhPV 5′UTR sequence. Further, IRES activity remained unchanged in all truncated RhPV 5′UTR constructs. Taken together, this novel 110 bp fragment having late promoter activity in baculovirus as well as IRES activity in mammalian cell, renders it a useful tool for the development of a “shuttle” bi-cistronic baculovirus gene expression and/or delivery vector.
“…The Spodoptera frugiperda IPBL-Sf21 (Sf21) cell line was cultured in TNM-FH insect medium containing 8% heat-inactivated fetal bovine serum (FBS) at 27 °C [ 68 ]. The U-2OS (human osteogenic sarcoma cell line, purchased from Bioresource Collection and Research Center 331, Shih-Pin Rd., Hsinchu 300193, Taiwan) cells were grown in McCoy’s medium supplemented with 10% fetal bovine serum [ 69 ]. Cellfectin (Invitrogen, Carlsbad, CA, USA) was used for transfection in Sf21 cells according to the manufacturer’s protocol.…”
Chikungunya virus (CHIKV) is a mosquito-transmitted infectious agent that causes an endemic or epidemic outbreak(s) of Chikungunya fever that is reported in almost all countries. This virus is an intense global threat, due to its high rate of contagion and the lack of effective remedies. In this study, we developed two baculovirus expression vector system (BEVS)-based approaches for the screening of anti-CHIKV drugs in Spodoptera frugiperda insect (Sf21) cells and U-2OS cells. First, structural protein of CHIKV was co-expressed through BEVS and thereby induced cell fusion in Sf21 cells. We used an internal ribosome entry site (IRES) to co-express the green fluorescent protein (EGFP) for identifying these fusion events. The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form syncytia. We identified that ursolic acid has potential anti-CHIKV activity in vitro, by using this approach. Second, BacMam virus-based gene delivery has been successfully applied for the transient expression of non-structural proteins with a subgenomic promoter-EGFP (SP-EGFP) cassette in U-2OS cells to act as an in vitro CHIKV replicon system. Our BacMam-based screening system has identified that the potential effects of baicalin and baicalein phytocompounds can inhibit the replicon activity of CHIKV in U-2OS cells. In conclusion, our results suggested that BEVS can be a potential tool for screening drugs against CHIKV.
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