A plasmid-based reverse genetics system for animal double-stranded RNA viruses.
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ABSTRACT:
SUBMITTER: Kobayashi T
PROVIDER: S-EPMC2034303 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.
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Project description:Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein-tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.
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Project description:Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. HuNoV replication studies have been hampered by the inability to grow the virus in cultured cells. The HuNoV genome is a positive-sense single-stranded RNA (ssRNA) molecule with three open reading frames (ORFs). We established a reverse genetics system driven by a mammalian promoter that functions without helper virus. The complete genome of the HuNoV genogroup II.3 U201 strain was cloned downstream of an elongation factor-1? (EF-1?) mammalian promoter. Cells transfected with plasmid containing the full-length genome (pHuNoVU201F) expressed the ORF1 polyprotein, which was cleaved by the viral protease to produce the mature nonstructural viral proteins, and the capsid proteins. Progeny virus produced from the transfected cells contained the complete NoV genomic RNA (VP1, VP2, and VPg) and exhibited the same density in isopycnic cesium chloride gradients as native infectious NoV particles from a patient's stool. This system also was applied to drive murine NoV RNA replication and produced infectious progeny virions. A GFP reporter construct containing the GFP gene in ORF1 produced complete virions that contain VPg-linked RNA. RNA from virions containing the encapsidated GFP-genomic RNA was successfully transfected back into cells producing fluorescent puncta, indicating that the encapsidated RNA is replication-competent. The EF-1? mammalian promoter expression system provides the first reverse genetics system, to our knowledge, generalizable for human and animal NoVs that does not require a helper virus. Establishing a complete reverse genetics system expressed from cDNA for HuNoVs now allows the manipulation of the viral genome and production of reporter virions.
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Project description:BACKGROUND:Post-transcriptional gene silencing (PTGS) by short interfering RNA has opened up new directions in the phenotypic mutation of cellular genes. However, its efficacy on non-nuclear genes and its effect on the interferon pathway remain unexplored. Since directed mutation of RNA genomes is not possible through conventional mutagenesis, we have tested sequence-specific 21-nucleotide long double-stranded RNAs (dsRNAs) for their ability to silence cytoplasmic RNA genomes. RESULTS:Short dsRNAs were generated against specific mRNAs of respiratory syncytial virus, a nonsegmented negative-stranded RNA virus with a cytoplasmic life cycle. At nanomolar concentrations, the dsRNAs specifically abrogated expression of the corresponding viral proteins, and produced the expected mutant phenotype ex vivo. The dsRNAs did not induce an interferon response, and did not inhibit cellular gene expression. The ablation of the viral proteins correlated with the loss of the specific mRNAs. In contrast, viral genomic and antigenomic RNA, which are encapsidated, were not directly affected. CONCLUSIONS:Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases. RNA-directed RNA gene silencing does not require cloning, expression, and mutagenesis of viral cDNA, and thus, will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle. This will, for the first time, permit functional genomic studies, attenuated infections, reverse genetic analysis, and studies of host-virus signaling pathways using a wild type RNA virus, unencumbered by any superinfecting virus.
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Project description:Mycoviral population dynamics in Spanish isolates of the entomopathogenic fungus Beauveria bassiana
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Project description:Reverse genetics is key technology for producing wild-type and genetically modified viruses. The ISA (Infectious Subgenomic Amplicons) method is a recent versatile and user-friendly reverse genetics method to rescue RNA viruses. The main constraint of its canonic protocol was the requirement to produce (e.g., by DNA synthesis or fusion PCR) 5' and 3' modified genomic fragments encompassing the human cytomegalovirus promoter (pCMV) and the hepatitis delta virus ribozyme/simian virus 40 polyadenylation signal (HDR/SV40pA), respectively. Here, we propose the ultimately simplified "Haiku" designs in which terminal pCMV and HDR/SV40pA sequences are provided as additional separate DNA amplicons. This improved procedure was successfully applied to the rescue of a wide range of viruses belonging to genera Flavivirus, Alphavirus and Enterovirus in mosquito or mammalian cells using only standard PCR amplification techniques and starting from a variety of original materials including viral RNAs extracted from cell supernatant media or animal samples. We also demonstrate that, in specific experimental conditions, the presence of the HDR/SV40pA is not necessary to rescue the targeted viruses. These ultimately simplified "Haiku" designs provide an even more simple, rapid, versatile and cost-effective tool to rescue RNA viruses since only generation of overlapping amplicons encompassing the entire viral genome is now required to generate infectious virus. This new approach may completely modify our capacity to obtain infectious RNA viruses.
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Project description:Dendrolimus punctatus cypovirus (DpCPV), belonging to the genus Cypovirus within the family Reoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed a vp80-knockout Autographa californica multiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of a HindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing an egfp gene flanked with the 5' and 3' untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infect Spodopetra exigua larvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.
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Project description:Reverse genetic systems are essential for the study of RNA viruses. Infectious clones remain the most widely used systems to manipulate viral genomes. Recently, a new PCR-based method called ISA (infectious subgenomic amplicons) has been developed. This approach has resulted in greater genetic diversity of the viral populations than that observed using infectious clone technology. However, for some studies, generation of clonal viral populations is necessary. In this study, we used the tick-borne encephalitis virus as model to demonstrate that utilization of a very high-fidelity, DNA-dependent DNA polymerase during the PCR step of the ISA procedure gives the possibility to reduce the genetic diversity of viral populations. We also concluded that the fidelity of the polymerase is not the only factor influencing this diversity. Studying the impact of genotype modification on virus phenotype is a crucial step for the development of reverse genetic methods. Here, we also demonstrated that the utilization of different PCR polymerases did not affect the phenotype (replicative fitness in cellulo and virulence in vivo) compared to the initial ISA procedure and the use of an infectious clone. In conclusion, we provide here an approach to control the genetic diversity of RNA viruses without modifying their phenotype.
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Project description:Bluetongue (BT) disease, caused by the non-enveloped bluetongue virus (BTV) belonging to the Reoviridae family, is an economically important disease that affects a wide range of wild and domestic ruminants. Currently, 26 different serotypes of BTV are recognized in the world, of which BTV-8 has been found to exhibit one of the most virulent manifestations of BT disease in livestock. In recent years incursions of BTV-8 in Europe have resulted in significant morbidity and mortality not only in sheep but also in cattle. The molecular and genetic basis of BTV-8 pathogenesis is not known. To understand the genetic basis of BTV-8 pathogenicity, we generated reassortant viruses by replacing the 3 most variable genes, S2, S6 and S10 of a recent isolate of BTV-8, in different combinations into the backbone of an attenuated strain of BTV-1. The growth profiles of these reassortant viruses were then analyzed in two different ovine cell lines derived from different organs, kidney and thymus. Distinct patterns for each reassortant virus in these two cell lines were observed. To determine the pathogenicity of these reassortant viruses, groups of BTV-susceptible sheep were infected with each of these viruses. The data suggested that the clinical manifestations of these two different serotypes, BTV-1 and BTV-8, were slightly distinct and BTV-1, when comprising all 3 genome segments of BTV-8, behaved differently to BTV-1. Our results also suggested that the molecular basis of BT disease is highly complex.
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