scholarly journals A Reverse Genetics System for Cypovirus Based on a Bacmid Expressing T7 RNA Polymerase

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 314 ◽  
Author(s):  
Gaobo Zhang ◽  
Jian Yang ◽  
Fujun Qin ◽  
Congrui Xu ◽  
Jia Wang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
T7 Rna Polymerase ◽  
Sf9 Cells ◽  
Genomic Segment ◽  
Egfp Gene ◽  
A Genome ◽  
Typical Morphology ◽  
Occlusion Bodies ◽  
Rescue System

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.

scholarly journals Recovery of Avian Metapneumovirus Subgroup C from cDNA: Cross-Recognition of Avian and Human Metapneumovirus Support Proteins

2006 ◽  
Vol 80 (12) ◽  
pp. 5790-5797 ◽  
Author(s):  
Dhanasekaran Govindarajan ◽  
Ursula J. Buchholz ◽  
Siba K. Samal
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Viral Vector ◽  
The United States ◽  
Human Metapneumovirus ◽  
T7 Rna Polymerase ◽  
Foreign Protein ◽  
Economic Losses ◽  
Avian Metapneumovirus

ABSTRACT Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with “swollen head syndrome” in chickens, contributing to significant economic losses for the U.S. poultry industry. With a long-term goal of developing a better vaccine for controlling AMPV in the United States, we established a reverse genetics system to produce infectious AMPV of subgroup C entirely from cDNA. A cDNA clone encoding the entire 14,150-nucleotide genome of AMPV subgroup C strain Colorado (AMPV/CO) was generated by assembling five cDNA fragments between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a transcription plasmid, pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1, and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. Characterization of the recombinant AMPV/CO showed that its growth properties in tissue culture were similar to those of the parental virus. The potential of AMPV/CO to serve as a viral vector was also assessed by generating another recombinant virus, rAMPV/CO-GFP, that expressed the enhanced green fluorescent protein (GFP) as a foreign protein. Interestingly, GFP-expressing AMPV and GFP-expressing human metapneumovirus (HMPV) could be recovered using the support plasmids of either virus, denoting that the genome promoters are conserved between the two metapneumoviruses and can be cross-recognized by the polymerase complex proteins of either virus. These results indicate a close functional relationship between AMPV/CO and HMPV.

scholarly journals A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei

2020 ◽  
Author(s):  
Sebastian Shaw ◽  
Sebastian Knüsel ◽  
Sarah Hoenner ◽  
Isabel Roditi
Keyword(s):  
Rna Polymerase ◽  
Cell Line ◽  
Genome Editing ◽  
Trypanosoma Brucei ◽  
Transient Expression ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
A Genome ◽  
Pcr Products

Abstract Objective Generation of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any trypanosome cell line. Results We describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes.

scholarly journals A reverse-genetics system for Influenza A virus using T7 RNA polymerase

2007 ◽  
Vol 88 (4) ◽  
pp. 1281-1287 ◽  
Author(s):  
Emmie de Wit ◽  
Monique I. J. Spronken ◽  
Gaby Vervaet ◽  
Guus F. Rimmelzwaan ◽  
Albert D. M. E. Osterhaus ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
T7 Rna Polymerase ◽  
Influenza Virus A ◽  
Sense Orientation ◽  
Polymerase I ◽  
Rna Polymerase Promoter

The currently available reverse-genetics systems for Influenza A virus are all based on transcription of genomic RNA by RNA polymerase I, but the species specificity of this polymerase is a disadvantage. A reverse-genetics vector containing a T7 RNA polymerase promoter, hepatitis delta virus ribozyme sequence and T7 RNA polymerase terminator sequence has been developed. To achieve optimal expression in minigenome assays, it was determined that viral RNA should be inserted in this vector in the negative-sense orientation with two additional G residues downstream of the T7 RNA polymerase promoter. It was also shown that expression of the minigenome was more efficient when a T7 RNA polymerase with a nuclear-localization signal was used. By using this reverse-genetics system, recombinant influenza virus A/PR/8/34 was produced more efficiently than by using a similar polymerase I-based reverse-genetics system. Furthermore, influenza virus A/NL/219/03 could be rescued from 293T, MDCK and QT6 cells. Thus, a reverse-genetics system for the rescue of Influenza A virus has been developed, which will be useful for fundamental research and vaccine seed strain production in a variety of cell lines.

scholarly journals Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus

2001 ◽  
Vol 75 (24) ◽  
pp. 12359-12369 ◽  
Author(s):  
Rosa Casais ◽  
Volker Thiel ◽  
Stuart G. Siddell ◽  
David Cavanagh ◽  
Paul Britton
Keyword(s):  
Molecular Biology ◽  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Infectious Bronchitis Virus ◽  
Reverse Genetics ◽  
Rna Virus ◽  
Full Length ◽  
T7 Rna Polymerase ◽  
Infectious Bronchitis

ABSTRACT Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.

scholarly journals A plasmid-based reverse genetics system for mammalian orthoreoviruses driven by a plasmid-encoded T7 RNA polymerase

2014 ◽  
Vol 196 ◽  
pp. 36-39 ◽  
Author(s):  
Satoshi Komoto ◽  
Takahiro Kawagishi ◽  
Takeshi Kobayashi ◽  
Mine Ikizler ◽  
Jason Iskarpatyoti ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
T7 Rna Polymerase

scholarly journals Rescue of Akabane virus (family Bunyaviridae) entirely from cloned cDNAs by using RNA polymerase I

2007 ◽  
Vol 88 (12) ◽  
pp. 3385-3390 ◽  
Author(s):  
Yohsuke Ogawa ◽  
Keita Sugiura ◽  
Kentaro Kato ◽  
Yukinobu Tohya ◽  
Hiroomi Akashi
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
T7 Rna Polymerase ◽  
Akabane Virus ◽  
Reverse Genetic ◽  
Structural Protein ◽  
Virus Family ◽  
The Family ◽  
Rescue System ◽  
Polymerase I

Reverse-genetic systems are often used to study different aspects of the viral life cycle. To date, three rescue systems have been developed for the family Bunyaviridae. These systems use T7 RNA polymerase, which is generally used in rescue systems for Mononegavirales. In the present study, we describe a rescue system for Akabane virus (family Bunyaviridae) that uses cDNAs and RNA polymerase I instead of T7 RNA polymerase. The utility of this system was demonstrated by the generation of a mutant with a deletion of the non-structural protein (NSs) on the S RNA segment. These results offer a new option for bunyavirus rescue.

scholarly journals African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Heather E. Eaton ◽  
Takeshi Kobayashi ◽  
Terence S. Dermody ◽  
Randal N. Johnston ◽  
Philippe H. Jais ◽  
...  
Keyword(s):  
Protein Expression ◽  
Rna Polymerase ◽  
Reverse Genetics ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Protein Translation ◽  
Fever Virus ◽  
T7 Rna Polymerase ◽  
Capping Enzyme ◽  
Reovirus Replication

ABSTRACT Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5′ nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure.

scholarly journals A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei

2020 ◽  
Author(s):  
Sebastian Shaw ◽  
Sebastian Knüsel ◽  
Sarah Hoenner ◽  
Isabel Roditi
Keyword(s):  
Rna Polymerase ◽  
Genome Editing ◽  
Trypanosoma Brucei ◽  
Transient Expression ◽  
Cell Lines ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
A Genome ◽  
Pcr Products

Abstract ObjectiveGeneration of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any cell line.ResultsWe describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes.

scholarly journals Recovery of genetically defined murine norovirus in tissue culture by using a fowlpox virus expressing T7 RNA polymerase

2007 ◽  
Vol 88 (8) ◽  
pp. 2091-2100 ◽  
Author(s):  
Yasmin Chaudhry ◽  
Michael A. Skinner ◽  
Ian G. Goodfellow
Keyword(s):  
Tissue Culture ◽  
Rna Polymerase ◽  
Reverse Genetics ◽  
Low Cost ◽  
T7 Rna Polymerase ◽  
Murine Norovirus ◽  
Fowlpox Virus ◽  
Negative Effect ◽  
Virus Recovery ◽  
First Time

Despite the significant disease burden caused by human norovirus infection, an efficient tissue-culture system for these viruses remains elusive. Murine norovirus (MNV) is an ideal surrogate for the study of norovirus biology, as the virus replicates efficiently in tissue culture and a low-cost animal model is readily available. In this report, a reverse-genetics system for MNV is described, using a fowlpox virus (FWPV) recombinant expressing T7 RNA polymerase to recover genetically defined MNV in tissue culture for the first time. These studies demonstrated that approaches that have proved successful for other members of the family Caliciviridae failed to lead to recovery of MNV. This was due to our observation that vaccinia virus infection had a negative effect on MNV replication. In contrast, FWPV infection had no deleterious effect and allowed the recovery of infectious MNV from cells previously transfected with MNV cDNA constructs. These studies also indicated that the nature of the 3′-terminal nucleotide is critical for efficient virus recovery and that inclusion of a hepatitis delta virus ribozyme at the 3′ end can increase the efficiency with which virus is recovered. This system now allows the recovery of genetically defined noroviruses and will facilitate the analysis of the effects of genetic variation on norovirus pathogenesis.

scholarly journals Rescue of Recombinant Thogoto Virus from Cloned cDNA

2001 ◽  
Vol 75 (19) ◽  
pp. 9282-9286 ◽  
Author(s):  
Elke Wagner ◽  
Othmar G. Engelhardt ◽  
Simone Gruber ◽  
Otto Haller ◽  
Georg Kochs
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
Infectious Virus ◽  
Rna Polymerase I ◽  
Structural Proteins ◽  
A Genome ◽  
Viral Genes ◽  
Cloned Cdna ◽  
Polymerase I ◽  
Expression Plasmids

ABSTRACT Thogoto virus (THOV) is a tick-transmitted orthomyxovirus with a genome consisting of six negative-stranded RNA segments. To rescue a recombinant THOV, the viral structural proteins were produced from expression plasmids by means of a vaccinia virus expressing the T7 RNA polymerase. Genomic virus RNAs (vRNAs) were generated from plasmids under the control of the RNA polymerase I promoter. Using this system, we could efficiently recover recombinant THOV following transfection of 12 plasmids into 293T cells. To verify the recombinant nature of the rescued virus, specific genetic tags were introduced into two vRNA segments. The availability of this efficient reverse genetics system will allow us to address hitherto-unanswered questions regarding the biology of THOV by manipulating viral genes in the context of infectious virus.

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