Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis

ABSTRACT The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2′-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

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An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

Viruses ◽

10.3390/v10100547 ◽

2018 ◽

Vol 10 (10) ◽

pp. 547 ◽

Cited By ~ 20

Author(s):

Silvia Márquez-Jurado ◽

Aitor Nogales ◽

Ginés Ávila-Pérez ◽

Francisco Iborra ◽

Luis Martínez-Sobrido ◽

...

Keyword(s):

Cdna Clone ◽

Zika Virus ◽

Rna Synthesis ◽

Infectious Clone ◽

Vero Cells ◽

Viral Rna ◽

Single Amino Acid ◽

Reverse Genetic System ◽

Infectious Clones

The recent outbreaks of Zika virus (ZIKV), its association with Guillain–Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies.

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Synthesis and Characterization of a Full-Length Infectious cDNA Clone of Tomato Mottle Mosaic Virus

Viruses ◽

10.3390/v13061050 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1050

Author(s):

Liqin Tu ◽

Shuhua Wu ◽

Danna Gao ◽

Yong Liu ◽

Yuelin Zhu ◽

...

Keyword(s):

Mosaic Virus ◽

Infection Rate ◽

Cdna Clone ◽

Infectious Clone ◽

Infectious Cdna Clone ◽

Full Length ◽

Economic Losses ◽

Chinese Isolate ◽

Solanum Lycopersicum L ◽

Rigid Rod

Tomato mottle mosaic virus (ToMMV) is a noteworthy virus which belongs to the Virgaviridae family and causes serious economic losses in tomato. Here, we isolated and cloned the full-length genome of a ToMMV Chinese isolate (ToMMV-LN) from a naturally infected tomato (Solanum lycopersicum L.). Sequence analysis showed that ToMMV-LN contains 6399 nucleotides (nts) and is most closely related to a ToMMV Mexican isolate with a sequence identity of 99.48%. Next, an infectious cDNA clone of ToMMV was constructed by a homologous recombination approach. Both the model host N. benthamiana and the natural hosts tomato and pepper developed severe symptoms upon agroinfiltration with pToMMV, which had a strong infectivity. Electron micrographs indicated that a large number of rigid rod-shaped ToMMV virions were observed from the agroinfiltrated N. benthamiana leaves. Finally, our results also confirmed that tomato plants inoculated with pToMMV led to a high infection rate of 100% in 4–5 weeks post-infiltration (wpi), while pepper plants inoculated with pToMMV led to an infection rate of 40–47% in 4–5 wpi. This is the first report of the development of a full-length infectious cDNA clone of ToMMV. We believe that this infectious clone will enable further studies of ToMMV genes function, pathogenicity and virus–host interaction.

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An Overlapping Bacterial Artificial Chromosome System That Generates Vectorless Progeny for Channel Catfish Herpesvirus

Journal of Virology ◽

10.1128/jvi.02152-07 ◽

2008 ◽

Vol 82 (8) ◽

pp. 3872-3881 ◽

Cited By ~ 8

Author(s):

Dusan Kunec ◽

Larry A. Hanson ◽

Sandra van Haren ◽

I. F. Nieuwenhuizen ◽

Shane C. Burgess

Keyword(s):

Channel Catfish ◽

Infectious Clone ◽

Artificial Chromosome ◽

Bacterial Artificial Chromosomes ◽

Bac Clone ◽

Artificial Chromosomes ◽

Targeted Mutation ◽

Infectious Clones ◽

Viral Progeny ◽

Herpesvirus 1

ABSTRACT Herpesviruses are important pathogens of humans and other animals. Herpesvirus infectious clones that can reconstitute phenotypically wild-type (wt) virus are extremely valuable tools for elucidating the roles of specific genes in virus pathophysiology as well as for making vaccines. Ictalurid herpesvirus 1 (channel catfish herpesvirus [CCV]) is economically very important and is the best characterized of the herpesviruses that occur primarily in bony fish and amphibians. Here, we describe the cloning of the hitherto recalcitrant CCV genome as three overlapping subgenomic bacterial artificial chromosomes (BACs). These clones allowed us to regenerate vectorless wt CCVs with a phenotype that is indistinguishable from that of the wt CCV from which the BACs were derived. To test the recombinogenic systems, we next used the overlapping BACs to construct a full-length CCV BAC by replacing the CCV ORF5 with the BAC cassette and cotransfecting CCO cells. The viral progeny that we used to transform Escherichia coli and the resulting BAC had only one of the 18-kb terminal repeated regions. Both systems suggest that one of the terminal repeat regions is lost during the replicative stage of the CCV life cycle. We also demonstrated the feasibility of introducing a targeted mutation into the CCV BAC infectious clone by constructing a CCV ORF12 deletion mutant and showed that ORF12 encodes a nonessential protein for virus replication. This is the first report of the generation of an infectious BAC clone of a member of the fish and amphibian herpesviruses and its use to generate recombinants.

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Characterization of Rose rosette virus and development of reverse genetic system for studying virus accumulation and movement in whole plants

10.1101/712000 ◽

2019 ◽

Cited By ~ 1

Author(s):

Jeanmarie Verchot ◽

Venura Herath ◽

Cesar D. Urrutia ◽

Mathieu Gayral ◽

Kelsey Lyle ◽

...

Keyword(s):

Infectious Clone ◽

Systemic Infection ◽

Genetic System ◽

Open Reading Frame ◽

Reverse Genetic System ◽

Reverse Genetic ◽

Glycoprotein Precursor ◽

Reading Frame ◽

Negative Strand ◽

Rose Rosette

ABSTRACTRose rosette virus (RRV) is an Emaravirus, a negative-sense RNA virus with a 7-segmented genome that is enclosed by a double membrane. While the genome sequences of many emaraviruses are reported, there is negligible information concerning virus replication and movement in host plants. Computational methods determined that RNA1 encoded the RNA dependent RNA polymerase (RdRp), RNA2 encoded glycoprotein precursor, and the RNA3 encoded the nucleocapsid (N), all share significant homologies with similar proteins of the Orthobunyavirus family. The RRV terminal UTR sequences are complementary and share significant identity with the UTR sequences of Bunyamwera virus. We report a minireplicon system and a full length infectious clone of RRV, which are the first for any emaravirus species. The photoreversible fluorescent iLOV protein was used to replace the RNA5 open reading frame (R5-iLOV). We demonstrate that agro-infiltration of Nicotiana benthamiana leaves to deliver RNA1, RNA3, and R5-iLOV cDNAs led to iLOV expression. A mutation was introduced into the RdRp active site and iLOV expression was eliminated. Delivery of four segments or seven segments of the RRV infectious clone produced systemic infection in N. benthamiana and rose plants. iLOV was also fused to the glycoprotein precursor (R2-iLOV). Using confocal microscopy, the R2-iLOV was seen in spherical bodies along membrane strands inside N. benthamiana epidermal cells. This new technology will enable future research to functionally characterize the RRV proteins, to study the virus-host interactions governing local and systemic infection, and examine the subcellular functions of the Gc.IMPORTANCERRV has emerged as a severe threat to cultivated roses, causing millions of dollars in losses to commercial producers. The majority of the viral gene products have not been researched or characterized until now. We constructed a minireplicon system and an infectious clone of the seven-segmented RRV genome that is contained in a binary vector and delivered by Agrobacterium. This technology has been slow to develop for viruses with negative-strand RNA genomes. It has been especially tricky for plant viruses with multicomponent negative-strand RNA genomes. We report the first reverse genetic system for a member of the genus Emaravirus, Rose rosette virus (RRV). We introduced the iLOV fluorescent protein as a fusion to the Gc protein and as a replacement for the open reading frame in genome segment 5. This game-changing reverse genetic system creates new opportunities for studying negative-strand RNA viruses in plants.

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Development of a Reverse Genetic System for Studying Rose Rosette Virus in Whole Plants

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-04-20-0094-r ◽

2020 ◽

Vol 33 (10) ◽

pp. 1209-1221 ◽

Cited By ~ 1

Author(s):

Jeanmarie Verchot ◽

Venura Herath ◽

Cesar D. Urrutia ◽

Mathieu Gayral ◽

Kelsey Lyle ◽

...

Keyword(s):

Catalytic Domain ◽

Rna Virus ◽

Infectious Clone ◽

Systemic Infection ◽

Genetic System ◽

Polymerase Activity ◽

Full Length ◽

Reverse Genetic System ◽

Reading Frame ◽

Rose Rosette

Rose rosette virus (RRV) is a negative-sense RNA virus with a seven-segmented genome that is enclosed by a double membrane. We constructed an unconventional minireplicon system encoding the antigenomic (ag)RNA1 (encoding the viral RNA-dependent RNA polymerase [RdRp]), agRNA3 (encoding the nucleocapsid protein [N]), and a modified agRNA5 containing the coding sequence for the iLOV protein in place of the P5 open reading frame (R5-iLOV). iLOV expression from the R5-iLOV template was amplified by activities of the RdRp and N proteins in Nicotiana benthamiana leaves. A mutation was introduced into the RdRp catalytic domain and iLOV expression was eliminated, indicating RNA1-encoded polymerase activity drives iLOV expression from the R5-iLOV template. Fluorescence from the replicon was highest at 3 days postinoculation (dpi) and declined at 7 and 13 dpi. Addition of the tomato bushy stunt virus (TBSV) P19 silencing-suppressor protein prolonged expression until 7 dpi. A full-length infectious clone system was constructed of seven binary plasmids encoding each of the seven genome segments. Agro-delivery of constructs encoding RRV RNAs 1 through 4 or RNAs 1 through 7 to N. benthamiana plants produced systemic infection. Finally, agro-delivery of the full-length RRV infectious clone including all segments produced systemic infection within 60 dpi. This advance opens new opportunities for studying RRV infection biology.

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Construction of an infectious clone of Zika virus stably expressing an EGFP marker in a eukaryotic expression system

Virology Journal ◽

10.1186/s12985-021-01622-z ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Jing Gao ◽

Jiayi Chen ◽

Weizhi Lu ◽

Jintai Cai ◽

Linjuan Shi ◽

...

Keyword(s):

Zika Virus ◽

Infectious Clone ◽

Expression System ◽

Plaque Assay ◽

Experimental System ◽

Genetic System ◽

Vero Cells ◽

Eukaryotic Expression ◽

Reverse Genetic System ◽

Cmv Promoter

Abstract Background Zika virus is becoming one of the most widely transmitted arboviruses in the world. Development of antiviral inhibitor and vaccine requires an experimental system that allows rapid monitoring of the virus infection. This is achievable with a reverse genetic system. In this study, we constructed an infectious clone for Zika virus that stably expressing EGFP. Methods A PCR-mediated recombination approach was used to assemble the full-length Zika virus genome containing the CMV promoter, intron, EGFP, hepatitis delta virus ribozyme, and SV40 terminator sequence for cloning into the pBAC11 vector to produce recombinant pBAC-ZIKA-EGFP. ZIKA-EGFP virus was rescued by transfection of pBAC-ZIKA-EGFP into 293T cells. The characterization of ZIKA-EGFP virus was determined by qPCR, plaque assay, CCK-8, and Western blot. Results Rescued ZIKA-EGFP virus exhibited stable replication for at least five generations in tissue culture. ZIKA-EGFP can effectively infect C6/36, SH-SY5Y and Vero cells, and cause cytopathic effects on SH-SY5Y and Vero cells. The inhibition of ZIKA-EGFP by NF-κB inhibitor, caffeic acid phenethyl ester was observed by fluorescence microscopy. Conclusion Our results suggested that Zika virus infectious clone with an EGFP marker retained it infectivity as wide-type Zika virus which could be used for drugs screening.

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Amino acid changes responsible for attenuation of virus neurovirulence in an infectious cDNA clone of the Oshima strain of Tick-borne encephalitis virus

Journal of General Virology ◽

10.1099/vir.0.19668-0 ◽

2004 ◽

Vol 85 (4) ◽

pp. 1007-1018 ◽

Cited By ~ 39

Author(s):

Daisuke Hayasaka ◽

Tamara S. Gritsun ◽

Kentarou Yoshii ◽

Tomotaka Ueki ◽

Akiko Goto ◽

...

Keyword(s):

Cell Culture ◽

Amino Acid ◽

Cdna Clone ◽

Infectious Clone ◽

Encephalitis Virus ◽

Infectious Cdna Clone ◽

Amino Acid Substitutions ◽

Parent Virus ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus

A stable full-length infectious cDNA clone of the Oshima strain of Tick-borne encephalitis virus (Far-Eastern subtype) was developed by a long high-fidelity RT-PCR and one-step cloning procedure. The infectious clone (O-IC) had four amino acid substitutions and produced smaller plaques when compared with the parent Oshima 5-10 strain. Using site-directed mutagenesis, the substitutions were reverted to restore the parent virus sequence (O-IC-pt). Although genetically identical, parent virus Oshima 5-10 and virus recovered from O-IC-pt demonstrated some biological differences that are possibly explained by the presence of quasispecies with differing virulence characteristics within the original virus population. These observations may have implications for vaccines based on modified infectious clones. It was also demonstrated that the amino acid substitution E-S40→P at position 40 in the envelope (E) glycoprotein was responsible for plaque size reduction, reduced infectious virus yields in cell culture and reduced mouse neurovirulence. Additionally, two amino acid substitutions in the non-structural (NS)5 protein (virus RNA-dependent RNA polymerase) NS5-V378→A and NS5-R674→K also contributed to attenuation of virulence in mice, but did not demonstrate a noticeable biological effect in baby hamster kidney cell culture. Comparative neurovirulence tests revealed how the accumulation of individual mutations (E-S40→P, NS5-V378→A and NS5-R674→K) can result in the attenuation of a virus.

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Molecular Identification of Prune Dwarf Virus (PDV) Infecting Sweet Cherry in Canada and Development of a PDV Full-Length Infectious cDNA Clone

Viruses ◽

10.3390/v13102025 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2025

Author(s):

Aaron J. Simkovich ◽

Yinzi Li ◽

Susanne E. Kohalmi ◽

Jonathan S. Griffiths ◽

Aiming Wang

Keyword(s):

Molecular Identification ◽

Cdna Clone ◽

Sweet Cherry ◽

Infectious Clone ◽

Infectious Cdna Clone ◽

Dwarf Virus ◽

Full Genome Sequence ◽

Small Interfering Rnas ◽

Prune Dwarf Virus ◽

Fruit Species

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses.

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Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase

Journal of Virology ◽

10.1128/jvi.78.11.5619-5632.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5619-5632 ◽

Cited By ~ 189

Author(s):

Konstantin A. Ivanov ◽

Volker Thiel ◽

Jessika C. Dobbe ◽

Yvonne van der Meer ◽

Eric J. Snijder ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Rna Synthesis ◽

Rna Virus ◽

Nonstructural Protein ◽

Enzymatic Activities ◽

Membrane Structures ◽

Processing Enzymes ◽

Cytoplasmic Rna ◽

Group 2 ◽

Rna And Dna

ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5′-to-3′ direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5′-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5′ cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5′-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.

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