scholarly journals Identification of 5′ and 3′ cis-Acting Elements of the Porcine Reproductive and Respiratory Syndrome Virus: Acquisition of Novel 5′ AU-Rich Sequences Restored Replication of a 5′-Proximal 7-Nucleotide Deletion Mutant

2006 ◽  
Vol 80 (2) ◽  
pp. 723-736 ◽  
Author(s):  
Yu-Jeong Choi ◽  
Sang-Im Yun ◽  
Shien-Young Kang ◽  
Young-Min Lee
Keyword(s):  
Reverse Genetics ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Heterologous Gene Expression ◽  
Artificial Chromosome ◽  
Expression Vectors ◽  
Respiratory Syndrome Virus ◽  
Genomic Rnas ◽  
Cis Acting ◽  
Green Fluorescent

ABSTRACT We here demonstrate the successful engineering of the RNA genome of porcine reproductive and respiratory syndrome virus (PRRSV) by using an infectious cDNA as a bacterial artificial chromosome. Runoff transcription from this cDNA by SP6 polymerase resulted in capped synthetic RNAs bearing authentic 5′ and 3′ ends of the viral genome that had specific infectivities of >5 × 105 PFU/μg of RNA. The synthetic viruses recovered from the transfected cells were genotypically and phenotypically indistinguishable from the parental virus. Using our system, a series of genomic RNAs with nucleotide deletions in their 5′ ends produced viruses with decreased or no infectivity. Various pseudorevertants were isolated, and acquisition of novel 5′ sequences of various sizes, composed predominantly of A and U bases, restored their infectivities, providing a novel insight into functional elements of the 5′ end of the PRRSV genome. In addition, our system was further engineered to generate a panel of self-replicating, self-limiting, luciferase-expressing PRRSV viral replicons bearing various deletions. Analysis of these replicons revealed the presence and location of a 3′ cis-acting element in the genome that was required for replication. Moreover, we produced enhanced green fluorescent protein-expressing infectious viruses, which indicates that the PRRSV cDNA/viral replicon/recombinant virus can be developed as a vector for the expression of a variety of heterologous genes. Thus, our PRRSV reverse genetics system not only offers a means of directly investigating the molecular mechanisms of PRRSV replication and pathogenesis but also can be used to generate new heterologous gene expression vectors and genetically defined antiviral vaccines.

scholarly journals Entirely plasmid-based reverse genetics system for rotaviruses

2017 ◽  
Vol 114 (9) ◽  
pp. 2349-2354 ◽  
Author(s):  
Yuta Kanai ◽  
Satoshi Komoto ◽  
Takahiro Kawagishi ◽  
Ryotaro Nouda ◽  
Naoko Nagasawa ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Transmembrane Protein ◽  
Gene Segment ◽  
Severe Diarrhea ◽  
Nsp1 Gene ◽  
Capping Enzyme ◽  
Green Fluorescent

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.

scholarly journals An infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene

2007 ◽  
Vol 88 (4) ◽  
pp. 1196-1205 ◽  
Author(s):  
Erwin van den Born ◽  
Clara C. Posthuma ◽  
Kèvin Knoops ◽  
Eric J. Snijder
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Heterologous Gene Expression ◽  
Equine Arteritis Virus ◽  
Expression Vectors ◽  
Wild Type Virus ◽  
Foreign Gene ◽  
Replicase Gene ◽  
Green Fluorescent

Thus far, systems developed for heterologous gene expression from the genomes of nidoviruses (arteriviruses and coronaviruses) have relied mainly on the translation of foreign genes from subgenomic mRNAs, whose synthesis is a key feature of the nidovirus life cycle. In general, such expression vectors often suffered from relatively low and unpredictable expression levels, as well as genome instability. In an attempt to circumvent these disadvantages, the possibility to express a foreign gene [encoding enhanced green fluorescent protein (eGFP)] from within the nidovirus replicase gene, which encodes two large polyproteins that are processed proteolytically into the non-structural proteins (nsps) required for viral RNA synthesis, has now been explored. A viable recombinant of the arterivirus Equine arteritis virus, EAV-GFP2, was obtained, which contained the eGFP insert at the site specifying the junction between the two most N-proximal replicase-cleavage products, nsp1 and nsp2. EAV-GFP2 replication could be launched by transfection of cells with either in vitro-generated RNA transcripts or a DNA launch plasmid. EAV-GFP2 displayed growth characteristics similar to those of the wild-type virus and was found to maintain the insert stably for at least eight passages. It is proposed that EAV-GFP2 has potential for arterivirus vector development and as a tool in inhibitor screening. It can also be used for fundamental studies into EAV replication, which was illustrated by the fact that the eGFP signal of EAV-GFP2, which largely originated from an eGFP–nsp2 fusion protein, could be used to monitor the formation of the membrane-bound EAV replication complex in real time.

scholarly journals Generation of Recombinant Rotaviruses Expressing Fluorescent Proteins by Using an Optimized Reverse Genetics System

2018 ◽  
Vol 92 (13) ◽  
pp. e00588-18 ◽  
Author(s):  
Satoshi Komoto ◽  
Saori Fukuda ◽  
Tomihiko Ide ◽  
Naoto Ito ◽  
Makoto Sugiyama ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Vectors ◽  
Next Generation ◽  
Efficient System ◽  
Molecular Virology ◽  
Highly Efficient ◽  
Group A Rotaviruses ◽  
Green Fluorescent

ABSTRACTAn entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (enhanced green fluorescent protein [EGFP] and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages. Our reverse genetics approach and recombinant rotaviruses carrying reporter genes will be great additions to the tool kit for studying the molecular virology of rotavirus and for developing future next-generation vaccines and expression vectors.IMPORTANCERotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. In this paper, we describe a robust and simple reverse genetics system based on only rotavirus cDNAs and its application for engineering infectious recombinant rotaviruses harboring bioluminescent (NanoLuc) and fluorescent (EGFP and mCherry) protein genes. This highly efficient reverse genetics system and recombinant group A rotaviruses expressing reporters could be powerful tools for the study of different aspects of rotavirus replication. Furthermore, they may be useful for next-generation vaccine production for this medically important virus.

scholarly journals Expression of Foreign Proteins by Poliovirus Polyprotein Fusion: Analysis of Genetic Stability Reveals Rapid Deletions and Formation of Cardioviruslike Open Reading Frames

1998 ◽  
Vol 72 (1) ◽  
pp. 20-31 ◽  
Author(s):  
Steffen Mueller ◽  
Eckard Wimmer
Keyword(s):  
Fluorescent Protein ◽  
Growth Cycle ◽  
Open Reading Frames ◽  
Expression Vectors ◽  
Green Fluorescent Protein Gene ◽  
Published Data ◽  
Gene Encoding ◽  
Genomic Rnas ◽  
Reading Frame ◽  
Green Fluorescent

ABSTRACT Using a strategy developed by R. Andino, D. Silvera, S. D. Suggett, P. I. Achacoso, C. J. Miller, D. Baltimore, and M. B. Feinberg (Science 265:1448–1451, 1994), we constructed recombinant polioviruses by fusing the open reading frame (ORF) of the green fluorescent protein gene (gfp) of Aequorea victoria or the gag gene (encoding p17-p24) of human immunodeficiency virus type 1 (HIV-1) to the N terminus of the poliovirus polyprotein. All poliovirus expression vectors constructed by us and those obtained from Andino et al. were found to be severely impaired in viral replication and genetically unstable. Upon replication, inserted sequences were rapidly deleted as early as the first growth cycle in HeLa cells. However, the vector viruses did not readily revert to the wild-type sequence but rather retained some of the insert plus the artificial 3Cpro/3CDprocleavage site, engineered between the heterologous sequence and the poliovirus polyprotein, to give rise to genotypes reminiscent of cardioviruses. These virus variants that carry a small leader polypeptide were now relatively stable, and they grew better than their progenitor strains. Reverse transcription followed by PCR and sequence analysis of the genomic RNAs reproducibly revealed a few preferred genotypes among the isolated deletion variants. The remaining truncated inserts were retained through subsequent passages. In the immediate vicinity of the deletion borders, we observed short direct sequence repeats that we propose are involved in aligning RNA strands for illegitimate (nonhomologous) RNA recombination during minus-strand synthesis. On the basis of our results, which are at variance with published data, the utility of poliovirus vectors to express proteins >10 kDa in size through fusion with the polyprotein needs to be reevaluated.

scholarly journals New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

2016 ◽  
Vol 82 (8) ◽  
pp. 2240-2246 ◽  
Author(s):  
Alex I. Kanno ◽  
Cibelly Goulart ◽  
Henrique K. Rofatto ◽  
Sergio C. Oliveira ◽  
Luciana C. C. Leite ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Expression Vectors ◽  
Content Type ◽  
Expression Levels ◽  
Wide Range ◽  
Mycobacteriophage L5 ◽  
Green Fluorescent ◽  
Selection Of

ABSTRACTThe expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

WZsGreen/+: a new green fluorescent protein knock-in mouse model for the study of KIT-expressing cells in gut and cerebellum

2005 ◽  
Vol 22 (3) ◽  
pp. 412-421 ◽  
Author(s):  
Mira Wouters ◽  
Karine Smans ◽  
Jean-Marie Vanderwinden
Keyword(s):  
Small Intestine ◽  
Green Fluorescent Protein ◽  
Gastrointestinal Tract ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Molecular Layer ◽  
Transgenic Animals ◽  
The Novel ◽  
Suitable Alternative ◽  
Green Fluorescent

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. KIT is currently the sole practical marker for ICC. Attempts to purify living ICC have so far largely failed, due to the loss of the KIT epitope during enzymatic dissociation. Aiming to identify and isolate living ICC, we designed a knock-in strategy to express a fluorescent tag in KIT-expressing cells by inserting the sequence of the novel green fluorescent protein ZsGreen into the first exon of the c-Kit gene, creating a null allele called WZsGreen. In the gastrointestinal tract of heterozygous WZsGreen/+ mice, tiny ZsGreen fluorescent dots were observed in all KIT-expressing ICC populations, with exception of ICC at the deep muscular plexus in small intestine. During development of the gastrointestinal tract, ZsGreen expression followed KIT expression in a spatiotemporal way. Stellate and basket KIT-expressing cells in the molecular layer of the cerebellum also exhibited ZsGreen dots, whereas no ZsGreen was detected in skin, testis, and bone marrow. ZsGreen dot-containing intestinal cells could be isolated from jejunum and maintained alive in culture for at least 3 days. ZsGreen is a suitable alternative to EGFP in transgenic animals. The novel WZsGreen/+ model reported here appears to be a promising tool for live studies of KIT-expressing cells in the gastrointestinal tract and cerebellum and for the further analysis of pacemaker mechanisms.

Induced Expression of the Heat Shock Protein Genes uspA and grpE during Starvation at Low Temperatures and Their Influence on Thermal Resistance of Escherichia coli O157:H7

2003 ◽  
Vol 66 (11) ◽  
pp. 2045-2050 ◽  
Author(s):  
YI ZHANG ◽  
MANSEL W. GRIFFITHS
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Stress Responses ◽  
Thermal Tolerance ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Bacterial Cells ◽  
Induced Expression ◽  
Green Fluorescent ◽  
Shock Proteins

Heat shock proteins play an important role in protecting bacterial cells against several stresses, including starvation. In this study, the promoters for two genes encoding heat shock proteins involved in many stress responses, UspA and GrpE, were fused with the green fluorescent protein (gfp) gene. Thus, the expression of the two genes could be quantified by measuring the fluorescence emitted by the cells under different environmental conditions. The heat resistance levels of starved and nonstarved cells during storage at 5, 10, and 37°C were compared with the levels of expression of the uspA and grpE genes. D52-values (times required for decimal reductions in count at 52°C) increased by 11.5, 14.6, and 18.5 min when cells were starved for 3 h at 37°C, for 24 h at 10°C, and for 2 days at 5°C, respectively. In all cases, these increases were significant (P < 0.01), indicating that the stress imposed by starvation altered the ability of E. coli O157:H7 to survive subsequent heat treatments. Thermal tolerance was correlative with the induction of UspA and GrpE. At 5°C, the change in the thermal tolerance of the pathogen was positively linked to the induced expression of the grpE gene but negatively related to the expression of the uspA gene. The results obtained in this study indicate that UspA plays an important role in starvation-induced thermal tolerance at 37°C but that GrpE may be more involved in regulating this response at lower temperatures. An improvement in our understanding of the molecular mechanisms involved in these cross-protection responses may make it possible to devise strategies to limit their effects.

scholarly journals Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 655 ◽  
Author(s):  
Yíngyún Caì ◽  
Masaharu Iwasaki ◽  
Brett Beitzel ◽  
Shuīqìng Yú ◽  
Elena Postnikova ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Quantitative Detection ◽  
Lassa Virus ◽  
Wild Type ◽  
Green Fluorescent

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000–300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

The Cdk and PCNA domains on p21Cip1 both function to inhibit G1/S progression during hyperoxia

2004 ◽  
Vol 286 (3) ◽  
pp. L506-L513 ◽  
Author(s):  
Christopher E. Helt ◽  
Rhonda J. Staversky ◽  
Yi-Jang Lee ◽  
Robert A. Bambara ◽  
Peter C. Keng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Nuclear Antigen ◽  
Cell Cycle Regulators ◽  
Amino Terminal ◽  
Binding Domains ◽  
Green Fluorescent

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G1 when exposed to 95% oxygen. Instead, cells arrested in S and G2. Stable expression of p53 restored induction of p21 and G1 arrest without affecting mRNA expression of the other Cip or INK4 G1 kinase inhibitors. To confirm the role of p21 in G1 arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G1 arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G1 during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G1 arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.

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