VLPs Derived from the CCMV Plant Virus Can Directly Transfect and Deliver Heterologous Genes for Translation into Mammalian Cells

Virus-like particles (VLPs) are being used for therapeutic developments such as vaccines and drug nanocarriers. Among these, plant virus capsids are gaining interest for the formation of VLPs because they can be safely handled and are noncytotoxic. A paradigm in virology, however, is that plant viruses cannot transfect and deliver directly their genetic material or other cargos into mammalian cells. In this work, we prepared VLPs with the CCMV capsid and the mRNA-EGFP as a cargo and reporter gene. We show, for the first time, that these plant virus-based VLPs are capable of directly transfecting different eukaryotic cell lines, without the aid of any transfecting adjuvant, and delivering their nucleic acid for translation as observed by the presence of fluorescent protein. Our results show that the CCMV capsid is a good noncytotoxic container for genome delivery into mammalian cells.

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Chromosomal Integration of Transduced Recombinant Baculovirus DNA in Mammalian Cells

Journal of Virology ◽

10.1128/jvi.75.2.903-909.2001 ◽

2001 ◽

Vol 75 (2) ◽

pp. 903-909 ◽

Cited By ~ 70

Author(s):

Raymond V. Merrihew ◽

William C. Clay ◽

J. Patrick Condreay ◽

Sam M. Witherspoon ◽

Walter S. Dallas ◽

...

Keyword(s):

Dna Sequencing ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Genetic Material ◽

Chinese Hamster ◽

Recombinant Baculovirus ◽

Single Copy ◽

Inverse Pcr ◽

Pcr Analysis ◽

Fish Analysis

ABSTRACT Our group and others have recently demonstrated the ability of recombinant baculoviruses to transduce mammalian cells at high frequency. To further characterize the use of baculovirus as a mammalian gene delivery system, we examined the status of transduced DNA stably maintained in Chinese hamster ovary (CHO) cells. Four independent clones carrying two introduced markers, the genes for neomycin resistance (Neo) and green fluorescent protein (GFP), were selected. PCR analysis, Southern blotting, and DNA sequencing showed that discrete portions of the 148-kb baculovirus DNA were present as single-copy fragments ranging in size from 5 to 18 kb. Integration into the CHO cell genome was confirmed by fluorescent in situ hybridization (FISH) analysis. For one clone, the left and right viral/chromosomal junctions were determined by DNA sequencing of inverse PCR products. Similarly, for a different clone, the left viral/chromosomal junction was determined; however, the right junction sequence revealed the joining to another viral fragment by a short homology (microhomology), a hallmark of illegitimate recombination. The random viral breakpoints and the lack of homology between the virus and flanking chromosomal sequences are also suggestive of an illegitimate integration mechanism. To examine the long-term stability of reporter gene expression, all four clones were grown continuously for 36 passages in either the presence or absence of selection for Neo. Periodic assays over a 5-month period showed no loss of GFP expression for at least two of the clones. This report represents the first detailed analysis of baculovirus integrants within mammalian cells. The potential advantages of the baculovirus system for the stable integration of genetic material into mammalian genomes are discussed.

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Transcription of 4′-thioDNA templates to natural RNA in vitro and in mammalian cells

Chemical Communications ◽

10.1039/c4cc08862j ◽

2015 ◽

Vol 51 (37) ◽

pp. 7887-7890 ◽

Cited By ~ 17

Author(s):

Hideto Maruyama ◽

Kazuhiro Furukawa ◽

Hiroyuki Kamiya ◽

Noriaki Minakawa ◽

Akira Matsuda

Keyword(s):

Nucleic Acids ◽

Mammalian Cells ◽

Genetic Material ◽

Rna Polymerases ◽

Chemically Modified ◽

Biological Studies ◽

Modified Nucleic Acids

Synthetic chemically modified nucleic acids, which are compatible with DNA/RNA polymerases, have great potential as a genetic material for synthetic biological studies.

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Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice

Metabolites ◽

10.3390/metabo11080481 ◽

2021 ◽

Vol 11 (8) ◽

pp. 481

Author(s):

Gemma G. Martínez-García ◽

Raúl F. Pérez ◽

Álvaro F. Fernández ◽

Sylvere Durand ◽

Guido Kroemer ◽

...

Keyword(s):

Skeletal Muscle ◽

Amino Acids ◽

Mammalian Cells ◽

Tissue Homeostasis ◽

In Vivo Studies ◽

Protective Mechanism ◽

Cardiac Damage ◽

Wide Range ◽

First Time

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

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Softening of Processed Plant Virus Infected Cucumis sativus Fruits

Agronomy ◽

10.3390/agronomy11081451 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1451

Author(s):

Anne-Katrin Kersten ◽

Sabrina Scharf ◽

Martina Bandte ◽

Peer Martin ◽

Peter Meurer ◽

...

Keyword(s):

Cucumis Sativus ◽

Plant Virus ◽

Plant Viruses ◽

Economic Losses ◽

Symptom Expression ◽

Considerable Influence ◽

Mechanical Inoculation ◽

Morphological Alterations ◽

Texture Properties ◽

Textural Changes

Texture softening of pickled cucumbers does not meet consumers’ quality expectations and leads to economic losses. The factor(s) triggering this phenomenon is still unknown. We investigated the importance of plant viruses such as Cucumber green mottle mosaic tobamovirus (CGMMV) and Zucchini yellow mosaic potyvirus (ZYMV) in the context of softening of pickles. Cucumber plants (Cucumis sativus) were infected by mechanical inoculation, grown under greenhouse conditions and tested positive for the viral infection by ELISA. The severity of virus infection was reflected in yield and symptom expression. Histological and morphological alterations were observed. All fruits were pasteurized, separately stored in jars and subjected to texture measurements after four, six and 12 months. CGMMV-infections were asymptomatic or caused mild symptoms on leaves and fruit, and texture quality was comparable to control. At the same time, fruits of ZYMV-infected plants showed severe symptoms like deformations and discoloration, as well as a reduction in firmness and crunchiness after pasteurization. In addition, histological alterations were detected in such fruits, possibly causing textural changes. We conclude that plant viruses could have a considerable influence on the firmness and crunchiness of pickled cucumbers after pasteurization. It is possible that the severity of symptom expression has an influence on texture properties.

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Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer

Scientific Reports ◽

10.1038/s41598-021-94551-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tomomi Kaku ◽

Kazunori Sugiura ◽

Tetsuyuki Entani ◽

Kenji Osabe ◽

Takeharu Nagai

Keyword(s):

Energy Transfer ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Color Change ◽

Yellow Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Bioluminescence Resonance Energy Transfer ◽

Bacterial Luciferase ◽

Bacterial Bioluminescence

AbstractUsing the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

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Novel Functional Genomics Approaches: A Promising Future in the Combat Against Plant Viruses

Phytopathology ◽

10.1094/phyto-03-16-0145-fi ◽

2016 ◽

Vol 106 (10) ◽

pp. 1231-1239 ◽

Cited By ~ 10

Author(s):

Vincent N. Fondong ◽

Ugrappa Nagalakshmi ◽

Savithramma P. Dinesh-Kumar

Keyword(s):

Functional Genomics ◽

Plant Virus ◽

Reverse Genetics ◽

Plant Viruses ◽

Micro Rna ◽

Viral Genomes ◽

Virus Control ◽

Local Lesions ◽

Short Palindromic Repeat ◽

Interfering Rna

Advances in functional genomics and genome editing approaches have provided new opportunities and potential to accelerate plant virus control efforts through modification of host and viral genomes in a precise and predictable manner. Here, we discuss application of RNA-based technologies, including artificial micro RNA, transacting small interfering RNA, and Cas9 (clustered regularly interspaced short palindromic repeat–associated protein 9), which are currently being successfully deployed in generating virus-resistant plants. We further discuss the reverse genetics approach, targeting induced local lesions in genomes (TILLING) and its variant, known as EcoTILLING, that are used in the identification of plant virus recessive resistance gene alleles. In addition to describing specific applications of these technologies in plant virus control, this review discusses their advantages and limitations.

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ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization

Molecular Biology of the Cell ◽

10.1091/mbc.e02-02-0078 ◽

2002 ◽

Vol 13 (9) ◽

pp. 3078-3095 ◽

Cited By ~ 30

Author(s):

Annette L. Boman ◽

Paul D. Salo ◽

Melissa J. Hauglund ◽

Nicole L. Strand ◽

Shelly J. Rensink ◽

...

Keyword(s):

Membrane Trafficking ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Protein Localization ◽

Carboxypeptidase Y ◽

Minor Role ◽

Temperature Sensitive ◽

Adp Ribosylation ◽

And Function ◽

Adp Ribosylation Factor

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

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