scholarly journals PostexitSurface Engineering of Retroviral/Lentiviral Vectors

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Christoph Metzner ◽  
Feliks Kochan ◽  
John A. Dangerfield
Keyword(s):  
Chemical Modification ◽  
Genetic Information ◽  
Viral Particle ◽  
Genetic Manipulation ◽  
Regulatory Elements ◽  
Safe Delivery ◽  
Advantages And Disadvantages ◽  
Genomic Level ◽  
Gene Delivery Vectors ◽  
Viral Particles

Gene delivery vectors based on retroviral or lentiviral particles are considered powerful tools for biomedicine and biotechnology applications. Such vectors require modification at the genomic level in the form of rearrangements to allow introduction of desired genes and regulatory elements (genotypic modification) as well as engineering of the physical virus particle (phenotypic modification) in order to mediate efficient and safe delivery of the genetic information to the target cell nucleus. Phenotypic modifications are typically introduced at the genomic level through genetic manipulation of the virus producing cells. However, this paper focuses on methods which allow modification of viral particle surfaces after they have exited the cell, that is, directly on the viral particles in suspension. These methods fall into three categories: (i) direct covalent chemical modification, (ii) membrane-topic reagents, and (iii) adaptor systems. Current applications of such techniques will be introduced and their advantages and disadvantages will be discussed.

scholarly journals Contribution of the Cytoplasmic Determinants of Vpu to the Expansion of Virus-Containing Compartments in HIV-1-Infected Macrophages

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Olivier Leymarie ◽  
Leslie Lepont ◽  
Margaux Versapuech ◽  
Delphine Judith ◽  
Sophie Abelanet ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Viral Particle ◽  
Transmembrane Domain ◽  
Immune Surveillance ◽  
Restriction Factor ◽  
Viral Particles ◽  
Protein Functions ◽  
Specific Accumulation ◽  
Model Cell ◽  
Hiv 1

ABSTRACTHIV-1 infection of macrophages leads to the sequestration of newly formed viruses in intracellular plasma membrane-connected structures termed virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The cellular restriction factor bone marrow stromal cell antigen 2 (BST2), which prevents HIV-1 dissemination by tethering budding viral particles at the plasma membrane, can be found in VCCs. The HIV-1 accessory protein Vpu counteracts the restriction factor BST2 by downregulating its expression and removing it from viral budding sites. Numerous studies described these Vpu countermeasures in CD4+T cells or model cell lines, but the interplay between Vpu and BST2 in VCC formation and HIV-1 production in macrophages is less explored. Here, we show that Vpu expression in HIV-1-infected macrophages enhances viral release. This effect is related to Vpu’s ability to circumvent BST2 antiviral activity. We show that in absence of Vpu, BST2 is enriched in VCCs and colocalizes with capsid p24, whereas Vpu expression significantly reduces the presence of BST2 in these compartments. Furthermore, our data reveal that BST2 is dispensable for the formation of VCCs and that Vpu expression impacts the volume of these compartments. This Vpu activity partly depends on BST2 expression and requires the integrity of the Vpu transmembrane domain, the dileucine-like motif E59XXXLV64and phosphoserines 52 and 56 of Vpu. Altogether, these results highlight that Vpu controls the volume of VCCs and promotes HIV-1 release from infected macrophages.IMPORTANCEHIV-1 infection of macrophages leads to the sequestration of newly formed viruses in virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The restriction factor BST2, which prevents HIV-1 dissemination by tethering budding viral particles, can be found in VCCs. The HIV-1 Vpu protein counteracts BST2. This study explores the interplay between Vpu and BST2 in the viral protein functions on HIV-1 release and viral particle sequestration in VCCs in macrophages. The results show that Vpu controls the volume of VCCs and favors viral particle release. These Vpu functions partly depend on Vpu’s ability to antagonize BST2. This study highlights that the transmembrane domain of Vpu and two motifs of the Vpu cytoplasmic domain are required for these functions. These motifs were notably involved in the control of the volume of VCCs by Vpu but were dispensable for the prevention of the specific accumulation of BST2 in these structures.

scholarly journals rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations

2019 ◽  
Vol 20 (22) ◽  
pp. 5702 ◽  
Author(s):  
Rebecca C. Feiner ◽  
Julian Teschner ◽  
Kathrin E. Teschner ◽  
Marco T. Radukic ◽  
Tobias Baumann ◽  
...  
Keyword(s):  
Biological Function ◽  
Genetic Manipulation ◽  
Full Potential ◽  
Concomitant Increase ◽  
Loop Region ◽  
Viral Particles ◽  
Genetic Level ◽  
Mosaic Approach ◽  
Thermal Perturbations ◽  
Protein Enzyme

Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme β-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with β-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology.

Artificially enhanced tolerance to fluoroacetate and its implications for wildlife conservation

2000 ◽  
Vol 6 (1) ◽  
pp. 9 ◽  
Author(s):  
Laurie E. Twigg ◽  
Laurie E. Twigg ◽  
Dennis R. King ◽  
Dennis R. King
Keyword(s):  
Wildlife Conservation ◽  
Genetic Manipulation ◽  
Research Programme ◽  
Field Trials ◽  
Advisory Council ◽  
Pest Species ◽  
Advantages And Disadvantages ◽  
Domestic Livestock ◽  
Potential Impact ◽  
Toxic Plant

It is with some concern that we have been following a research programme aimed at developing non-specific, genetically modified ruminant bacteria capable of detoxifying fluoroacetate (Compound 1080) to protect domestic livestock from fluoroacetate-poisoning (Gregg et al. 1994; Gregg et al. 1998; Synergy 1998). The main thrust of this research programme is the prevention of cattle losses in the Georgina Basin in the Northern Territory and Queensland where the toxic plant, Gidgee Acacia georginae occurs. Although rarely mentioned in formal reports of this work, 1080 is an important vertebrate pesticide that is widely used throughout Australia and New Zealand. It is the first defence against a number of pest species that impact on agricultural production and conservation efforts in both countries. The main concerns about this work relate to the potential lack of target specificity of these modified microorganisms, and also, if released, their potential impact on conservation biology. We raise these questions now because the Genetic Manipulation Advisory Council has been approached for approval to conduct field trials in Western Australia using the modified bacteria. We believe the perceived advantages and disadvantages of these modified organisms need informed debate before such approval, or any general release of these modified rumen bacteria, could be considered.

scholarly journals An exploration of synthetic biology: A preliminary Christian ethical assessment of the advantages and disadvantages of synthetic biology

2014 ◽  
Vol 48 (2) ◽  
Author(s):  
Riaan A.L. Rheeder
Keyword(s):  
Synthetic Biology ◽  
Genetic Manipulation ◽  
Positive Outcomes ◽  
Ethical Assessment ◽  
Advantages And Disadvantages ◽  
Natural Function ◽  
Mycoplasma Capricolum ◽  
Mycoplasma Mycoides ◽  
Point Of Departure

On 20 May 2010, the Venter Institute in America announced that they have fully synthesised the genome of the organism Mycoplasma mycoides whilst in vitro by using a computer connected to a machine that synthesises genes. Thereafter, the genome was placed back into the casing of another organism (Mycoplasma capricolum) and it was reported that the synthesised organism and the genome functioned normally. This synthesised organism was reconstructed to function as a minute little factory with the aim of producing and secreting fuel and medicine − something that is not the natural function of this organism. There are certain potential dangers inherent in this kind of technology. Scientists fear that this technology may contaminate or infect humans, animals or the environment, and that it can as such be extremely harmful, or even lead to the destruction of humans. Other scientists are concerned that terrorists can use this technology to kill innocent citizens. Some ethicists are of the opinion that the consequences of synthetic biology is currently unpredictable and that it is therefore risky. In opposition to the potential dangers, one has to mention that synthetic biology indeed can result in far-reaching positive outcomes such as the manufacturing of biofuel and medication. Most scientists and ethicists are of the opinion that the potential dangers involved in synthetic biology should be evaluated in light of the fact that genetic manipulation has not caused any biological devastation over the last 30 years. From a Christian point of departure, the opinion is currently that synthetic biology is not an irresponsible science and technology.’n Verkenning van sintetiese biologie: ’n Voorlopige Christelik-etiese beoordeling van die voor- en nadele van sintetiese biologie. Op 20 Mei 2010 het die Venter-instituut (in Amerika) aangekondig dat hulle die genoom van die organisme Mycoplasma mycoides ten volle in vitro gesintetiseer het (deur middel van ’n rekenaar, gekoppel aan ’n masjien wat gene sintetiseer). Die berig het verder gelei dat die genoom daarna teruggeplaas is in die omhulsel van ’n ander organisme (Mycoplasma capricolum) − en dat die gesintetiseerde genoom en organisme normaal gefunksioneer het. Hierdie gesintetiseerde organisme word gerekonstrueer om as minuskule fabriek te funksioneer met die doel om brandstof en medisyne te produseer en te sekreer − wat nie die natuurlike funksie van die organisme is nie. Aan hierdie tegnologie is daar ook bepaalde potensiële gevare verbonde. Wetenskaplikes is bang dat hierdie tegnologie mens, dier en omgewing kan kontamineer of infekteer en op dié wyse groot skade kan aanrig − en selfs tot mense se dood kan lei. Ander wetenskaplikes is weer bekommerd dat hierdie tegnologie deur terroriste gebruik kan word om onskuldige burgers dood te maak. Sommige etici is oortuig dat die gevolge van sintetiese biologie tans onvoorspelbaar, en daarom riskant is. Teenoor die potensiële gevare moet gestel word dat sintetiese biologie inderdaad omvangryke positiewe uitkomste soos die vervaardiging van biobrandstof en medisyne tot gevolg kan hê. Meeste wetenskaplikes en etici is van mening dat die potensiële gevare verbonde aan sintetiese biologie beoordeel moet word in die lig van die feit dat genetiese manipulasie in die afgelope 30 jaar geen biologiese ramp veroorsaak het nie. Uit ’n Christelike oogpunt word voorlopig geoordeel dat sintetiese biologie nie ’n onverantwoordelike wetenskap en tegnologie is nie.

scholarly journals Sindbis Virus with a Tricomponent Genome

2005 ◽  
Vol 79 (1) ◽  
pp. 637-643 ◽  
Author(s):  
Rafik Fayzulin ◽  
Rodion Gorchakov ◽  
Olga Petrakova ◽  
Evgenia Volkova ◽  
Ilya Frolov
Keyword(s):  
Tissue Culture ◽  
Virus Replication ◽  
Genetic Information ◽  
Sindbis Virus ◽  
Viral Particles ◽  
Rna Fragments

ABSTRACT We established a system for propagation of Sindbis virus (SIN)-based replicons in tissue culture in the form of a tricomponent genome virus. Three RNA fragments containing complementing genetic information required for virus replication are packaged into separate viral particles, and each cell produces at least 1,000 packaged replicons and the number of packaged helpers sufficient to perform the next passage. This system can be used to generate large stocks of packaged replicons. The formation of infectious recombinant SIN virus was not detected in any experiments. These features make multicomponent genome SIN an attractive system for a variety of research and biotechnology applications.

scholarly journals Caveolae Are Involved in the Trafficking of Mouse Polyomavirus Virions and Artificial VP1 Pseudocapsids toward Cell Nuclei

2001 ◽  
Vol 75 (22) ◽  
pp. 10880-10891 ◽  
Author(s):  
Zuzana Richterová ◽  
David Liebl ◽  
Martin Horák ◽  
Zdena Palková ◽  
Jitka Štokrová ◽  
...  
Keyword(s):  
Viral Particle ◽  
Stress Fibers ◽  
Particle Movement ◽  
Cell Nuclei ◽  
Membrane Structures ◽  
Caveolin 1 ◽  
Strong Signal ◽  
Artificial Virus ◽  
Viral Particles ◽  
Perinuclear Area

ABSTRACT Electron and confocal microscopy were used to observe the entry and the movement of polyomavirus virions and artificial virus-like particles (VP1 pseudocapsids) in mouse fibroblasts and epithelial cells. No visible differences in adsorption and internalization of virions and VP1 pseudocapsids (“empty” or containing DNA) were observed. Viral particles entered cells internalized in smooth monopinocytic vesicles, often in the proximity of larger, caveola-like invaginations. Both “empty” vesicles derived from caveolae and vesicles containing viral particles were stained with the anti-caveolin-1 antibody, and the two types of vesicles often fused in the cytoplasm. Colocalization of VP1 with caveolin-1 was observed during viral particle movement from the plasma membrane throughout the cytoplasm to the perinuclear area. Empty vesicles and vesicles with viral particles moved predominantly along microfilaments. Particle movement was accompanied by transient disorganization of actin stress fibers. Microfilaments decorated by the VP1 immunofluorescent signal could be seen as concentric curves, apparently along membrane structures that probably represent endoplasmic reticulum. Colocalization of VP1 with tubulin was mostly observed in areas close to the cell nuclei and on mitotic tubulin structures. By 3 h postinfection, a strong signal of the VP1 (but no viral particles) had accumulated in the proximity of nuclei, around the outer nuclear membrane. However, the vast majority of VP1 pseudocapsids did not enter the nuclei.

scholarly journals Colorimetric Test for Fast Detection of SARS-CoV-2 in Nasal and Throat Swabs

2020 ◽  
Author(s):  
Bartolomeo Della Ventura ◽  
Michele Cennamo ◽  
Antonio Minopoli ◽  
Raffaele Campanile ◽  
Sergio Bolletti Censi ◽  
...  
Keyword(s):  
Viral Particle ◽  
Extinction Spectrum ◽  
Colorimetric Method ◽  
Surface Proteins ◽  
Mixed Solution ◽  
Colorimetric Test ◽  
The People ◽  
Viral Particles ◽  
New Perspective ◽  
Viral Target

Mass testing is fundamental to face the pandemic caused by the coronavirus SARS-CoV-2 discovered at the end of 2019. To this aim, it is necessary to establish reliable, fast and cheap tools to detect viral particles in biological material so to identify the people capable to spread the infection. We demonstrate that a colorimetric biosensor based on gold nanoparticle (AuNP) interaction induced by SARS-CoV-2 lends itself as an outstanding tool for detecting viral particles in nasal and throat swabs. The extinction spectrum of a colloidal solution of multiple viral-target gold nanoparticles - AuNPs functionalized with antibodies targeting three surface proteins of SARS-CoV-2 (spike, envelope and membrane) - is redshifted in few minutes when mixed to a solution containing the viral particle. The optical density of the mixed solution measured at 560 nm was compared to the threshold cycle (Ct) of a Real Time-PCR (gold standard for detecting the presence of viruses) finding that the colorimetric method is able to detect very low viral load with a detection limit approaching that of RT-PCR. Since the method is sensitive to the infecting viral particle rather than to its RNA, the achievements reported here open new perspective not only in the context of the current and possible future pandemics, but also in microbiology as the biosensor proves itself to be a powerful though simple tool for measuring the viral particle concentration.

scholarly journals Functional Correlation between Subcellular Localizations of Japanese Encephalitis Virus Capsid Protein and Virus Production

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Kotaro Ishida ◽  
Simon Goto ◽  
Marina Ishimura ◽  
Misato Amanuma ◽  
Yumiko Hara ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Virus Particle ◽  
Capsid Protein ◽  
Viral Particle ◽  
Particle Formation ◽  
Molecular Surface ◽  
Functional Correlation ◽  
Viral Particles ◽  
Detergent Treatment ◽  
Virus Capsid Protein

ABSTRACT The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles. IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

Virus Particles Infected the Erythrocytes of Cultured Eel in Taiwan

1981 ◽  
Vol 39 ◽  
pp. 608-609
Author(s):  
Kuo-chun Liu
Keyword(s):  
Viral Particle ◽  
Electron Microscopic Observation ◽  
Field Trips ◽  
Fish Farms ◽  
Electron Microscopic ◽  
Internal Organs ◽  
Virus Particles ◽  
Pathological Conditions ◽  
Viral Particles ◽  
Electron Lucent

The erythrocytes infected by viral particles and the pathological conditions by the presence of those viral particles were reported in cod, herring and few other species of fish in the western waters. This report will present the evidence that the erythrocytes of some of the cultured eel in Taiwan were infected by virus particles.In the summer of 1980, in one of the field trips to the fish farms in Lotung county, northeastern of Taiwan, the author was informed that dead eels from unknown cause were picked up from eel ponds every morning. Samples were collected the following morning. There was no observable external symptom.The internal organs showed a red coloration (Fig. 1,2). Tissues from the livers and gills were processed for electron microscopic observation. Virus particles were found in some of the erythrocytes in both organs (Fig.3).The viral particles aggregated together as an inclusion body in the cytoplasm only. No intranuclear viral particle was found. In a single viral aggregate 1 to 40 particles were observed. A electron lucent ring separated the viral aggregate from the hemoglobin cytoplasm.

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