scholarly journals Establishment of a Parvovirus B19 NS1-Expressing Recombinant Adenoviral Vector for Killing Megakaryocytic Leukemia Cells

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 820 ◽  
Author(s):  
Xu ◽  
Wang ◽  
Li ◽  
Qiu
Keyword(s):  
Gene Delivery ◽  
Nonstructural Protein ◽  
Parvovirus B19 ◽  
Shuttle Vector ◽  
Leukemic Cells ◽  
Transduction Efficiency ◽  
The Novel ◽  
Ns1 Protein ◽  
M Phase ◽  
Serotype 5

Adenoviral viral vectors have been widely used for gene-based therapeutics, but commonly used serotype 5 shows poor transduction efficiency into hematopoietic cells. In this study, we aimed to generate a recombinant adenovirus serotype 5 (rAd5) vector that has a high efficiency in gene transfer to megakaryocytic leukemic cells with anticancer potential. We first modified the rAd5 backbone vector with a chimeric fiber gene of Ad5 and Ad11p (rAd5F11p) to increase the gene delivery efficiency. Then, the nonstructural protein NS1 of human parvovirus B19 (B19V), which induces cell cycle arrest at the G2/M phase and apoptosis, was cloned into the adenoviral shuttle vector. As the expression of parvoviral NS1 protein inhibited Ad replication and production, we engineered the cytomegalovirus (CMV) promoter, which governs NS1 expression, with two tetracycline operator elements (TetO2). Transfection of the rAd5F11p proviral vectors in Tet repressor-expressing T-REx-293 cells produced rAd in a large quantity. We further evaluated this chimeric rAd5F11p vector in gene delivery in human leukemic cells, UT7/Epo-S1. Strikingly, the novel rAd5F11p-B19NS1-GFP vector, exhibited a transduction efficiency much higher than the original vector, rAd5-B19NS1-GFP, in UT7/Epo-S1 cells, in particular, when they were transduced at a relatively low multiplicity of infection (100 viral genome copies/cell). After the transduction of rAd5F11p-B19NS1-GFP, over 90% of the UT7/Epo-S1 cells were arrested at the G2/M phase, and approximately 40%–50% of the cells were undergoing apoptosis, suggesting the novel rAd5F11P-B19NS1-GFP vector holds a promise in therapeutic potentials of megakaryocytic leukemia.

A cytotoxic nonstructural protein, NS1, of human parvovirus B19 induces activation of interleukin-6 gene expression.

1996 ◽  
Vol 70 (12) ◽  
pp. 8485-8491 ◽  
Author(s):  
S Moffatt ◽  
N Tanaka ◽  
K Tada ◽  
M Nose ◽  
M Nakamura ◽  
...  
Keyword(s):  
Gene Expression ◽  
Interleukin 6 ◽  
Nonstructural Protein ◽  
Parvovirus B19 ◽  
Human Parvovirus B19

Inhibition by CTP and UTP analogues of uridine kinase from mouse leukemic cells

1982 ◽  
Vol 47 (12) ◽  
pp. 3464-3469 ◽  
Author(s):  
Jiří Veselý ◽  
Ivan Rosenberg ◽  
Antonín Holý
Keyword(s):  
Leukemic Cells ◽  
The Novel ◽  
Uridine Kinase ◽  
Phosphate Donor

The new phosphonate analogues of CTP and UTP (CTPc and UTPc) inhibit the phosphorylation of uridine catalysed by uridine kinase in the presence of ATP and Mg2+-ions. The inhibition is competitive with respect to phosphate donor, and non-competitive with respect to phosphate acceptor. With respect to uridine the Ki constants for CTPc and UTPc are 7.5 . 10-5 mol l-1 and 1.0 . 10-4 mol l-1, respectively. With respect to ATP the Ki value for CTPc (3.6 . 10-6 mol l-1) is 3x lower than that for CTP. The novel analogues could be useful for further study of uridine kinase.

scholarly journals Development of an HTS Assay for the Search of Anti-influenza Agents Targeting the Interaction of Viral RNA with the NS1 Protein

2008 ◽  
Vol 13 (7) ◽  
pp. 581-590 ◽  
Author(s):  
Marta Maroto ◽  
Yolanda Fernandez ◽  
Juan Ortin ◽  
Fernando Pelaez ◽  
M. Angerles Cabello
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Cytopathic Effect ◽  
Nonstructural Protein ◽  
Specific Interaction ◽  
Chemical Compounds ◽  
Viral Rna ◽  
Ns1 Protein ◽  
Rna Molecules ◽  
Novel Target

The NS1 protein is a nonstructural protein encoded by the influenza A virus. It is responsible for many alterations produced in the cellular metabolism upon infection by the virus and for modulation of virus virulence. The NS1 protein is able to perform a large variety of functions due to its ability to bind various types of RNA molecules, from both viral and nonviral origin, and to interact with several cell factors. With the aim of exploring whether the binding of NS1 protein to viral RNA (vRNA) could constitute a novel target for the search of anti-influenza drugs, a filter-binding assay measuring the specific interaction between the recombinant His-NS1 protein from influenza A virus and a radiolabeled model vRNA ( 32P-vNSZ) was adapted to a format suitable for screening and easy automation. Flashplate® technology (PerkinElmer, Waltham, MA), either in 96- or 384-well plates, was used. The Flashplate® wells were precoated with the recombinant His-NS1 protein, and the binding of His-NS1 to a 35S-vNSZ probe was measured. A pilot screening of a collection of 27,520 mixtures of synthetic chemical compounds was run for inhibitors of NS1 binding to vRNA. We found 3 compounds in which the inhibition of NS1 binding to vRNA, observed at submicromolar concentrations, was correlated with a reduction of the cytopathic effect during the infection of cell cultures with influenza virus. These results support the hypothesis that the binding of NS1 to vRNA could be a novel target for the development of anti-influenza drugs. ( Journal of Biomolecular Screening 2008:581-590)

scholarly journals Unexpected Functional Divergence of Bat Influenza Virus NS1 Proteins

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Hannah L. Turkington ◽  
Mindaugas Juozapaitis ◽  
Nikos Tsolakos ◽  
Eugenia Corrales-Aguilar ◽  
Martin Schwemmle ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Virulence Factor ◽  
Influenza A ◽  
Nonstructural Protein ◽  
Functional Divergence ◽  
Protein A ◽  
Regulatory Subunit ◽  
Ns1 Protein ◽  
Influenza A Viruses ◽  
Hydrophobic Patch

ABSTRACT Recently, two influenza A virus (FLUAV) genomes were identified in Central and South American bats. These sequences exhibit notable divergence from classical FLUAV counterparts, and functionally, bat FLUAV glycoproteins lack canonical receptor binding and destroying activity. Nevertheless, other features that distinguish these viruses from classical FLUAVs have yet to be explored. Here, we studied the viral nonstructural protein NS1, a virulence factor that modulates host signaling to promote efficient propagation. Like all FLUAV NS1 proteins, bat FLUAV NS1s bind double-stranded RNA and act as interferon antagonists. Unexpectedly, we found that bat FLUAV NS1s are unique in being unable to bind host p85β, a regulatory subunit of the cellular metabolism-regulating enzyme, phosphoinositide 3-kinase (PI3K). Furthermore, neither bat FLUAV NS1 alone nor infection with a chimeric bat FLUAV efficiently activates Akt, a PI3K effector. Structure-guided mutagenesis revealed that the bat FLUAV NS1-p85β interaction can be reengineered (in a strain-specific manner) by changing two to four NS1 residues (96L, 99M, 100I, and 145T), thereby creating a hydrophobic patch. Notably, ameliorated p85β-binding is insufficient for bat FLUAV NS1 to activate PI3K, and a chimeric bat FLUAV expressing NS1 with engineered hydrophobic patch mutations exhibits cell-type-dependent, but species-independent, propagation phenotypes. We hypothesize that bat FLUAV hijacking of PI3K in the natural bat host has been selected against, perhaps because genes in this metabolic pathway were differentially shaped by evolution to suit the unique energy use strategies of this flying mammal. These data expand our understanding of the enigmatic functional divergence between bat FLUAVs and classical mammalian and avian FLUAVs. IMPORTANCE The potential for novel influenza A viruses to establish infections in humans from animals is a source of continuous concern due to possible severe outbreaks or pandemics. The recent discovery of influenza A-like viruses in bats has raised questions over whether these entities could be a threat to humans. Understanding unique properties of the newly described bat influenza A-like viruses, such as their mechanisms to infect cells or how they manipulate host functions, is critical to assess their likelihood of causing disease. Here, we characterized the bat influenza A-like virus NS1 protein, a key virulence factor, and found unexpected functional divergence of this protein from counterparts in other influenza A viruses. Our study dissects the molecular changes required by bat influenza A-like virus NS1 to adopt classical influenza A virus properties and suggests consequences of bat influenza A-like virus infection, potential future evolutionary trajectories, and intriguing virus-host biology in bat species.

Abstract 17357: Direct Cardiac Reprogramming Using Combinatorial Modified mRNA

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Keerat Kaur ◽  
Asharee Mahmoud ◽  
Hanna Girard ◽  
Ann Anu Kurian ◽  
Magdalena Zak ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Gene Delivery ◽  
High Efficiency ◽  
De Novo ◽  
Heart Function ◽  
Lineage Tracing ◽  
Dominant Negative ◽  
Post Myocardial Infarction ◽  
Transduction Efficiency

Introduction: Despite various clinical modalities, ischemic heart disease remains among the leading causes of mortality and morbidity worldwide. The elemental problem is the immense loss of cardiomyocytes (CMs) post-myocardial infarction (MI). Reprogramming non- cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells in vivo is a promising strategy for cardiac regeneration: however, the traditional viral delivery method hampered its application into clinical settings due to low and erratic transduction efficiency. Methods: We used a modified mRNA (modRNA) gene delivery platform to deliver different stoichiometry of cardiac-reprogramming genes (Gata4, Mef2c, Tbx5 and Hand2) together with reprogramming helper genes (Dominant Negative (DN)-TGFβ, DN- Wnt8a and Acid ceramidase (AC)), named 7G, to induce direct cardiac reprogramming post myocardial infarction (MI). Results: Here, we identified 7G modRNA cocktail as an important regulator ofthe cardiac reprogramming. Cardiac transfection with 7G modRNA doubled cardiac reprogramming efficiency (57%) in comparison to Gata4, Mef2C and Tbx5 (GMT) alone (28%) in vitro . By inducing MI in our lineage tracing model, we showed that one-time delivery of the 7G-modRNA cocktail reprogrammed ~25% of the non-CMs in the scar area to CM- like cells. Furthermore, 7G modRNA treated mice showed significantly improved cardiac function, longer survival, reduced scar size and greater capillary density than control mice 28 days post-MI. We attributed the improvement in heart function post modRNA delivery of 7G or 7G with increased Hand2 ratio (7G-GMT Hx2) to significant upregulation of 15 key angiogenic factors without any signs of angioma or edema. Conclusions: 7G or 7G GMT HX2 modRNA cocktails boosts de novo CM-like cells and promotes cardiovascular regeneration post-MI. Thus, we highlight that this gene delivery approach not only has high efficiency but also high margin of safety for translation to clinics.

Structurally Mapping Antigenic Epitopes of Adeno-Associated Virus 9: Development of Antibody Escape Variants

2021 ◽  
Author(s):  
Shanan N. Emmanuel ◽  
J. Kennon Smith ◽  
Jane Hsi ◽  
Yu-Shan Tseng ◽  
Matias Kaplan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Gene Delivery ◽  
General Population ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Transduction Efficiency ◽  
Viral Neutralization ◽  
Patient Cohort ◽  
Antigenic Epitopes

Adeno-associated viruses (AAV) serve as vectors for therapeutic gene delivery. AAV9 vectors have been FDA approved, as Zolgensma®, for the treatment of spinal muscular atrophy and is being evaluated in clinical trials for the treatment of neurotropic and musculotropic diseases. A major hurdle for AAV-mediated gene delivery is the presence of pre-existing neutralizing antibodies in 40 to 80% of the general population. These pre-existing antibodies can reduce therapeutic efficacy through viral neutralization, and the size of the patient cohort eligible for treatment. In this study, cryo-electron microscopy and image reconstruction was used to define the epitopes of five anti-AAV9 monoclonal antibodies (MAbs); ADK9, HL2368, HL2370, HL2372, and HL2374, on the capsid surface. Three of these, ADK9, HL2370, and HL2374, bound on or near the icosahedral 3-fold axes, HL2368 to the 2/5-fold wall, and HL2372 to the region surrounding the 5-fold axes. Pseudo-atomic modeling enabled the mapping and identification of antibody contact amino acids on the capsid, including S454 and P659. These epitopes overlap with previously defined parvovirus antigenic sites. Capsid amino acids critical for the interactions were confirmed by mutagenesis followed by biochemical assays testing recombinant AAV9 (rAAV9) variants capable of escaping recognition and neutralization by the parental MAbs. These variants retained parental tropism and had similar or improved transduction efficiency compared to AAV9. These engineered rAAV9 variants could expand the patient cohort eligible for AAV9-mediated gene delivery by avoiding pre-existing circulating neutralizing antibodies. IMPORTANCE The use of recombinant AAVs (rAAVs) as delivery vectors for therapeutic genes is becoming increasingly popular, especially following the FDA approval of Luxturna® and Zolgensma®, based on serotypes AAV2 and AAV9, respectively. However, high titer anti-AAV neutralizing antibodies in the general population, exempts patients from treatment. The goal of this study is to circumvent this issue by creating AAV variant vectors not recognized by pre-existing neutralizing antibodies. The mapping of the antigenic epitopes of five different monoclonal antibodies (MAbs) on AAV9, to recapitulate a polyclonal response, enabled the rational design of escape variants with minimal disruption to cell tropism and gene expression. This study, which included four newly developed and now commercially available MAbs, provides a platform for the engineering of rAAV9 vectors that can be used to deliver genes to patients with pre-exiting AAV antibodies.

scholarly journals Threshold for Pre-existing Antibody Levels Limiting Transduction Efficiency of Systemic rAAV9 Gene Delivery: Relevance for Translation

2019 ◽  
Vol 13 ◽  
pp. 453-462 ◽  
Author(s):  
Aaron S. Meadows ◽  
Ricardo J. Pineda ◽  
Laurie Goodchild ◽  
Tierra A. Bobo ◽  
Haiyan Fu
Keyword(s):  
Gene Delivery ◽  
Transduction Efficiency ◽  
Antibody Levels

scholarly journals Mutations in the NS1 Protein of Swine Influenza Virus Impair Anti-Interferon Activity and Confer Attenuation in Pigs

2005 ◽  
Vol 79 (12) ◽  
pp. 7535-7543 ◽  
Author(s):  
Alicia Solórzano ◽  
Richard J. Webby ◽  
Kelly M. Lager ◽  
Bruce H. Janke ◽  
Adolfo García-Sastre ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Nonstructural Protein ◽  
Swine Influenza Virus ◽  
Swine Influenza ◽  
Influenza Viruses ◽  
Model Systems ◽  
Ns1 Protein ◽  
Wild Type ◽  
Virulence Attenuation

ABSTRACT It has been shown previously that the nonstructural protein NS1 of influenza virus is an alpha/beta interferon (IFN-α/β) antagonist, both in vitro and in experimental animal model systems. However, evidence of this function in a natural host has not yet been obtained. Here we investigated the role of the NS1 protein in the virulence of a swine influenza virus (SIV) isolate in pigs by using reverse genetics. The virulent wild-type A/Swine/Texas/4199-2/98 (TX/98) virus and various mutants encoding carboxy-truncated NS1 proteins were rescued. Growth properties of TX/98 viruses with mutated NS1, induction of IFN in tissue culture, and virulence-attenuation in pigs were analyzed and compared to those of the recombinant wild-type TX/98 virus. Our results indicate that deletions in the NS1 protein decrease the ability of the TX/98 virus to prevent IFN-α/β synthesis in pig cells. Moreover, all NS1 mutant viruses were attenuated in pigs, and this correlated with the amount of IFN-α/β induced in vitro. These data suggest that the NS1 protein of SIV is a virulence factor. Due to their attenuation, NS1-mutated swine influenza viruses might have a great potential as live attenuated vaccine candidates against SIV infections of pigs.

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