Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Xu P

PROVIDER: S-EPMC6783920 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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

Xu Peng P Wang Xiaomei X Li Yi Y Qiu Jianming J

Viruses 20190904 9

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 <i>Ad5</i> and <i>Ad11p</i> (rAd5F11p) to increase the gene delivery ef ...[more]

PMID: 31487941

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Project description:Human parvovirus B19 (B19V) has been strongly associated with a variety of inflammatory disorders, such as rheumatoid arthritis (RA), inflammatory bowel disease and systemic lupus erythematosus. Non‑structural protein 1 (NS1) of B19V has been demonstrated to play essential roles in the pathological processes of B19V infection due to its regulatory properties on inflammatory cytokines. Celastrol, a quinone methide isolated from Tripterygium wilfordii, has displayed substantial potential in treating inflammatory diseases, such as psoriasis and RA. However, little is known about the effects of celastrol on B19V NS1‑induced inflammation. Therefore, cell viability assay, migration assay, phagocytosis analysis, zymography assay, ELISA and immunoblotting were conducted to verify the influences of celastrol on macrophages. The present study reported the attenuating effects of celastrol on B19V NS1‑induced inflammatory responses in macrophages derived from human acute monocytic leukemia cell lines, U937 and THP‑1. Although the migration was not significantly decreased by celastrol in both U937 and THP‑1 macrophages, significantly decreased viability, migration and phagocytosis were detected in both B19V NS1‑activated U937 and THP‑1 macrophages in the presence of celastrol. Additionally, celastrol significantly decreased MMP‑9 activity and the levels of inflammatory cytokines, including IL‑6, TNF‑α and IL‑1β, in B19V NS1‑activated U937 and THP‑1 cells. Notably, significantly decreased levels of NLR family pyrin domain‑containing 3, apoptosis‑associated speck‑like, caspase‑1 and IL‑18 proteins were observed in both B19V NS1‑activated U937 and THP‑1 cells in the presence of celastrol, indicating the involvement of the inflammasome pathway. To the best of our knowledge, the present study is the first to report on the attenuating effects of celastrol on B19V NS1‑induced inflammatory responses in macrophages, suggesting a therapeutic role for celastrol in B19V NS1‑related inflammatory diseases.

Project description:Parvovirus B19 infections are associated with diverse clinical manifestations, ranging from no symptoms to severe symptoms. The virus shows an extreme tropism for replication in erythroid progenitor cells, possibly due to the activity of the only functional promoter (p6) of the B19 virus genome in combination with both cell- and cell cycle-specific factors and the trans-activator protein NS1. As presented here, p6 promoter sequences derived from several B19 virus isolates proved to be highly conserved. Furthermore, mutations did not affect any of the potential binding sites for transcription factors. One variation of the base at position 223 was identified only in B19 virus isolates derived from patients with persistent infection or chronic arthritis. To determine promoter activity and to characterize regulatory elements, sequences spanning the total p6 promoter and subfragments of them were introduced into a eukaryotic expression vector upstream of the luciferase gene (from Photinus pyralis). After transfection into HeLa, CEM, BJAB, and K562 cells, the p6 promoter was found to be highly active. When introduced into the erythroid cell line K562, p6-controlled transcription exceeded that of the simian virus 40 promoter-enhancer used as a control by more than 25-fold. Sequence elements relevant for promoter activity mapped to the regions from nucleotides (nt) 100 to 190 and 233 to 298. Also, the segment from nt 343 to 400 downstream of the TATA box was important for transcriptional activity in HeLa and K562 cells. By transfecting the promoter-luciferase constructs into a HeLa cell line stably carrying the viral NS1 gene under the control of an inducible promoter, transcriptional activity mediated by the p6 promoter rose significantly after induction of NS1 expression. The region from nt 100 to 160 proved to be essential for NS1-mediated transcriptional activation. Furthermore, NS1-mediated transactivation was dependent on the presence of two GC-rich elements arranged in tandem upstream of the TATA box. These data indicate that NS1-mediated p6 transactivation is dependent on a multicomponent complex combining NS1 with ATF, NF-kappaB/c-Rel, and GC-box binding cellular factors.

Dataset Information

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

Publications

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

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