scholarly journals A Recombinant Adenoviral Vector With a Specific Tropism to CD4-positive Cells: a New Tool for HIV-1 Inhibition

2021 ◽  
Author(s):  
Abtin Behmardi ◽  
Touraj Farazmandfar
Keyword(s):  
Expression System ◽  
Inducible Promoter ◽  
Protein Domain ◽  
Hek293 Cells ◽  
Cd4 Cells ◽  
Shrna Expression ◽  
Viral Binding ◽  
Extracellular Region ◽  
Fiber Protein ◽  
Hiv 1

Abstract Gene therapy can be an option to overcoming the side effects of chemotherapy and preventing the development of drug-resistant HIV viruses in HIV-infected patients. The need to develop a safe and efficient vector for gene transfer is always necessary and an appropriate option might be adenovirus (Ad). the use of Ad vectors in the gene delivery applications is limited due to the semi-specific tropism. A strategy to overcome this tropism limitation may be the modification of fiber protein domain involved in the viral binding to cells. Therefore, we designed an Ad5 vector with a specific tropism to CD4+ cells containing an expression system limited to HIV-infected cells. We replaced the knob region of Ad5 fiber protein with the extracellular region of HIV-1 envelope. We also used a specific Tat-inducible promoter to express two anti-HIV-1 shRNAs. Tropism of recombinant Ad5 was assayed by comparison of shRNA expression level in CEM and PBMC cells (as CD4+ cells) and HEK293 cells (as CD4- cells). HIV-1 inhibition was assayed by determination of p24 antigen in the HIV-infected CEM cells transduced with the recombinant Ad5 vector. Our results showed that shRNA expression was significantly higher in CEM and PBMC cells than HEK293 cells when were transduced with recombinant Ad5 vector. This new Ad5 vector also inhibited HIV-1 proliferation in a Tat-inducible manner. Our new recombinant Ad5 vector has a specific tropism to CD4-positive cells that can effectively suppress the HIV-1 replication.

Inhibition of HIV-1 Replication by an HIV-1 Dependent Ribozyme Expression Vector with the Cre/loxP (ON/OFF) System

2002 ◽  
Vol 13 (5) ◽  
pp. 273-281 ◽  
Author(s):  
Yuichiro Habu ◽  
Naoko Miyano-Kurosaki ◽  
Takashi Nagawa ◽  
Norihiko Matsumoto ◽  
Hiroaki Takeuchi ◽  
...  
Keyword(s):  
Expression Vector ◽  
Inducible Promoter ◽  
Protein Product ◽  
Gene Products ◽  
Cd4 Cells ◽  
High Background ◽  
Recombination System ◽  
Recombinase Gene ◽  
Ltr Promoter ◽  
Hiv 1

Antiviral strategies to inhibit HIV-1 replication have included the generation of gene products that provide the intracellular inhibition of an essential viral protein or RNA. When used in conjunction with the HIV-1 long terminal repeat (LTR), an inducible promoter dependent on the virus-encoded trans-activator ( tat), relatively high background activity is still observed in the absence of tat (Caruso & Klatzmann, 1992; Dinges et al., 1995). In order to circumvent this problem, we used the Cre/loxP (ON/OFF) recombination system as a tool for our investigation. In the present study, we constructed a loxP-cassette vector with the ribozyme (Rz) expression portion under the control of the tRNAiMet promoter between two loxP sequences (plox-Rz-U5). We also constructed an HIV-1 LTR promoter-driven Cre recombinase gene (pLTR-Cre). These vectors were triple-transfected into HeLa CD4 cells with the HIV-1 pseudo-type viral expression vector. Basal activity was not detectable before HIV-1 infection. The LTR-dependent Cre protein product in HIV-1 infected HeLa CD4 cells expressed the ribozyme by inducing loxP homologous recombination, which strongly inhibited the HIV-1 gene expression. These results demonstrate the potential of an anti-ribozyme with the Cre/loxP system for controlling HIV-1 infection via gene therapy.

Production of HIV-1 gp120 in Packed-Bed Bioreactor Using the Vaccinia Virus/T7 Expression System

2000 ◽  
Vol 16 (5) ◽  
pp. 744-750 ◽  
Author(s):  
Y.-C. Hu ◽  
J. Kaufman ◽  
M.W. Cho ◽  
H. Golding ◽  
J. Shiloach
Keyword(s):  
Vaccinia Virus ◽  
Expression System ◽  
Packed Bed ◽  
Packed Bed Bioreactor ◽  
T7 Expression System ◽  
Hiv 1

Conditional expression of the FTO gene product in rat INS-1 cells reveals its rapid turnover and a role in the profile of glucose-induced insulin secretion

2011 ◽  
Vol 120 (9) ◽  
pp. 403-413 ◽  
Author(s):  
Mark A. Russell ◽  
Noel G. Morgan
Keyword(s):  
Insulin Secretion ◽  
Expression System ◽  
Proteasome Inhibitors ◽  
Inducible Promoter ◽  
Insulin Biosynthesis ◽  
Second Phase ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Peripheral Tissues ◽  
Conditional Expression

Common polymorphisms within the FTO (fat mass and obesity-associated) gene correlate with increased BMI (body mass index) and a rising risk of Type 2 diabetes. FTO is highly expressed in the brain but has also been detected in peripheral tissues, including the endocrine pancreas, although its function there is unclear. The aim of the present study was to investigate the role of FTO protein in pancreatic β-cells using a conditional expression system developed in INS-1 cells. INS-1 cells were stably transfected with FTO–HA (haemagluttinin) incorporated under the control of a tetracycline-inducible promoter. Induction of FTO protein resulted in localization of the tagged protein to the nucleus. The level of FTO–HA protein achieved in transfected cells was tightly regulated, and experiments with selective inhibitors revealed that FTO–HA is rapidly degraded via the ubiquitin/proteasome pathway. The nuclear localization was not altered by proteasome inhibitors, although following treatment with PYR-41, an inhibitor of ubiquitination, some of the protein adopted a perinuclear localization. Unexpectedly, modestly increased expression of FTO–HA selectively enhanced the first phase of insulin secretion when INS-1 monolayers or pseudoislets were stimulated with 20 mM glucose, whereas the second phase remained unchanged. The mechanism responsible for the potentiation of glucose-induced insulin secretion is unclear; however, further experiments revealed that it did not involve an increase in insulin biosynthesis or any changes in STAT3 (signal transducer and activator of transcription 3) expression. Taken together, these results suggest that the FTO protein may play a hitherto unrecognized role in the control of first-phase insulin secretion in pancreatic β-cells.

scholarly journals Expression-System-Dependent Modulation of HIV-1 Envelope Glycoprotein Antigenicity and Immunogenicity

2010 ◽  
Vol 403 (1) ◽  
pp. 131-147 ◽  
Author(s):  
Leopold Kong ◽  
Neil C. Sheppard ◽  
Guillaume B.E. Stewart-Jones ◽  
Cynthia L. Robson ◽  
Hongying Chen ◽  
...  
Keyword(s):  
Envelope Glycoprotein ◽  
Expression System ◽  
Hiv 1

A multiplex real-time PCR for quantification of HIV-1 DNA and the human albumin gene in CD4+ cells

Apmis ◽  
2003 ◽  
Vol 111 (6) ◽  
pp. 625-633 ◽  
Author(s):  
LARS E. ERIKSSON ◽  
THOMAS LEITNER ◽  
BRITTA WAHREN ◽  
ANN-CHARLOTTE BOSTROM ◽  
KERSTIN I. FALK
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Human Albumin ◽  
Cd4 Cells ◽  
Albumin Gene ◽  
Hiv 1

Abstract 1503: Knockdown of Mog1 Expression Reduces Sodium Currents by Reducing the Trafficking of Cardiac Sodium Channel Na V 1.5 to the Cell Membrane

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Susmita Chakrabarti ◽  
Sandro Yong ◽  
Shin Yoo ◽  
Ling Wu ◽  
Qing Kenneth Wang
Keyword(s):  
Sodium Channel ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Expression System ◽  
Heart Association ◽  
Hek293 Cells ◽  
Similar Reduction ◽  
Mammalian Expression ◽  
Ventricular Cells ◽  
Cardiac Sodium Channel

The cardiac sodium channel (Na v 1.5) plays a significant role in cardiac physiology and leads to cardiac arrhythmias and sudden death when mutated. Modulation of Na v 1.5 activity can also arise from changes to accessory subunits or proteins. Our laboratory has recently reported that MOG1, a small protein that is highly conserved from yeast to humans, is a co-factor of Na v 1.5. Increased MOG1 expression has been shown to increase Na v 1.5 current density. In adult mouse ventricular myocytes, these two proteins were found to be co-localized at the intercalated discs. Here, we further characterize the regulatory role of MOG1 using the RNA interference technique. Sodium current was recorded in voltage-clamp mode from a holding potential of −100 mV and activated to −20 mV. In 3-day old mouse neonatal ventricular cells transfected with siRNA against mouse MOG1 decreased sodium current densities (pA/pF) compared to control or scramble siRNA treated cells (−10.2±3.3, n=11 vs. −165±16, n=20 or −117.9±11.7, n=11). A similar reduction in sodium current was observed in mammalian expression system consisting of HEK293 cells stably expressing human Na v 1.5, by transfecting siRNAs against either human or mouse MOG1 (−41.7±8.3, n=7 or, −82.6±9.6, n=7 vs. −130.6±11.5, n=7; −111.5±8.5, n=7, respectively). Immunocytochemistry revealed that the expression of MOG1 and Na v 1.5 were decreased in both HEK and neonatal cells when compared to scramble siRNAs or control groups. These results show that MOG1 is an essential co-factor for Na v 1.5 by way of a channel trafficking. Such interactions between MOG1 and Na v 1.5 suggest that early localization of MOG1 on the membrane of neonatal cardiomyocytes may be necessary for proper localization and the distribution of Na v 1.5 during cardiac development. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Database Study on the Expression and Purification of Membrane Proteins

2021 ◽  
Vol 28 ◽  
Author(s):  
Chen-Yan china Zhang ◽  
Shi-Qi Zhao ◽  
Shi-Long Zhang ◽  
Li-Heng Luo ◽  
Ding-Chang Liu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Expression ◽  
Drug Targets ◽  
Expression System ◽  
Data Bank ◽  
Hek293 Cells ◽  
Expression And Purification ◽  
Beta Barrel ◽  
Membrane Protein Expression

: Membrane proteins are crucial for biological processes, and many of them are important to drug targets. Understanding the three-dimensional structures of membrane proteins are essential to evaluate their bio function and drug design. High-purity membrane proteins are important for structural determination. Membrane proteins have low yields and are difficult to purify because they tend to aggregate. We summarized membrane protein expression systems, vectors, tags, and detergents, which have deposited in the Protein Data Bank (PDB) in recent four-and-a-half years. Escherichia coli is the most expression system for membrane proteins, and HEK293 cells are the most commonly cell lines for human membrane protein expression. The most frequently vectors are pFastBac1 for alpha-helical membrane proteins, pET28a for beta-barrel membrane proteins, and pTRC99a for monotopic membrane proteins. The most used tag for membrane proteins is the 6×His-tag. FLAG commonly used for alpha-helical membrane proteins, Strep and GST for beta-barrel and monotopic membrane proteins, respectively. The detergents and their concentrations used for alpha-helical, beta-barrel, and monotopic membrane proteins are different, and DDM is commonly used for membrane protein purification. It can guide the expression and purification of membrane proteins, thus contributing to their structure and bio function studying.

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