scholarly journals Super-rapid quantitation of the production of HIV-1 harboring a luminescent peptide tag

2020 ◽  
Vol 295 (37) ◽  
pp. 13023-13030
Author(s):  
Seiya Ozono ◽  
Yanzhao Zhang ◽  
Minoru Tobiume ◽  
Satoshi Kishigami ◽  
Kenzo Tokunaga
Keyword(s):  
Virus Production ◽  
Integrase Inhibitor ◽  
Luciferase Assay ◽  
Protein Levels ◽  
C Terminus ◽  
P24 Protein ◽  
Luminescent Signal ◽  
Routine Handling ◽  
Hiv 1 ◽  
Peptide Tag

In studies of HIV-1, virus production is normally monitored by either a reverse transcriptase assay or a p24 antigen capture ELISA. However, these assays are costly and time-consuming for routine handling of a large number of HIV-1 samples. For example, sample dilution is always required in the ELISA procedure to determine p24 protein levels because of the very narrow range of detectable concentrations in this assay. Here, we establish a novel HIV-1 production assay system to solve the aforementioned problems by using a recently developed small peptide tag called HiBiT. This peptide is a fragment of NanoLuc luciferase and generates a strong luminescent signal when complemented with the remaining subunit. To employ this technology, we constructed a novel full-length proviral HIV-1 DNA clone and a lentiviral packaging vector in which the HiBiT tag was added to the C terminus of the integrase. Tagging the integrase with the HiBiT sequence did not impede the resultant virus production, infectivity, or susceptibility to an integrase inhibitor. EM revealed normal morphology of the virus particles. Most importantly, by comparing between ELISA and the HiBiT luciferase assay, we successfully obtained an excellent linear correlation between p24 concentrations and HiBiT-based luciferase activity. Overall, we conclude that HiBiT-tagged viruses can replace the parental HIV-1 and lentiviral vectors, which enables us to perform a super-rapid, inexpensive, convenient, simple, and highly accurate quantitative assay for HIV-1/lentivirus production. This system can be widely applied to a variety of virological studies, along with screening for candidates of future antiviral drugs.

scholarly journals Super-rapid quantitation of the production of HIV-1 harboring a luminescent peptide tag

2020 ◽  
Author(s):  
Seiya Ozono ◽  
Yanzhao Zhang ◽  
Minoru Tobiume ◽  
Satoshi Kishigami ◽  
Kenzo Tokunaga
Keyword(s):  
Virus Production ◽  
Luciferase Assay ◽  
Virus Particles ◽  
Protein Levels ◽  
C Terminus ◽  
P24 Protein ◽  
P24 Antigen ◽  
Routine Handling ◽  
Hiv 1 ◽  
Peptide Tag

ABSTRACTIn virological studies using HIV-1 proviral clones, virus production is normally monitored by either an RT assay or a p24 antigen capture ELISA. However, these assays are costly and time-consuming for routine handling of a large number of HIV-1 samples. In addition, sample dilution is always required in ELISA to determine p24 protein levels because of the very narrow range of detectable concentrations in this assay. Here, we establish a novel HIV-1 production assay system to solve the aforementioned problems by using a recently developed small peptide tag called HiBiT. First, we constructed a novel full-length proviral HIV-1 DNA clone and a lentiviral packaging vector in which the HiBiT tag was added to the C terminus of the integrase. Tagging the integrase with the HiBiT sequence did not impede the production or infectivity of the resultant viruses. Electron microscopy revealed normal morphology of the virus particles. Most importantly, by comparing between ELISA and the HiBiT luciferase assay, we successfully obtained an excellent linear correlation between p24 concentrations and HiBiT-based luciferase activity. Overall, we conclude that HiBiT-tagged viruses can replace the parental HIV-1 and lentiviral vectors, which enables us to perform a super-rapid, inexpensive, convenient, simple, and highly accurate quantitative assay for HIV-1 production.

scholarly journals Flavonol 7-O-Glucoside Herbacitrin Inhibits HIV-1 Replication through Simultaneous Integrase and Reverse Transcriptase Inhibition

2019 ◽  
Vol 2019 ◽  
pp. 1-6 ◽  
Author(s):  
Éva Áy ◽  
Attila Hunyadi ◽  
Mária Mezei ◽  
János Minárovits ◽  
Judit Hohmann
Keyword(s):  
Antiviral Activity ◽  
Reverse Transcriptase ◽  
Cell Cultures ◽  
Core Protein ◽  
Integrase Inhibitor ◽  
Host Cells ◽  
Cytotoxic Activities ◽  
Protein Levels ◽  
Hiv 1 ◽  
Antiretroviral Activity

Here we report the evaluation of the antiretroviral effect of two flavonoid 7-O-glucosides, herbacitrin (1) and gossypitrin (2), together with quercetin (3), a well-studied flavonol. Antiviral activity of the flavonoids was assessed by analyzing HIV-1 p24 core protein levels in the supernatants of HIV-1 infected MT-4 and MT-2 cell cultures. The compounds showed mild to weak cytotoxic activities on the host cells; herbacitrin was the strongest in this regard (CC50=27.8 and 63.64 μM on MT-4 and MT-2 cells, respectively). In nontoxic concentrations, herbacitrin and quercetin reduced HIV-1 replication, whereas gossypitrin was ineffective. Herbacitrin was found to inhibit reverse transcriptase at 21.5 μM, while it was a more potent integrase inhibitor already active at 2.15 μM. Therefore, our observations suggest that herbacitrin exerts antiretroviral activity through simultaneously acting on these two targets of HIV-1 and that integrase inhibition might play a major role in this activity.

scholarly journals C-Terminal HIV-1 Transframe p6* Tetrapeptide Blocks Enhanced Gag Cleavage Incurred by Leucine Zipper Replacement of a Deleted p6* Domain

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Fu-Hsien Yu ◽  
Kuo-Jung Huang ◽  
Chin-Tien Wang
Keyword(s):  
Leucine Zipper ◽  
Virus Assembly ◽  
Virus Production ◽  
Supporting Evidence ◽  
Dependent Manner ◽  
Dimer Formation ◽  
Reading Frame ◽  
Virus Maturation ◽  
C Terminus ◽  
Hiv 1

ABSTRACT HIV-1 protease (PR) functions as a homodimer mediating virus maturation following virus budding. Gag-Pol dimerization is believed to trigger embedded PR activation by promoting PR dimer formation. Early PR activation can lead to markedly reduced virus yields due to premature Gag cleavage. The p6* peptide, located between Gag and PR, is believed to ensure virus production by preventing early PR maturation. Studies aimed at finding supporting evidence for this proposal are limited due to a reading frame overlap between p6* and the p6gag budding domain. To determine if p6* affects virus production via the modulation of PR activation, we engineered multiple constructs derived from Dp6*PR (an assembly- and processing-competent construct with Pol fused at the inactivated PR C terminus). The data indicated that a p6* deletion adjacent to active PR significantly impaired virus processing. We also observed that the insertion of a leucine zipper (LZ) dimerization motif in the deleted region eliminated virus production in a PR activity-dependent manner, suggesting that the LZ insertion triggered premature PR activation by facilitating PR dimer formation. As few as four C-terminal p6* residues remaining at the p6*/PR junction were sufficient to restore virus yields, with a Gag processing profile similar to that of the wild type. Our study provides supporting evidence in a virus assembly context that the C-terminal p6* tetrapeptide plays a role in preventing premature PR maturation. IMPORTANCE Supporting evidence for the assumption that p6* retards PR maturation in the context of virus assembly is lacking. We found that replacing p6* with a leucine zipper peptide abolished virus assembly due to the significant enhancement of Gag cleavage. However, as few as four C-terminal p6* residues remaining in the deleted region were sufficient for significant PR release, as well as for counteracting leucine zipper-incurred premature Gag cleavage. Our data provide evidence that (i) p6* ensures virus assembly by preventing early PR activation and (ii) four C-terminal p6* residues are critical for modulating PR activation. Current PR inhibitor development efforts are aimed largely at mature PR, but there is a tendency for HIV-1 variants that are resistant to multiple protease inhibitors to emerge. Our data support the idea of modulating PR activation by targeting PR precursors as an alternative approach to controlling HIV-1/AIDS.

scholarly journals Zinc Finger Endonuclease TargetingPSIP1Inhibits HIV-1 Integration

2014 ◽  
Vol 58 (8) ◽  
pp. 4318-4327 ◽  
Author(s):  
Roger Badia ◽  
Eduardo Pauls ◽  
Eva Riveira-Munoz ◽  
Bonaventura Clotet ◽  
José A. Esté ◽  
...  
Keyword(s):  
Viral Replication ◽  
Zinc Finger ◽  
Viral Entry ◽  
Antiviral Agent ◽  
Integrase Inhibitor ◽  
Replication Cycle ◽  
Zinc Finger Nucleases ◽  
Coding Region ◽  
Protein Levels ◽  
Hiv 1

ABSTRACTGenome editing using zinc finger nucleases (ZFNs) has been successfully applied to disrupt CCR5 or CXCR4 host factors and inhibit viral entry and infection. Gene therapy using ZFNs to modify thePSIP1gene, which encodes the lens epithelium-derived growth factor (LEDGF) protein, might restrain an early step of the viral replication cycle at the integration level. ZFNs targeting thePSIP1gene (ZFNLEDGF) were designed to specifically recognize the sequence after the integrase binding domain (IBD) of the LEDGF/p75 protein. ZFNLEDGFsuccessfully recognized the target region of thePSIP1gene in TZM-bl cells by heteroduplex formation and DNA sequence analysis. Gene editing induced a frameshift of the coding region and resulted in the abolishment of LEDGF expression at the mRNA and protein levels. Functional assays revealed that infection with the HIV-1 R5 BaL or X4 NL4-3 viral strains was impaired in LEDGF/p75 knockout cells regardless of entry tropism due to a blockade in HIV-1 proviral integration into the host genome. However, residual infection was detected in the LEDGF knockout cells. Indeed, LEDGF knockout restriction was overcome at a high multiplicity of infection, suggesting alternative mechanisms for HIV-1 genome integration rather than through LEDGF/p75. However, the observed residual integration was sensitive to the integrase inhibitor raltegravir. These results demonstrate that the described ZFNLEDGFeffectively targets thePSIP1gene, which is involved in the early steps of the viral replication cycle; thus, ZFNLEDGFmay become a potential antiviral agent for restricting HIV-1 integration. Moreover, LEDGF knockout cells represent a potent tool for elucidating the role of HIV integration cofactors in virus replication.

scholarly journals Chimeric Cyanovirin-MPER Recombinantly Engineered Proteins Cause Cell-Free Virolysis of HIV-1

2013 ◽  
Vol 57 (10) ◽  
pp. 4743-4750 ◽  
Author(s):  
Mark Contarino ◽  
Arangassery R. Bastian ◽  
Ramalingam Venkat Kalyana Sundaram ◽  
Karyn McFadden ◽  
Caitlin Duffy ◽  
...  
Keyword(s):  
Leukemia Virus ◽  
Gel Filtration ◽  
Etiologic Agent ◽  
Host Cells ◽  
Dependent Manner ◽  
Cell Infection ◽  
C Terminus ◽  
P24 Protein ◽  
Env Protein ◽  
Hiv 1

ABSTRACTHuman immunodeficiency virus (HIV) is the primary etiologic agent responsible for the AIDS pandemic. In this work, we used a chimeric recombinant protein strategy to test the possibility of irreversibly destroying the HIV-1 virion using an agent that simultaneously binds the Env protein and viral membrane. We constructed a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly4Ser)xlinker (wherexis 4 or 8) between the C terminus of the former and N terminus of the latter. The His-tagged recombinant proteins, expressed in BL21(DE3)pLysS cells and purified by immobilized metal affinity chromatography followed by gel filtration, were found to display a nanomolar efficacy in blocking BaL-pseudotyped HIV-1 infection of HOS.T4.R5 cells. This antiviral activity was HIV-1 specific, since it did not inhibit cell infection by vesicular stomatitis virus (VSV) or amphotropic-murine leukemia virus. Importantly, the chimeric proteins were found to release intraviral p24 protein from both BaL-pseudotyped HIV-1 and fully infectious BaL HIV-1 in a dose-dependent manner in the absence of host cells. The addition of either MPER or CVN was found to outcompete this virolytic effect, indicating that both components of the chimera are required for virolysis. The finding that engaging the Env protein spike and membrane using a chimeric ligand can destabilize the virus and lead to inactivation opens up a means to investigate virus particle metastability and to evaluate this approach for inactivation at the earliest stages of exposure to virus and before host cell encounter.

Ultrastructural observations of human monocytes infected with HIV-1

1991 ◽  
Vol 49 ◽  
pp. 108-109
Author(s):  
James K. Koehler ◽  
Steven G. Reed ◽  
Joao S. Silva
Keyword(s):  
Gradient Centrifugation ◽  
Virus Production ◽  
Human Monocyte ◽  
Red Cross ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Hiv Replication ◽  
Electron Microscopic ◽  
Ultrastructural Studies ◽  
Hiv 1

As part of a larger study involving the co-infection of human monocyte cultures with HIV and protozoan parasites, electron microscopic observations were made on the course of HIV replication and infection in these cells. Although several ultrastructural studies of the cytopathology associated with HIV infection have appeared, few studies have shown the details of virus production in “normal,” human monocytes/macrophages, one of the natural targets of the virus, and suspected of being a locus of quiescent virus during its long latent period. In this report, we detail some of the interactions of developing virons with the membranes and organelles of the monocyte host.Peripheral blood monocytes were prepared from buffy coats (Portland Red Cross) by Percoll gradient centrifugation, followed by adherence to cover slips. 90-95% pure monocytes were cultured in RPMI with 5% non-activated human AB serum for four days and infected with 100 TCID50/ml of HIV-1 for four hours, washed and incubated in fresh medium for 14 days.

PROTEIN 24 HIV DAN LIMFOSIT T-CD4+ DI INFEKSI HIV TAHAP I

2016 ◽  
Vol 21 (3) ◽  
pp. 273
Author(s):  
I Made Sila Darmana ◽  
Endang Retnowati ◽  
Erwin Astha Triyono
Keyword(s):  
T Lymphocytes ◽  
Hiv Infection ◽  
T Lymphocyte ◽  
Negative Correlation ◽  
Stage I ◽  
Cross Sectional ◽  
Protein Levels ◽  
P24 Protein ◽  
Persistent Generalized Lymphadenopathy ◽  
Spearman Test

Measuring HIV p24 protein is a test which is more practical than determination of CD4+ T-lymphocyte counts and viral load, as it does not require a very sophisticated instrument and requires a lower cost. Independent predictive value of p24 to the decline of CD4+ T-lymphocytes, clinical progression and survival in HIV-infected patients have been reported. In this study, HIV-infected patients were found to have HIV p24 protein levels inversely proportional to CD4+ T-lymphocyte counts by using Spearman test (R2=0.225; p=0.0331). Studies on the correlation between HIV p24 protein levels and CD4+ T-lymphocyte counts in stage I HIV infection have not yet been reported. The aim of this study was to prove the correlation between HIV p24 protein levels and CD4+ T-lymphocytes in stage I HIV infection. Research issue was whether a correlation between HIV p24 protein levels and CD4+ T-lymphocyte counts in stage I HIVinfection existed ? The hypothesis was that a correlation between HIV p24 protein levels and CD4+ T-lymphocyte counts in stage I HIV infection existed. The study design was cross sectional observational. Subjects consisted of 30 stage I HIV-infected patients treated at the Infectious Disease Intermediate Care Unit, Dr. Soetomo Hospital and VCT Clinic of the Dr. Ramelan Naval Hospital, Surabaya from May to July 2014. Stage I HIV infection is an asymptomatic HIV infection or with persistent generalized lymphadenopathy and the patient is able to perform normal activities. Levels of p24 were measured by ELISA method and CD4+ T-lymphocyte counts using flowcytometry(BD FACSCaliburTM). The results were statistically analyzed using Pearson’s correlation test. HIV p24 protein levels in stage I of HIV infection ranged from 1.8 to 10.8 pg/mL, mean of 5.14 pg/mL and a standard deviation of 2.08 pg/mL. CD4+ T-lymphocyte counts decreased with a range of 49-559 cells /uL for absolute values and 4.42–26.02% for percentage values Correlations between blood p24 levels and CD4+ T-lymphocyte counts either absolute (r=–0.392, p=0.032) or percentage (r=–0.363, p=0.049) were found. In stage I HIV-infected patients, a negative correlation was found between p24 levels and CD4+ T-lymphocyte counts, in both CD4+T-lymphocyte counts as absolute and as well as percentage values. This negative correlation showed that the p24 HIV levels were inversely proportional to the CD4+ T-lymphocyte counts. HIV p24 protein levels have a possibility to be used predicting CD4+ T-lymphocyte counts

The Natural Flavonoid Naringenin Inhibits the Cell Growth of WilmsTumorinChildren by Suppressing TLR4/NF-κB Signaling

2020 ◽  
Vol 20 ◽  
Author(s):  
Hongtao Li ◽  
Peng Chen ◽  
Lei Chen ◽  
Xinning Wang
Keyword(s):  
Cell Growth ◽  
Western Blot ◽  
Wilms Tumor ◽  
Critical Role ◽  
Time Dependent ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Tlr4 Expression ◽  
Protein Levels

Background: Nuclear factor kappa B (NF-κB) is usually activated in Wilms tumor (WT) cells and plays a critical role in WT development. Objective: The study purpose was to screen a NF-κB inhibitor from natural product library and explore its effects on WT development. Methods: Luciferase assay was employed to assess the effects of natural chemical son NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Results: Naringenin displayed significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SK-NEP-1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α-induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in dose-and time-dependent manner, whereas Toll-like receptor 4 (TLR4) over expression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in dose-and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Conclusion: Naringenin inhibits WT development viasuppressing TLR4/NF-κB signaling

Contribution of Growth Arrest-Specific 5/miR-674 to the Hypothalamus Pituitary Adrenal Axis Regulation Effect by Electroacupuncture following Trauma

2021 ◽  
pp. 1-13
Author(s):  
Jing Zhu ◽  
Chunxia Guo ◽  
Pingping Lu ◽  
Shuijin Shao ◽  
Bing Tu
Keyword(s):  
Hpa Axis ◽  
Interleukin 1 ◽  
Growth Arrest ◽  
Luciferase Assay ◽  
Function Evaluation ◽  
Protein Levels ◽  
Adrenal Axis ◽  
Post Surgery ◽  
Hypothalamus Pituitary Adrenal Axis ◽  
Pituitary Adrenal Axis

<b><i>Background:</i></b> Electroacupuncture (EA) can improve trauma-induced hypothalamus pituitary adrenal axis (HPA) hyperactivity. However, the mechanism underlying the EA effect has not been fully understood. <b><i>Methods and Study Design:</i></b> This study was undertaken to explore the role of hypothalamic growth arrest-specific 5 (Gas5) in the regulation of EA on HPA axis function post-surgery. Paraventricular nuclear Gas5 levels were upregulated in rats using an intracerebroventricular injection of pAAV-Gas5. Primary hypothalamic neurons and 293T cells were cultured for miRNA and siRNAs detection. Radioimmunoassay, PCR, Western blot, and immunohistochemistry were used for HPA axis function evaluation. <b><i>Results:</i></b> The overexpression of Gas5 abolished the effect of EA on the regulation of trauma-induced HPA axis hyperactivity. Using a bioinformatics analysis and dual luciferase assay, we determined that miRNA-674 was a target of Gas5. Additionally, miRNA-674 levels were found to have decreased in trauma rats, and this effect was reversed after EA intervention. TargetScan analysis showed that serum and glucocorticoid inducible kinase 1 (SGK1) were targets of miR-674. Moreover, we found that SGK1 protein levels increased in trauma rats and SGK1 expression inhibition alleviated HPA axis abnormality post-surgery. EA could improve the number of hypothalamus iba-1 positive cells and hypothalamic interleukin 1 beta protein expression. <b><i>Conclusions:</i></b> Our study demonstrated the involvement of the hypothalamic Gas5/miRNA-674/SGK1 signaling pathway in EA regulation of HPA axis function after trauma.

scholarly journals No difference in HIV-1 integrase inhibitor resistance between CSF and blood compartments

2021 ◽  
Author(s):  
Basma Abdi ◽  
Mouna Chebbi ◽  
Marc Wirden ◽  
Elisa Teyssou ◽  
Sophie Sayon ◽  
...  
Keyword(s):  
Integrase Inhibitor ◽  
Routine Care ◽  
Biological Data ◽  
Recombinant Form ◽  
Hiv Integrase ◽  
Resistance Mutations ◽  
Clinical Routine ◽  
Inhibitor Resistance ◽  
Hiv 1

Abstract Background Little is known about HIV-1 integrase inhibitor resistance in the CNS. Objectives This study aimed to evaluate integrase inhibitor resistance in CSF, as a marker of the CNS, and compare it with the resistance in plasma. Methods HIV integrase was sequenced both in plasma and CSF for 59 HIV-1 patients. The clinical and biological data were collected from clinical routine care. Results Among the 59 HIV-1 patients, 32 (54.2%) were under antiretroviral (ARV) treatment. The median (IQR) HIV-1 RNA in the plasma of viraemic patients was 5.32 (3.85–5.80) and 3.59 (2.16–4.50) log10 copies/mL versus 4.79 (3.56–5.25) and 3.80 (2.68–4.33) log10 copies/mL in the CSF of ARV-naive and ARV-treated patients, respectively. The patients were mainly infected with non-B subtypes (72.2%) with the most prevalent recombinant form being CRF02_AG (42.4%). The HIV-1 integrase sequences from CSF presented resistance mutations for 9/27 (33.3%) and 8/32 (25.0%) for ARV-naive (L74I, n = 3; L74I/M, n = 1; T97A, n = 1; E157Q, n = 4) and ARV-treated (L74I, n = 6; L74M, n = 1; T97A, n = 1; N155H, n = 1) patients, respectively. Integrase inhibitor resistance mutations in CSF were similar to those in plasma, except for 1/59 patients. Conclusions This work shows similar integrase inhibitor resistance profiles in the CNS and plasma in a population of HIV-1 viraemic patients.

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