scholarly journals Tracking the Transcription Kinetic of SARS-CoV-2 in Human Cells by Reverse Transcription-Droplet Digital PCR

Pathogens ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1274
Author(s):  
Ka Ki Au ◽  
Chunke Chen ◽  
Yee Man Chan ◽  
Winsome Wing Sum Wong ◽  
Huibin Lv ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Digital Pcr ◽  
Human Cells ◽  
Target Cells ◽  
Viral Transcription ◽  
Structural Genes ◽  
Specific Primers ◽  
Transcription Process ◽  
Viral Genes ◽  
Kinetics Of

Viral transcription is an essential step of SARS-CoV-2 infection after invasion into the target cells. Antiviral drugs such as remdesivir, which is used to treat COVID-19 patients, targets the viral RNA synthesis. Understanding the mechanism of viral transcription may help to develop new therapeutic treatment by perturbing virus replication. In this study, we established 28 ddPCR assays and designed specific primers/probe sets to detect the RNA levels of 15 NSP, 9 ORF, and 4 structural genes of SARS-CoV-2. The transcriptional kinetics of these viral genes were determined longitudinally from the beginning of infection to 12 hours postinfection in Caco-2 cells. We found that SARS-CoV-2 takes around 6 hours to hijack the cells before the initiation of viral transcription process in human cells. Our results may contribute to a deeper understanding of the mechanisms of SARS-CoV-2 infection.

scholarly journals Detailed Characterization of Early HIV-1 Replication Dynamics in Primary Human Macrophages

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 620 ◽  
Author(s):  
David Bejarano ◽  
Maria Puertas ◽  
Kathleen Börner ◽  
Javier Martinez-Picado ◽  
Barbara Müller ◽  
...  
Keyword(s):  
Viral Replication ◽  
Reverse Transcription ◽  
Human Monocyte ◽  
Digital Pcr ◽  
Productive Infection ◽  
Target Cells ◽  
Post Entry ◽  
Early Replication ◽  
Kinetics Of ◽  
Hiv 1

Macrophages are natural target cells of human immunodeficiency virus type 1 (HIV-1). Viral replication appears to be delayed in these cells compared to lymphocytes; however, little is known about the kinetics of early post-entry events. Time-of-addition experiments using several HIV-1 inhibitors and the detection of reverse transcriptase (RT) products with droplet digital PCR (ddPCR) revealed that early replication was delayed in primary human monocyte-derived macrophages of several donors and peaked late after infection. Direct imaging of reverse-transcription and pre-integration complexes (RTC/PIC) by click-labeling of newly synthesized DNA further confirmed our findings and showed a concomitant shift to the nuclear stage over time. Altering the entry pathway enhanced infectivity but did not affect kinetics of viral replication. The addition of viral protein X (Vpx) enhanced productive infection and accelerated completion of reverse transcription and nuclear entry. We propose that sterile alpha motif (SAM) and histidine/aspartate (HD) domain-containing protein 1 (SAMHD1) activity lowering deoxyribonucleotide triphosphate (dNTP) pools is the principal factor delaying early HIV-1 replication in macrophages.

scholarly journals Continuous HIV-1 Escape from Autologous Neutralization and Development of Cross-Reactive Antibody Responses Characterizes Slow Disease Progression of Children

Vaccines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 260
Author(s):  
Stefania Dispinseri ◽  
Mariangela Cavarelli ◽  
Monica Tolazzi ◽  
Anna Maria Plebani ◽  
Marianne Jansson ◽  
...  
Keyword(s):  
Disease Progression ◽  
Antibody Responses ◽  
Viral Escape ◽  
Target Cells ◽  
Transmitted Virus ◽  
Slow Progressors ◽  
Vaccine Antigens ◽  
Kinetics Of ◽  
Hiv 1

The antibodies with different effector functions evoked by Human Immunodeficiency Virus type 1 (HIV-1) transmitted from mother to child, and their role in the pathogenesis of infected children remain unresolved. So, too, the kinetics and breadth of these responses remain to be clearly defined, compared to those developing in adults. Here, we studied the kinetics of the autologous and heterologous neutralizing antibody (Nab) responses, in addition to antibody-dependent cellular cytotoxicity (ADCC), in HIV-1 infected children with different disease progression rates followed from close after birth and five years on. Autologous and heterologous neutralization were determined by Peripheral blood mononuclear cells (PBMC)- and TZMbl-based assays, and ADCC was assessed with the GranToxiLux assay. The reactivity to an immunodominant HIV-1 gp41 epitope, and childhood vaccine antigens, was assessed by ELISA. Newborns displayed antibodies directed towards the HIV-1 gp41 epitope. However, antibodies neutralizing the transmitted virus were undetectable. Nabs directed against the transmitted virus developed usually within 12 months of age in children with slow progression, but rarely in rapid progressors. Thereafter, autologous Nabs persisted throughout the follow-up of the slow progressors and induced a continuous emergence of escape variants. Heterologous cross-Nabs were detected within two years, but their subsequent increase in potency and breadth was mainly a trait of slow progressors. Analogously, titers of antibodies mediating ADCC to gp120 BaL pulsed target cells increased in slow progressors during follow-up. The kinetics of antibody responses to the immunodominant viral antigen and the vaccine antigens were sustained and independent of disease progression. Persistent autologous Nabs triggering viral escape and an increase in the breadth and potency of cross-Nabs are exclusive to HIV-1 infected slowly progressing children.

scholarly journals Changes in J chain and mu chain RNA expression as a function of B cell differentiation.

1984 ◽  
Vol 160 (3) ◽  
pp. 877-892 ◽  
Author(s):  
G Lamson ◽  
M E Koshland
Keyword(s):  
B Cell ◽  
Time Course ◽  
Rna Synthesis ◽  
Rna Expression ◽  
B Cell Differentiation ◽  
Activated Lymphocytes ◽  
J Chain ◽  
Pattern Characteristic ◽  
Kinetics Of ◽  
Lymphoid Cell Lines

The time course of differentiative events in the pentamer IgM response was examined by following the expression of J chain and mu chain RNA and their protein products in mitogen-stimulated lymphocytes. The analyses showed that the shift to mus RNA synthesis begins shortly after stimulation and precedes proliferation of the cells and any increase in mu RNA levels. In contrast, expression of J chain RNA and the amplification of J chain and mus message are late events that coincide with a phase of rapid proliferation and with the secretion of pentamer IgM antibody. The kinetics of J and mu chain RNA expression observed in normal lymphocytes were supported by analyses of lymphoid cell lines. B lymphomas were found to display the RNA pattern characteristic of early-activated lymphocytes, i.e., a partial shift to mus RNA production and no J chain RNA, whereas IgM-secreting lines resembled late-activated lymphocytes in their expression of high levels of both mus and J chain mRNA. Moreover, the kinetics of J and mus chain RNA expression correlates with the sequential action of B cell lymphokines in the induction of the pentamer IgM response. This correlation suggests that the successive differentiative changes are triggered by successive membrane stimuli.

scholarly journals Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL.

1996 ◽  
Vol 184 (2) ◽  
pp. 485-492 ◽  
Author(s):  
M A Alexander-Miller ◽  
G R Leggatt ◽  
A Sarin ◽  
J A Berzofsky
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocyte ◽  
Target Cells ◽  
High Dose ◽  
High Avidity ◽  
Apoptotic Pathway ◽  
Peptide Antigen ◽  
Kinetics Of ◽  
Selection Of

Experimental data suggest that negative selection of thymocytes can occur as a result of supraoptimal antigenic stimulation. It is unknown, however, whether such mechanisms are at work in mature CD8+ T lymphocytes. Here, we show that CD8+ effector cytotoxic T lymphocytes (CTL) are susceptible to proliferative inhibition by high dose peptide antigen, leading to apoptotic death mediated by TNF-alpha release. Such inhibition is not reflected in the cytolytic potential of the CTL, since concentrations of antigen that are inhibitory for proliferation promote efficient lysis of target cells. Thus, although CTL have committed to the apoptotic pathway, the kinetics of this process are such that CTL function can occur before death of the CTL. The concentration of antigen required for inhibition is a function of the CTL avidity, in that concentrations of antigen capable of completely inhibiting high avidity CTL maximally stimulate low avidity CTL. Importantly, the inhibition can be detected in both activated and resting CTL. Blocking studies demonstrate that the CD8 molecule contributes significantly to the inhibitory signal as the addition of anti-CD8 antibody restores the proliferative response. Thus, our data support the model that mature CD8+ CTL can accommodate an activation signal of restricted intensity, which, if surpassed, results in deletion of that cell.

scholarly journals Effect of DNA-interacting drugs on phage T7 RNA polymerase.

1998 ◽  
Vol 45 (1) ◽  
pp. 127-132 ◽  
Author(s):  
M Piestrzeniewicz ◽  
K Studzian ◽  
D Wilmańska ◽  
G Płucienniczak ◽  
M Gniazdowski
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
T7 Rna Polymerase ◽  
Distamycin A ◽  
E Coli ◽  
Phage T7 ◽  
Drug Dependent ◽  
Dna Complex ◽  
Kinetics Of

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.

Effect of the Vinca Alkaloids on RNA Synthesis in Human Cells in vitro

Science ◽  
1968 ◽  
Vol 162 (3853) ◽  
pp. 569-570 ◽  
Author(s):  
E. K. Wagner ◽  
B. Roizman
Keyword(s):  
Rna Synthesis ◽  
Human Cells ◽  
Vinca Alkaloids

scholarly journals The essential activities of the bacterial sigma factor

2017 ◽  
Vol 63 (2) ◽  
pp. 89-99 ◽  
Author(s):  
Maria C. Davis ◽  
Christopher A. Kesthely ◽  
Emily A. Franklin ◽  
Shawn R. MacLellan
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Rna Synthesis ◽  
Gene Expression Regulation ◽  
Structure And Function ◽  
Structural Studies ◽  
General Transcription Factors ◽  
Transcription Process ◽  
Promoter Dna ◽  
And Function

Transcription is the first and most heavily regulated step in gene expression. Sigma (σ) factors are general transcription factors that reversibly bind RNA polymerase (RNAP) and mediate transcription of all genes in bacteria. σ Factors play 3 major roles in the RNA synthesis initiation process: they (i) target RNAP holoenzyme to specific promoters, (ii) melt a region of double-stranded promoter DNA and stabilize it as a single-stranded open complex, and (iii) interact with other DNA-binding transcription factors to contribute complexity to gene expression regulation schemes. Recent structural studies have demonstrated that when σ factors bind promoter DNA, they capture 1 or more nucleotides that are flipped out of the helical DNA stack and this stabilizes the promoter open-complex intermediate that is required for the initiation of RNA synthesis. This review describes the structure and function of the σ70 family of σ proteins and the essential roles they play in the transcription process.

scholarly journals G100R Mutation within 4070A Murine Leukemia Virus Env Increases Virus Receptor Binding, Kinetics of Entry, and Viral Transduction Efficiency

2003 ◽  
Vol 77 (1) ◽  
pp. 739-743 ◽  
Author(s):  
Chi-Wei Lu ◽  
Lucille O'Reilly ◽  
Monica J. Roth
Keyword(s):  
Viral Entry ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Cell Types ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Multiple Cell ◽  
Viral Transduction ◽  
Kinetics Of ◽  
Murine Leukemia

ABSTRACT Passage of 4070A murine leukemia virus (MuLV) in D17 cells resulted in a G-to-R change at position 100 within the VRA of the envelope protein (Env). Compared with 4070A MuLV, virus with the G100R Env displayed enhanced binding on target cells, internalized the virus more rapidly, and increased the overall viral titer in multiple cell types. This provides a direct correlation between binding strength and efficiency of viral entry. Deletion of a His residue at the SU N terminus eliminated the transduction efficiency by the G100R virus, suggesting that the G100R virus maintains the regulatory characteristics of 4070A viral entry. The improved transduction efficiency of G100R Env would be an asset for gene delivery systems.

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