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HIV/AIDS
Published in Patricia G. Melloy, Viruses and Society, 2023
Reverse transcriptase was mentioned in Chapter 1 of this book as an enzyme first discovered by cancer biologists studying RNA tumor viruses. These researchers also confirmed the existence of the proviral form of the virus, where its genome is stably integrated into the genome of the host cell (Weinberg 2014; Nobel Media AB 2021). Reverse transcriptase is known for lacking “proofreading” capability, meaning that errors in the transferring of the code from RNA to DNA are not corrected (Walker 2008a; Ojikutu 2008a). These errors become a source of variation in the genetic code of the HIV virus. Overall, the HIV life cycle will become important later because antiretroviral drugs have been developed that block many steps of the pathway, including preventing spike proteins from binding to cells, blocking reverse transcriptase activity, and disabling the protease needed to mature HIV proteins (Lostroh 2019).
The Viruses
Published in Julius P. Kreier, Infection, Resistance, and Immunity, 2022
In 1970 Temin and Baltimore independently demonstrated a virally-encoded enzyme that transcribes RNA into DNA. This enzyme, RNA-dependent DNA polymerase, is a reverse transcriptase. Prior to this, it had generally been accepted that cellular and viral DNA was the template for production of messenger RJMA. The only known exception to this pattern occurred in the nontumorigenic RNA viruses, which produced their new genomic RNA on their old genomic RNA and transcribed their messenger RNA by the virus-specific, RNA-dependent RNA polymerase.
Antiviral Agents and Rational Drug Design
Published in Nathan Keighley, Miraculous Medicines and the Chemistry of Drug Design, 2020
The enzyme reverse transcriptase is a DNA polymerase exclusively associated with the virus, but caution should nevertheless be taken to ensure that there is no inhibitory effect on cellular DNA polymerases when designing nucleoside reverse transcriptase inhibitors. These molecules mimic the nucleoside structure and become phosphorylated to the active drug, as explained in the previous example, acyclovir. The important difference here though is that the phosphorylation steps must all be carried out by cellular enzymes because, unlike Herpesvirus, HIV does not have the necessary kinase enzyme.
A Study on the Effect of Vitamins A and C to Modulate the Expression of NKG2D Ligands in Hepatic and Colon Cancer Cells
Published in Nutrition and Cancer, 2021
Mazin A. Zamzami, Mohammad Nasrullah, Hani Choudhry, Mohammad Imran Khan
High-Capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Lithuania, Catalog no. 4368814) uses the random primer scheme for initiating cDNA synthesis. The reverse transcription process is usually used to synthesize DNA from an RNA template to produce complementary DNA (cDNA). Reverse transcriptase is an RNA-dependent DNA polymerase and is directed the synthesis of the first standard DNA by the RNA template and short primer complementary to the 3-end of the RNA. To synthesize cDNA, the reagents of the kit were combined to form a reverse transcription master mix. RNA samples (300 ng) were added after the necessary normalization step with nuclease-free water. The reactions were prepared and thermocycler reactions were programmed following the manufacturer's protocol. Primers for RT-PCR reaction (Table 1) were designed to measure the expression of NKG2DLs, DNA methyltransferases (DNMTs), and TETs. The primer sequences were obtained using the USCS browser. mRNA sequence (only exons) were taken and by the primer three web tool (http://bioinfo.ut.ee/primer3-0.4.0/), primers were designed after confirming all primer characteristics. Moreover, sequences of primers were confirmed (in silico PCR) by the UCSC browser (https://genome.ucsc.edu/cgi-bin/hgPcr). RPL0 was used as a housekeeping gene to compare the expression of other genes.
False-positive and false-negative COVID-19 cases: respiratory prevention and management strategies, vaccination, and further perspectives
Published in Expert Review of Respiratory Medicine, 2021
Dimitra S. Mouliou, Konstantinos I. Gourgoulianis
PCR inhibitors act on one or more essential stages of the PCR testing procedure, from nucleic acid binding, capture or degradation, DNA polymerase inhibition, or ionic buffer alteration which may increase the Ct value and give rise to false-negative test results. When referring to RT-PCR, reverse transcriptase can be inhibited, too. Schrader et al. [50], Wilson et al. [51] and Sidstedt et al. [52,53] present several substances (including hemoglobin, lactoferrin, melanin, IgG, myoglobin, NaCl, tannic acids, urea, bile salts, bilirubin, cellulose, heparin, free radicals and ethanol) which, when present in high concentrations, have been found to affect test performance. Combs et al. [54] and Kuffel et al. [55] report metal ions that affect PCR performance, and Marino-Merlo et al. [56] show that reverse transcriptase can be inhibited from antiretroviral drugs. The capacity of each PCR test kit to perform in the presence of different inhibitors have been presented in interim guidance documents. Some PCR kits have controls to detect inhibitors, while others cannot detect these substances.
Macrophage targeted nanocarrier delivery systems in HIV therapeutics
Published in Expert Opinion on Drug Delivery, 2020
Tabassum Khan, Mayuresh Mayuresh Patkar, Munira Momin, Abdelwahab Omri
The human immunodeficiency virus (HIV) targets the immune system of the host and modulates the function of immune cells resulting in severe immunodeficiency. The immune status of HIV infected individuals is assessed by the CD4+ T cell count in the blood [1]. In the early stage of infection, the virus affects CD4+ T cells and macrophages. This infection stimulates intracellular signal cascades and assists viral replication [2]. The viral glycoprotein 120 (gp120) binds to CD4 receptor facilitating a conformational change [3,4]. This leads to entry of viral RNA into the host cell cytoplasm. Subsequently, the enzyme reverse transcriptase assists incorporation of viral RNA into the host DNA. This replication and transcription of viral proteins are dependent on integrase binding host factor, and lens epithelium-derived growth factor (LEDGF/p75) [5]. This binding later facilitates the host cell to produce virion particles. The viral proteins are stored in the cell membrane and subsequent assembly and release of the virus leads to lysis of CD4+ memory T cells in the lymph node. Thus, T cell count forms the hallmark of HIV-1 infection. Often, the dramatic decrease in T cell count leads to decrease in the immune capacity of the individual, leading to a stage of immune suppression. This condition is often termed acquired immunodeficiency syndrome (AIDS) and on an average takes about 10–15 years [6,7].