ABSTRACT
[Figure: see text].
Subject(s)
Antiviral Agents/pharmacology , Cytidine/analogs & derivatives , Hydroxylamines/pharmacology , Mutagenesis , RNA Viruses/drug effects , SARS-CoV-2/drug effects , Animals , Antiviral Agents/adverse effects , Antiviral Agents/therapeutic use , COVID-19/virology , Cytidine/adverse effects , Cytidine/metabolism , Cytidine/pharmacology , Cytidine/therapeutic use , Cytidine/toxicity , DNA/biosynthesis , Evolution, Molecular , Genome, Viral , Humans , Hydroxylamines/adverse effects , Hydroxylamines/metabolism , Hydroxylamines/therapeutic use , Mutagenicity Tests , Phosphorylation , RNA Virus Infections/drug therapy , RNA Virus Infections/virology , RNA Viruses/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , Ribonucleosides/metabolism , SARS-CoV-2/genetics , COVID-19 Drug TreatmentABSTRACT
Higher selenium status has been shown to improve the clinical outcome of infections caused by a range of evolutionally diverse viruses, including SARS-CoV-2. However, the impact of SARS-CoV-2 on host-cell selenoproteins remains elusive. The present study investigated the influence of SARS-CoV-2 on expression of selenoprotein mRNAs in Vero cells. SARS-CoV-2 triggered an inflammatory response as evidenced by increased IL-6 expression. Of the 25 selenoproteins, SARS-CoV-2 significantly suppressed mRNA expression of ferroptosis-associated GPX4, DNA synthesis-related TXNRD3 and endoplasmic reticulum-resident SELENOF, SELENOK, SELENOM and SELENOS. Computational analysis has predicted an antisense interaction between SARS-CoV-2 and TXNRD3 mRNA, which is translated with high efficiency in the lung. Here, we confirmed the predicted SARS-CoV-2/TXNRD3 antisense interaction in vitro using DNA oligonucleotides, providing a plausible mechanism for the observed mRNA knockdown. Inhibition of TXNRD decreases DNA synthesis which is thereby likely to increase the ribonucleotide pool for RNA synthesis and, accordingly, RNA virus production. The present findings provide evidence for a direct inhibitory effect of SARS-CoV-2 replication on the expression of a specific set of selenoprotein mRNAs, which merits further investigation in the light of established evidence for correlations between dietary selenium status and the outcome of SARS-CoV-2 infection.
Subject(s)
DNA/biosynthesis , Endoplasmic Reticulum Stress/physiology , Ferroptosis/physiology , RNA, Messenger/metabolism , SARS-CoV-2/physiology , Selenoproteins/metabolism , Animals , Chlorocebus aethiops , Gene Expression Regulation/physiology , RNA, Messenger/genetics , Selenoproteins/genetics , Vero CellsABSTRACT
Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.
Subject(s)
Benzothiazoles/chemistry , COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Diamines/chemistry , Intercalating Agents/chemistry , Quinolines/chemistry , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , DNA/analysis , DNA/biosynthesis , DNA Primers/chemistry , DNA Primers/metabolism , Humans , Nasopharynx/virology , Real-Time Polymerase Chain Reaction/standards , Sensitivity and SpecificityABSTRACT
Specificity of DNA polymerization plays a critical role in DNA replication and storage of genetic information. Likewise, biotechnological applications, such as nucleic acid detection, DNA amplification, and gene cloning, require high specificity in DNA synthesis catalyzed by DNA polymerases. However, errors in DNA polymerization (such as mis-incorporation and mis-priming) can significantly jeopardize the specificity. Herein, we report our discovery that the specificity of DNA enzymatic synthesis can be substantially enhanced (up to 100-fold higher) by attenuating DNA polymerase kinetics via the phosphorothioate dNTPs. This specificity enhancement allows convenient and sensitive nucleic acid detection, polymerization, PCR, and gene cloning with complex systems (such as human cDNA and genomic DNA). Further, we found that the specificity enhancement offered higher sensitivity (up to 50-fold better) for detecting nucleic acids, such as COVID-19 viral RNAs. Our findings have revealed a simple and convenient strategy for facilitating specificity and sensitivity of nucleic acid detection, amplification, and gene cloning.