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1.
Sci Rep ; 11(1): 24442, 2021 12 24.
Article in English | MEDLINE | ID: covidwho-1577650

ABSTRACT

Therapeutic interventions targeting viral infections remain a significant challenge for both the medical and scientific communities. While specific antiviral agents have shown success as therapeutics, viral resistance inevitably develops, making many of these approaches ineffective. This inescapable obstacle warrants alternative approaches, such as the targeting of host cellular factors. Respiratory syncytial virus (RSV), the major respiratory pathogen of infants and children worldwide, causes respiratory tract infection ranging from mild upper respiratory tract symptoms to severe life-threatening lower respiratory tract disease. Despite the fact that the molecular biology of the virus, which was originally discovered in 1956, is well described, there is no vaccine or effective antiviral treatment against RSV infection. Here, we demonstrate that targeting host factors, specifically, mTOR signaling, reduces RSV protein production and generation of infectious progeny virus. Further, we show that this approach can be generalizable as inhibition of mTOR kinases reduces coronavirus gene expression, mRNA transcription and protein production. Overall, defining virus replication-dependent host functions may be an effective means to combat viral infections, particularly in the absence of antiviral drugs.


Subject(s)
Coronavirus/metabolism , Respiratory Syncytial Virus, Human/metabolism , TOR Serine-Threonine Kinases/metabolism , Viral Proteins/metabolism , A549 Cells , Coronavirus/drug effects , Coronavirus/genetics , Gene Expression Regulation, Viral/drug effects , Humans , Protein Biosynthesis/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , RNA Interference , RNA, Small Interfering/metabolism , Rapamycin-Insensitive Companion of mTOR Protein/antagonists & inhibitors , Rapamycin-Insensitive Companion of mTOR Protein/genetics , Rapamycin-Insensitive Companion of mTOR Protein/metabolism , Regulatory-Associated Protein of mTOR/antagonists & inhibitors , Regulatory-Associated Protein of mTOR/genetics , Regulatory-Associated Protein of mTOR/metabolism , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Virus Infections/pathology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/drug effects , Respiratory Syncytial Virus, Human/isolation & purification , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/genetics , Viral Proteins/genetics
2.
Antiviral Res ; 197: 105232, 2022 01.
Article in English | MEDLINE | ID: covidwho-1588314

ABSTRACT

We report the in vitro antiviral activity of DZNep (3-Deazaneplanocin A; an inhibitor of S-adenosylmethionine-dependent methyltransferase) against SARS-CoV-2, besides demonstrating its protective efficacy against lethal infection of infectious bronchitis virus (IBV, a member of the Coronaviridae family). DZNep treatment resulted in reduced synthesis of SARS-CoV-2 RNA and proteins without affecting other steps of viral life cycle. We demonstrated that deposition of N6-methyl adenosine (m6A) in SARS-CoV-2 RNA in the infected cells recruits heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), an RNA binding protein which serves as a m6A reader. DZNep inhibited the recruitment of hnRNPA1 at m6A-modified SARS-CoV-2 RNA which eventually suppressed the synthesis of the viral genome. In addition, m6A-marked RNA and hnRNPA1 interaction was also shown to regulate early translation to replication switch of SARS-CoV-2 genome. Furthermore, abrogation of methylation by DZNep also resulted in defective synthesis of the 5' cap of viral RNA, thereby resulting in its failure to interact with eIF4E (a cap-binding protein), eventually leading to a decreased synthesis of viral proteins. Most importantly, DZNep-resistant mutants could not be observed upon long-term sequential passage of SARS-CoV-2 in cell culture. In summary, we report the novel role of methylation in the life cycle of SARS-CoV-2 and propose that targeting the methylome using DZNep could be of significant therapeutic value against SARS-CoV-2 infection.


Subject(s)
Adenosine/analogs & derivatives , Genome, Viral/drug effects , Methyltransferases/antagonists & inhibitors , SARS-CoV-2/drug effects , Adenosine/pharmacology , Animals , Chick Embryo , Chlorocebus aethiops , Chromatin Immunoprecipitation Sequencing , DNA Methylation/drug effects , DNA Methylation/physiology , Drug Resistance, Viral/drug effects , Genome, Viral/genetics , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Humans , Lethal Dose 50 , Mice , Protein Biosynthesis/drug effects , RNA, Viral/drug effects , RNA, Viral/metabolism , Rabbits , SARS-CoV-2/genetics , Specific Pathogen-Free Organisms , Transcription, Genetic/drug effects , Vero Cells
3.
Acc Chem Res ; 55(1): 24-34, 2022 01 04.
Article in English | MEDLINE | ID: covidwho-1569196

ABSTRACT

Over just the last 2 years, mRNA therapeutics and vaccines have undergone a rapid transition from an intriguing concept to real-world impact. However, whereas some aspects of mRNA therapeutics, such as the use of chemical modifications to increase stability and reduce immunogenicity, have been extensively optimized for over two decades, other aspects, particularly the selection and design of the noncoding leader and trailer sequences which control translation efficiency and stability, have received comparably less attention. In practice, such 5' and 3' untranslated regions (UTRs) are often borrowed from highly expressed human genes with few or no modifications, as in the case for the Pfizer/BioNTech Covid vaccine. Focusing on the 5'UTR, we here argue that model-driven design is a promising alternative that provides unprecedented control over 5'UTR function. We review recent work that combines synthetic biology with machine learning to build quantitative models that relate ribosome loading, and thus translation efficiency, to the 5'UTR sequence. We first introduce an experimental approach that uses polysome profiling and high-throughput sequencing to quantify ribosome loading for hundreds of thousands of 5'UTRs in parallel. We apply this approach to measure ribosome loading in synthetic RNA libraries with a random sequence inserted into the 5'UTR. We then review Optimus 5-Prime, a convolutional neural network model trained on the experimental data. We highlight that very accurate models of biological regulation can be learned from synthetic data sets with degenerate 5'UTRs. We validate model predictions not only on held-out data sets from our random library but also on a large library of over 30 000 human 5'UTR fragments and using translation reporter data collected independently by other groups. Both the experiment and model are compatible with commonly used chemically modified nucleosides, in particular, pseudouridine (Ψ) and 1-methyl-pseudouridine (m1Ψ). We find that, in general, 5'UTRs have very similar impacts when combined with different protein-coding sequences and even in the context of different chemical modifications. We demonstrate that Optimus 5-Prime can be combined with design algorithms to generate de novo sequences with precisely defined translation efficiencies. We emphasize recent developments in design algorithms that rely on activation maximization and generative modeling to improve both the fitness and diversity of designed sequences. Compared with prior approaches such as genetic algorithms, we show that these approaches are not only faster but also less likely to get stuck in local sequence optima. Finally, we discuss how the approach reviewed here can be generalized to other gene regions and applications.


Subject(s)
COVID-19 , Protein Biosynthesis , COVID-19 Vaccines , Humans , Machine Learning , RNA, Messenger/genetics , RNA, Messenger/metabolism , SARS-CoV-2
4.
Cell Rep ; 37(2): 109806, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1466094

ABSTRACT

Tactical disruption of protein synthesis is an attractive therapeutic strategy, with the first-in-class eIF4A-targeting compound zotatifin in clinical evaluation for cancer and COVID-19. The full cellular impact and mechanisms of these potent molecules are undefined at a proteomic level. Here, we report mass spectrometry analysis of translational reprogramming by rocaglates, cap-dependent initiation disruptors that include zotatifin. We find effects to be far more complex than simple "translational inhibition" as currently defined. Translatome analysis by TMT-pSILAC (tandem mass tag-pulse stable isotope labeling with amino acids in cell culture mass spectrometry) reveals myriad upregulated proteins that drive hitherto unrecognized cytotoxic mechanisms, including GEF-H1-mediated anti-survival RHOA/JNK activation. Surprisingly, these responses are not replicated by eIF4A silencing, indicating a broader translational adaptation than currently understood. Translation machinery analysis by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) identifies rocaglate-specific dependence on specific translation factors including eEF1ε1 that drive translatome remodeling. Our proteome-level interrogation reveals that the complete cellular response to these historical "translation inhibitors" is mediated by comprehensive translational landscape remodeling.


Subject(s)
Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , Animals , Benzofurans/pharmacology , Cell Line, Tumor , Eukaryotic Initiation Factor-4A/drug effects , Eukaryotic Initiation Factor-4A/metabolism , Humans , Male , Mice , Mice, Inbred NOD , Primary Cell Culture , Protein Biosynthesis/physiology , Proteomics/methods , Ribosomes/metabolism , Transcriptome/drug effects , Transcriptome/genetics , Triterpenes/pharmacology
5.
PLoS Pathog ; 17(10): e1009412, 2021 10.
Article in English | MEDLINE | ID: covidwho-1448584

ABSTRACT

Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.


Subject(s)
COVID-19/metabolism , Host-Parasite Interactions/physiology , SARS-CoV-2/metabolism , Viral Proteins/metabolism , Humans , Protein Biosynthesis/physiology , Proteome/metabolism
6.
Cell Rep ; 37(3): 109841, 2021 10 19.
Article in English | MEDLINE | ID: covidwho-1439922

ABSTRACT

Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus.


Subject(s)
COVID-19/genetics , Gene Expression Regulation, Viral , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Anisotropy , COVID-19/immunology , DNA Mutational Analysis , Female , Gene Expression Profiling , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Kinetics , Mutation , Phenotype , Point Mutation , Protein Biosynthesis , Protein Domains , RNA Stability , Ribosome Subunits, Small, Eukaryotic/metabolism , Ribosomes/metabolism
7.
Nat Commun ; 12(1): 5536, 2021 09 20.
Article in English | MEDLINE | ID: covidwho-1428813

ABSTRACT

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.


Subject(s)
Endoplasmic Reticulum Stress , SARS-CoV-2/physiology , Virus Replication/physiology , Animals , Autophagy/drug effects , Bronchi/pathology , COVID-19/pathology , COVID-19/virology , Cell Differentiation/drug effects , Cell Extracts , Cell Line , Cell Survival/drug effects , Chlorocebus aethiops , Coronavirus 229E, Human/physiology , Down-Regulation/drug effects , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/genetics , Endoplasmic Reticulum-Associated Degradation/drug effects , Epithelial Cells/drug effects , Epithelial Cells/virology , Heat-Shock Proteins/metabolism , Humans , Macrolides/pharmacology , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/physiology , Protein Biosynthesis/drug effects , Proteome/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , SARS-CoV-2/drug effects , Thapsigargin/pharmacology , Unfolded Protein Response/drug effects , Vero Cells , Virus Replication/drug effects
8.
Mol Genet Genomics ; 296(1): 113-118, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-1384446

ABSTRACT

To better understand the interaction between SARS-CoV-2 and human host and find potential ways to block the pandemic, one of the unresolved questions is that how the virus economically utilizes the resources of the hosts. Particularly, the tRNA pool has been adapted to the host genes. If the virus intends to translate its own RNA, then it has to compete with the abundant host mRNAs for the tRNA molecules. Translation initiation is the rate-limiting step during protein synthesis. The tRNAs carrying the initiation Methionine (iMet) recognize the start codon termed initiation ATG (iATG). Other normal Met-carrying tRNAs recognize the internal ATGs. The tAI of virus genes is significantly lower than the tAI of human genes. This disadvantage in translation elongation of viral RNAs must be compensated by more efficient initiation rates. In the human genome, the abundance of iMet-tRNAs to Met-tRNAs is five times higher than the iATG to ATG ratio. However, when SARS-CoV-2 infects human cells, the iMet has an 8.5-time enrichment to iATG. We collected 58 virus species and found that the enrichment of iMet is higher in all viruses compared to human. Our study indicates that the genome sequences of viruses like SARS-CoV-2 have the advantage of competing for the iMet-tRNAs with host mRNAs. The capture of iMet-tRNAs allows the fast translation initiation and the reproduction of virus itself, which compensates the lower tAI of viral genes. This might explain why the virus could rapidly translate its own RNA and reproduce itself from the sea of host mRNAs. Meanwhile, our study reminds the researchers not to ignore the mutations related to ATGs.


Subject(s)
Peptide Chain Initiation, Translational , RNA, Transfer, Met/metabolism , SARS-CoV-2/physiology , COVID-19/virology , Codon , Evolution, Molecular , Genome, Human , Host-Pathogen Interactions , Humans , Mutation , Protein Biosynthesis , SARS-CoV-2/genetics
9.
Angew Chem Int Ed Engl ; 60(24): 13280-13286, 2021 06 07.
Article in English | MEDLINE | ID: covidwho-1384109

ABSTRACT

Eukaryotic mRNAs are emerging modalities for protein replacement therapy and vaccination. Their 5' cap is important for mRNA translation and immune response and can be naturally methylated at different positions by S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases (MTases). We report on the cosubstrate scope of the MTase CAPAM responsible for methylation at the N6 -position of adenosine start nucleotides using synthetic AdoMet analogs. The chemo-enzymatic propargylation enabled production of site-specifically modified reporter-mRNAs. These cap-propargylated mRNAs were efficiently translated and showed ≈3-fold increased immune response in human cells. The same effects were observed when the receptor binding domain (RBD) of SARS-CoV-2-a currently tested epitope for mRNA vaccination-was used. Site-specific chemo-enzymatic modification of eukaryotic mRNA may thus be a suitable strategy to modulate translation and immune response of mRNAs for future therapeutic applications.


Subject(s)
RNA Caps/immunology , RNA, Messenger/immunology , COVID-19/pathology , COVID-19/virology , Chromatography, High Pressure Liquid , Genes, Reporter , HEK293 Cells , Humans , Mass Spectrometry , Methylation , Methyltransferases/metabolism , Protein Biosynthesis , Protein Domains/genetics , Protein Domains/immunology , RNA Caps/analysis , RNA Caps/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/immunology , S-Adenosylmethionine/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
10.
Commun Biol ; 4(1): 715, 2021 06 10.
Article in English | MEDLINE | ID: covidwho-1387495

ABSTRACT

While SARS-CoV-2 is causing modern human history's most serious health crisis and upending our way of life, clinical and basic research on the virus is advancing rapidly, leading to fascinating discoveries. Two studies have revealed how the viral virulence factor, nonstructural protein 1 (Nsp1), binds human ribosomes to inhibit host cell translation. Here, we examine the main conclusions on the molecular activity of Nsp1 and its role in suppressing innate immune responses. We discuss different scenarios potentially explaining how the viral RNA can bypass its own translation blockage and speculate on the suitability of Nsp1 as a therapeutic target.


Subject(s)
Host-Pathogen Interactions/physiology , Ribosomes/virology , SARS-CoV-2/pathogenicity , Viral Nonstructural Proteins/metabolism , 5' Untranslated Regions , Gene Expression Regulation, Viral , Humans , Immunity, Innate , Protein Biosynthesis , RNA, Messenger/metabolism , Ribosomes/metabolism , SARS-CoV-2/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
11.
Biochemistry ; 60(24): 1869-1875, 2021 06 22.
Article in English | MEDLINE | ID: covidwho-1387102

ABSTRACT

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro, and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Coronavirus RNA-Dependent RNA Polymerase/genetics , Ebolavirus/drug effects , SARS-CoV-2/drug effects , Virus Replication/drug effects , Adenosine Monophosphate/genetics , Adenosine Monophosphate/metabolism , Alanine/genetics , Alanine/metabolism , Antiviral Agents/metabolism , Base Pairing , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/metabolism , Models, Molecular , Protein Biosynthesis/drug effects , RNA/genetics , RNA/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism
12.
Nat Commun ; 12(1): 5120, 2021 08 25.
Article in English | MEDLINE | ID: covidwho-1373414

ABSTRACT

COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which infected >200 million people resulting in >4 million deaths. However, temporal landscape of the SARS-CoV-2 translatome and its impact on the human genome remain unexplored. Here, we report a high-resolution atlas of the translatome and transcriptome of SARS-CoV-2 for various time points after infecting human cells. Intriguingly, substantial amount of SARS-CoV-2 translation initiates at a novel translation initiation site (TIS) located in the leader sequence, termed TIS-L. Since TIS-L is included in all the genomic and subgenomic RNAs, the SARS-CoV-2 translatome may be regulated by a sophisticated interplay between TIS-L and downstream TISs. TIS-L functions as a strong translation enhancer for ORF S, and as translation suppressors for most of the other ORFs. Our global temporal atlas provides compelling insight into unique regulation of the SARS-CoV-2 translatome and helps comprehensively evaluate its impact on the human genome.


Subject(s)
COVID-19/virology , Protein Biosynthesis , SARS-CoV-2/genetics , Transcriptome , Gene Expression Regulation, Viral , Genome, Human , Humans , Open Reading Frames , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism
13.
Viruses ; 13(6)2021 05 21.
Article in English | MEDLINE | ID: covidwho-1359299

ABSTRACT

Viral RNAs contain the information needed to synthesize their own proteins, to replicate, and to spread to susceptible cells. However, due to their reduced coding capacity RNA viruses rely on host cells to complete their multiplication cycle. This is largely achieved by the concerted action of regulatory structural elements on viral RNAs and a subset of host proteins, whose dedicated function across all stages of the infection steps is critical to complete the viral cycle. Importantly, not only the RNA sequence but also the RNA architecture imposed by the presence of specific structural domains mediates the interaction with host RNA-binding proteins (RBPs), ultimately affecting virus multiplication and spreading. In marked difference with other biological systems, the genome of positive strand RNA viruses is also the mRNA. Here we focus on distinct types of positive strand RNA viruses that differ in the regulatory elements used to promote translation of the viral RNA, as well as in the mechanisms used to evade the series of events connected to antiviral response, including translation shutoff induced in infected cells, assembly of stress granules, and trafficking stress.


Subject(s)
Host-Pathogen Interactions , RNA Viruses/physiology , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Response Elements , Biological Transport , Cytoplasmic Granules/metabolism , Gene Expression Regulation, Viral , Humans , Protein Biosynthesis , RNA Virus Infections/metabolism , RNA Virus Infections/virology , RNA, Viral/chemistry , Stress, Physiological , Transport Vesicles/metabolism , Virus Replication
14.
Trends Biochem Sci ; 46(4): 270-283, 2021 04.
Article in English | MEDLINE | ID: covidwho-958915

ABSTRACT

RNA G-quadruplexes (RG4s) are four-stranded structures known to control gene expression mechanisms, from transcription to protein synthesis, and DNA-related processes. Their potential impact on RNA biology allows these structures to shape cellular processes relevant to disease development, making their targeting for therapeutic purposes an attractive option. We review here the current knowledge on RG4s, focusing on the latest breakthroughs supporting the notion of transient structures that fluctuate dynamically in cellulo, their interplay with RNA modifications, their role in cell compartmentalization, and their deregulation impacting the host immune response. We emphasize RG4-binding proteins as determinants of their transient conformation and effectors of their biological functions.


Subject(s)
G-Quadruplexes , Biology , DNA , Protein Biosynthesis , RNA/metabolism
15.
Adv Exp Med Biol ; 1332: 167-187, 2021.
Article in English | MEDLINE | ID: covidwho-1305141

ABSTRACT

As a functional amino acid (AA), L-arginine (Arg) serves not only as a building block of protein but also as an essential substrate for the synthesis of nitric oxide (NO), creatine, polyamines, homoarginine, and agmatine in mammals (including humans). NO (a major vasodilator) increases blood flow to tissues. Arg and its metabolites play important roles in metabolism and physiology. Arg is required to maintain the urea cycle in the active state to detoxify ammonia. This AA also activates cellular mechanistic target of rapamycin (MTOR) and focal adhesion kinase cell signaling pathways in mammals, thereby stimulating protein synthesis, inhibiting autophagy and proteolysis, enhancing cell migration and wound healing, promoting spermatogenesis and sperm quality, improving conceptus survival and growth, and augmenting the production of milk proteins. Although Arg is formed de novo from glutamine/glutamate and proline in humans, these synthetic pathways do not provide sufficient Arg in infants or adults. Thus, humans and other animals do have dietary needs of Arg for optimal growth, development, lactation, and fertility. Much evidence shows that oral administration of Arg within the physiological range can confer health benefits to both men and women by increasing NO synthesis and thus blood flow in tissues (e.g., skeletal muscle and the corpora cavernosa of the penis). NO is a vasodilator, a neurotransmitter, a regulator of nutrient metabolism, and a killer of bacteria, fungi, parasites, and viruses [including coronaviruses, such as SARS-CoV and SARS-CoV-2 (the virus causing COVID-19). Thus, Arg supplementation can enhance immunity, anti-infectious, and anti-oxidative responses, fertility, wound healing, ammonia detoxification, nutrient digestion and absorption, lean tissue mass, and brown adipose tissue development; ameliorate metabolic syndromes (including dyslipidemia, obesity, diabetes, and hypertension); and treat individuals with erectile dysfunction, sickle cell disease, muscular dystrophy, and pre-eclampsia.


Subject(s)
COVID-19 , Nitric Oxide , Animals , Arginine/metabolism , Female , Humans , Male , Pregnancy , Protein Biosynthesis , SARS-CoV-2
16.
Nutrients ; 13(7)2021 Jul 06.
Article in English | MEDLINE | ID: covidwho-1295894

ABSTRACT

Angiotensin converting enzyme 2 (ACE2) is a key entry point of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus known to induce Coronavirus disease 2019 (COVID-19). We have recently outlined a concept to reduce ACE2 expression by the administration of glycyrrhizin, a component of Glycyrrhiza glabra extract, via its inhibitory activity on 11beta hydroxysteroid dehydrogenase type 2 (11betaHSD2) and resulting activation of mineralocorticoid receptor (MR). We hypothesized that in organs such as the ileum, which co-express 11betaHSD2, MR and ACE2, the expression of ACE2 would be suppressed. We studied organ tissues from an experiment originally designed to address the effects of Glycyrrhiza glabra extract on stress response. Male Sprague Dawley rats were left undisturbed or exposed to chronic mild stress for five weeks. For the last two weeks, animals continued with a placebo diet or received a diet containing extract of Glycyrrhiza glabra root at a dose of 150 mg/kg of body weight/day. Quantitative PCR measurements showed a significant decrease in gene expression of ACE2 in the small intestine of rats fed with diet containing Glycyrrhiza glabra extract. This effect was independent of the stress condition and failed to be observed in non-target tissues, namely the heart and the brain cortex. In the small intestine we also confirmed the reduction of ACE2 at the protein level. Present findings provide evidence to support the hypothesis that Glycyrrhiza glabra extract may reduce an entry point of SARS-CoV-2. Whether this phenomenon, when confirmed in additional studies, is linked to the susceptibility of cells to the virus requires further studies.


Subject(s)
Angiotensin-Converting Enzyme 2/antagonists & inhibitors , COVID-19/drug therapy , Dietary Supplements , Glycyrrhiza , Plant Extracts/therapeutic use , Protein Biosynthesis/drug effects , Angiotensin-Converting Enzyme 2/metabolism , Animals , Glycyrrhizic Acid/administration & dosage , Glycyrrhizic Acid/therapeutic use , Male , Plant Extracts/administration & dosage , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley
17.
Viruses ; 13(7)2021 06 27.
Article in English | MEDLINE | ID: covidwho-1289026

ABSTRACT

Many viruses, especially RNA viruses, utilize programmed ribosomal frameshifting and/or stop codon readthrough in their expression, and in the decoding of a few a UGA is dynamically redefined to specify selenocysteine. This recoding can effectively increase viral coding capacity and generate a set ratio of products with the same N-terminal domain(s) but different C-terminal domains. Recoding can also be regulatory or generate a product with the non-universal 21st directly encoded amino acid. Selection for translation speed in the expression of many viruses at the expense of fidelity creates host immune defensive opportunities. In contrast to host opportunism, certain viruses, including some persistent viruses, utilize recoding or adventitious frameshifting as part of their strategy to evade an immune response or specific drugs. Several instances of recoding in small intensively studied viruses escaped detection for many years and their identification resolved dilemmas. The fundamental importance of ribosome ratcheting is consistent with the initial strong view of invariant triplet decoding which however did not foresee the possibility of transitory anticodon:codon dissociation. Deep level dynamics and structural understanding of recoding is underway, and a high level structure relevant to the frameshifting required for expression of the SARS CoV-2 genome has just been determined.


Subject(s)
DNA Viruses/genetics , DNA Viruses/immunology , Histocompatibility Antigens Class I/immunology , Immune Evasion , RNA Viruses/genetics , Antiviral Agents/pharmacology , Codon, Terminator , DNA Viruses/drug effects , Frameshifting, Ribosomal , Histocompatibility Antigens Class I/genetics , Nucleic Acid Conformation , Peptides/immunology , Protein Biosynthesis , RNA Viruses/drug effects , RNA Viruses/immunology
18.
Microbiol Spectr ; 9(1): e0016921, 2021 09 03.
Article in English | MEDLINE | ID: covidwho-1270881

ABSTRACT

Nonstructural protein 1 (Nsp1) of severe acute respiratory syndrome coronaviruses (SARS-CoVs) is an important pathogenic factor that inhibits host protein translation by means of its C terminus. However, its N-terminal function remains elusive. Here, we determined the crystal structure of the N terminus (amino acids [aa] 11 to 125) of SARS-CoV-2 Nsp1 at a 1.25-Å resolution. Further functional assays showed that the N terminus of SARS-CoVs Nsp1 alone loses the ability to colocalize with ribosomes and inhibit protein translation. The C terminus of Nsp1 can colocalize with ribosomes, but its protein translation inhibition ability is significantly weakened. Interestingly, fusing the C terminus of Nsp1 with enhanced green fluorescent protein (EGFP) or other proteins in place of its N terminus restored the protein translation inhibitory ability to a level equivalent to that of full-length Nsp1. Thus, our results suggest that the N terminus of Nsp1 is able to stabilize the binding of the Nsp1 C terminus to ribosomes and act as a nonspecific barrier to block the mRNA channel, thus abrogating host mRNA translation.


Subject(s)
SARS-CoV-2/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , COVID-19 , Crystallography, X-Ray , HEK293 Cells , Humans , Protein Biosynthesis , Protein Conformation , Protein Domains , RNA, Messenger , Sequence Analysis, Protein , Viral Nonstructural Proteins/metabolism
19.
RNA ; 27(9): 1025-1045, 2021 09.
Article in English | MEDLINE | ID: covidwho-1269913

ABSTRACT

Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a nonstructural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation levels. We discover that a functionally coherent subset of human genes is preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5' terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5' UTRs from TOP transcripts can drive preferential expression in the presence of Nsp1. Finally, we found that LARP1, a key effector protein in the mTOR pathway, may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine-tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.


Subject(s)
Host-Pathogen Interactions/genetics , Protein Biosynthesis , Proteins/chemistry , Proteins/genetics , Viral Nonstructural Proteins/genetics , 5' Untranslated Regions , Autoantigens/genetics , Autoantigens/metabolism , Gene Expression Regulation , HEK293 Cells , Humans , Protein Folding , Pyrimidines , RNA, Messenger/genetics , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Ribosomes/genetics , Ribosomes/virology , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Viral Nonstructural Proteins/metabolism
20.
Biochemistry ; 60(24): 1869-1875, 2021 06 22.
Article in English | MEDLINE | ID: covidwho-1263454

ABSTRACT

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro, and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Coronavirus RNA-Dependent RNA Polymerase/genetics , Ebolavirus/drug effects , SARS-CoV-2/drug effects , Virus Replication/drug effects , Adenosine Monophosphate/genetics , Adenosine Monophosphate/metabolism , Alanine/genetics , Alanine/metabolism , Antiviral Agents/metabolism , Base Pairing , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/metabolism , Models, Molecular , Protein Biosynthesis/drug effects , RNA/genetics , RNA/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism
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