Characterization of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification.

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp14/nsp10 exoribonuclease.

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp14 RNA cap methyltransferase.

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.

Structure-based virtual screening and molecular dynamics simulation of SARS-CoV-2 Guanine-N7 methyltransferase (nsp14) for identifying antiviral inhibitors against COVID-19.

The recent pandemic caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) calls the whole world into a medical emergency. For tackling Coronavirus Disease 2019 (COVID-19), researchers from around the world are swiftly working on designing and identifying inhibitors against all possible viral key protein targets. One of the attractive drug targets is guanine-N7 methyltransferase which plays the main role in capping the 5'-ends of viral genomic RNA and sub genomic RNAs, to escape the host's innate immunity. We performed homology modeling and molecular dynamic (MD) simulation, in order to understand the molecular architecture of Guanosine-P3-Adenosine-5',5'-Triphosphate (G3A) binding with C-terminal N7-MTase domain of nsp14 from SARS-CoV-2. The residue Asn388 is highly conserved in present both in N7-MTase from SARS-CoV and SARS-CoV-2 and displays a unique function in G3A binding. For an in-depth understanding of these substrate specificities, we tried to screen and identify inhibitors from the Traditional Chinese Medicine (TCM) database. The combination of several computational approaches, including screening, MM/GBSA, MD simulations, and PCA calculations, provides the screened compounds that readily interact with the G3A binding site of homology modeled N7-MTase domain. Compounds from this screening will have strong potency towards inhibiting the substrate-binding and efficiently hinder the viral 5'-end RNA capping mechanism. We strongly believe the final compounds can become COVID-19 therapeutics, with huge international support.[Formula: see text]The focus of this study is to screen for antiviral inhibitors blocking guanine-N7 methyltransferase (N7-MTase), one of the key drug targets involved in the first methylation step of the SARS-CoV-2 RNA capping mechanism. Compounds binding the substrate-binding site can interfere with enzyme catalysis and impede 5'-end cap formation, which is crucial to mimic host RNA and evade host cellular immune responses. Therefore, our study proposes the top hit compounds from the Traditional Chinese Medicine (TCM) database using a combination of several computational approaches.Communicated by Ramaswamy H. Sarma.

Bufalin induces cell death in human lung cancer cells through disruption of DNA damage response pathways.

Bufalin is a key component of a Chinese medicine (Chan Su) and has been proved effective in killing various cancer cells. Its role in inducing DNA damage and the inhibition of the DNA damage response (DDR) has been reported, but none have studied such action in lung cancer in detail. In this study, we demonstrated bufalin-induced DNA damage and condensation in NCI-H460 cells through a comet assay and DAPI staining, respectively. Western blotting indicated that bufalin suppressed the protein levels associated with DNA damage and repair, such as a DNA dependent serine/threonine protein kinase (DNA-PK), DNA repair proteins breast cancer 1, early onset (BRCA1), 14-3-3 σ (an important checkpoint keeper of DDR), mediator of DNA damage checkpoint 1 (MDC1), O6-methylguanine-DNA methyltransferase (MGMT) and p53 (tumor suppressor protein). Bufalin could activate phosphorylated p53 in NCI-H460 cells. DNA damage in NCI-H460 cells after treatment with bufalin up-regulated its ATM and ATR genes, which encode proteins functioning as sensors in DDR, and also up-regulated the gene expression (mRNA) of BRCA1 and DNA-PK. But bufalin suppressed the gene expression (mRNA) of p53 and 14-3-3 σ, however, bufalin did not significantly affect the mRNA of MGMT. In conclusion, bufalin induced DNA damage in NCI-H460 cells and also inhibited its DNA repair and checkpoint function.

Mutations defective in ribonucleotide reductase activity interfere with pollen plastid DNA degradation mediated by DPD1 exonuclease.

Organellar DNAs in mitochondria and plastids are present in multiple copies and make up a substantial proportion of total cellular DNA despite their limited genetic capacity. We recently demonstrated that organellar DNA degradation occurs during pollen maturation, mediated by the Mg(2+) -dependent organelle exonuclease DPD1. To further understand organellar DNA degradation, we characterized a distinct mutant (dpd2). In contrast to the dpd1 mutant, which retains both plastid and mitochondrial DNAs, dpd2 showed specific accumulation of plastid DNAs. Multiple abnormalities in vegetative and reproductive tissues of dpd2 were also detected. DPD2 encodes the large subunit of ribonucleotide reductase, an enzyme that functions at the rate-limiting step of de novo nucleotide biosynthesis. We demonstrated that the defects in ribonucleotide reductase indirectly compromise the activity of DPD1 nuclease in plastids, thus supporting a different regulation of organellar DNA degradation in pollen. Several lines of evidence provided here reinforce our previous conclusion that the DPD1 exonuclease plays a central role in organellar DNA degradation, functioning in DNA salvage rather than maternal inheritance during pollen development.

Tissue-specific organelle DNA degradation mediated by DPD1 exonuclease.

Organelle DNA in plastids and mitochondria is present in multiple copies and undergoes degradation developmentally. For example, organelle DNA that is detectable cytologically using DNA-fluorescent dye disappears during pollen development. Nevertheless, nucleases involved in this degradation process remain unknown. Our recent study identified the organelle nuclease, DPD1, which has Mg2+ -dependent exonuclease activity in vitro. The discovery of DPD1 emerged from Arabidopsis mutant screening and concomitant isolation of dpd1 mutants that retain organelle DNA in mature pollen. DPD1 is conserved only in angiosperms: not in other photosynthetic organisms. Despite these findings, the physiological significance of organelle DNA degradation during pollen development remains unclear because dpd1 exhibits no apparent defects in pollen viability or in the maternal inheritance of organelle DNA. We discuss a possible role of organelle DNA degradation mediated by DPD1, based on a DPD1 expression profile studied using in silico analyses.

Arabidopsis cell-free extract, ACE, a new in vitro translation system derived from Arabidopsis callus cultures.

The analysis of post-transcriptional regulatory mechanisms in plants has benefited greatly from the use of cell-free extract systems. Arabidopsis as a model system provides extensive genetic resources; however, to date a suitable cell-free translation system from Arabidopsis has not been available. In this study, we devised an Arabidopsis cell-free extract (ACE) to be used for in vitro translation studies. Protoplasts were prepared from callus cultures derived from Arabidopsis seedlings, and cell-free extracts were prepared after evacuolation of the protoplasts by Percoll gradient centrifugation. The new ACE system exhibits translation activity comparable with that of the wheat germ extract system. We demonstrated that ACE prepared from the 5'-3' exoribonuclease-deficient mutant of Arabidopsis, xrn4-5, exhibited increased stability of an uncapped mRNA as compared with that from wild-type Arabidopsis. We applied the ACE system to study post-transcriptional regulation of AtCGS1. AtCGS1 codes for cystathionine γ-synthase (CGS) that catalyzes the first committed step of methionine and S-adenosyl-l-methionine (AdoMet) biosynthesis in plants, and is feedback regulated by mRNA degradation coupled with translation elongation arrest. The ACE system was capable of reproducing translation elongation arrest and subsequent AtCGS1 mRNA degradation that are induced by AdoMet. The ACE system described here can be prepared in a month after seed sowing and will make it possible to study post-transcriptional regulation of plant genes while taking advantage of the genetics of Arabidopsis.

A conserved, Mg²+-dependent exonuclease degrades organelle DNA during Arabidopsis pollen development.

In plant cells, mitochondria and plastids contain their own genomes derived from the ancestral bacteria endosymbiont. Despite their limited genetic capacity, these multicopy organelle genomes account for a substantial fraction of total cellular DNA, raising the question of whether organelle DNA quantity is controlled spatially or temporally. In this study, we genetically dissected the organelle DNA decrease in pollen, a phenomenon that appears to be common in most angiosperm species. By staining mature pollen grains with fluorescent DNA dye, we screened Arabidopsis thaliana for mutants in which extrachromosomal DNAs had accumulated. Such a recessive mutant, termed defective in pollen organelle DNA degradation1 (dpd1), showing elevated levels of DNAs in both plastids and mitochondria, was isolated and characterized. DPD1 encodes a protein belonging to the exonuclease family, whose homologs appear to be found in angiosperms. Indeed, DPD1 has Mg²âº-dependent exonuclease activity when expressed as a fusion protein and when assayed in vitro and is highly active in developing pollen. Consistent with the dpd phenotype, DPD1 is dual-targeted to plastids and mitochondria. Therefore, we provide evidence of active organelle DNA degradation in the angiosperm male gametophyte, primarily independent of maternal inheritance; the biological function of organellar DNA degradation in pollen is currently unclear.

An intact unfolded protein response in Trpt1 knockout mice reveals phylogenic divergence in pathways for RNA ligation.

Unconventional mRNA splicing by an endoplasmic reticulum stress-inducible endoribonuclease, IRE1, is conserved in all known eukaryotes. It controls the expression of a transcription factor, Hac1p/XBP-1, that regulates gene expression in the unfolded protein response. In yeast, the RNA fragments generated by Ire1p are ligated by tRNA ligase (Trl1p) in a process that leaves a 2'-PO4(2-) at the splice junction, which is subsequently removed by an essential 2'-phosphotransferase, Tpt1p. However, animals, unlike yeast, have two RNA ligation/repair pathways that could potentially rejoin the cleaved Xbp-1 mRNA fragments. We report that inactivation of the Trpt1 gene, encoding the only known mammalian homolog of Tpt1p, eliminates all detectable 2'-phosphotransferase activity from cultured mouse cells but has no measurable effect on spliced Xbp-1 translation. Furthermore, the relative translation rates of tyrosine-rich proteins is unaffected by the Trpt1 genotype, suggesting that the pool of (normally spliced) tRNA(Tyr) is fully functional in the Trpt1-/- mouse cells. These observations argue against the presence of a 2'-PO4(2-) at the splice junction of ligated RNA molecules in Trpt1-/- cells, and suggest that Xbp-1 and tRNA ligation proceed by distinct pathways in yeast and mammals.

Functional and phylogenetic analysis of a plant-inducible oligoribonuclease (orn) gene from an indigenous Pseudomonas plasmid.

Application of a promoter-trapping strategy to identify plant-inducible genes carried on an indigenous Pseudomonas plasmid, pQBR103, revealed the presence of a putative oligoribonuclease (orn) gene that encodes a highly conserved 3' to 5' exoribonuclease specific for small oligoribonucleotides. The deduced amino acid sequence of the plasmid-derived orn (orn(pl)) showed three conserved motifs characteristic of Orn from both prokaryotes and eukaryotes. Deletion of orn(pl) generated no observable phenotype, but inactivation of the chromosomal copy caused slow growth in Pseudomonas putida KT2440. This defect was fully restored by complementation with orn from Escherichia coli (orn(E.coli)). Plasmid-derived orn(pl) was capable of partially complementing the P. putida orn mutant, demonstrating functionality of orn(pl). Phylogenetic analysis showed that plasmid-encoded Orn was distinct from Orn encoded by the chromosome of proteobacteria. A survey of orn(pl) from related Pseudomonas plasmids showed a sporadic distribution but no sequence diversity. These data suggest that the orn(pl) was acquired by pQBR103 in a single gene-transfer event: the donor is unknown, but is unlikely to be a member of the Proteobacteria.

The yin and yang of the exosome.

Recent studies of the eukaryotic ribosomal RNA processing pathway have identified a complex of ten riboexonucleases called the exosome that plays a central role in the precise formation of the 3' ends of several types of RNAs. The exosome also destroys excess ribosomal RNA precursors and unused intermediates and degrades poly(A)-mRNAs in the cytoplasm. In the nucleus, the complex appears to function in a regulated mRNA surveillance system that degrades transcripts in response to defects in the mRNA processing and export pathways. How the cell regulates the nucleolytic prowess of the exosome to ensure correct and timely synthesis and destruction of RNAs is a central focus of current research.

Human immunodeficiency virus type-1 reverse transcription can be inhibited in vitro by oligonucleotides that target both natural and synthetic tRNA primers.

Reverse transcription of human immunodeficiency virus type-1 is primed by cellular tRNA(Lys3), which is selectively packaged into viral particles where it is bound at its 3' terminus to a complementary sequence of viral RNA termed the primer binding site (PBS). Since cellular tRNA(Lys3) is highly conserved, it might conceivably serve as a good target for novel antagonists to block reverse transcriptase (RT) activity. In this study, we have examined a number of antisense oligodeoxyribonucleotides (ODNs) that are complementary to different parts of the tRNA primer and, therefore, may interfere with the initiation of RT-mediated DNA synthesis. We found that the stability of complexes between synthetic tRNA(Lys3 )and ODNs was significantly increased when binding occurred via sequences involved in tertiary interactions of the tRNA. In particular, ODNs with complementarity to both the variable and TPsiC stem-loop of tRNA(Lys3 )bound with high affinity to both free tRNA(Lys3 )as well as to the binary tRNA(Lys3)/RNA complex. As a result, the initiation of DNA synthesis was severely compromised under these conditions. Moreover, RT-associated RNase H activity recognized the tRNA within this ternary tRNA(Lys3)/RNA/ODN complex as an RNA template and initiated its degradation. Both this RNase H degradation of tRNA(Lys3 )as well as the altered structure of the tRNA/RNA complex, due to the binding of the ODN, contributed to the inhibition of synthesis of viral DNA. The initiation of RT activity was almost completely blocked when using ODNs that interfered with intermolecular tRNA/RNA interactions that involved both the PBS and sequences outside the PBS. Similar findings were obtained with natural preparation of tRNA(Lys3).

The DNase activity of RNase T and its application to DNA cloning.

RNase T is one of eight distinct 3'-->5' exoribonucleases present in Escherichia coli. The enzyme plays an important role in stable RNA metabolism, including tRNA end turnover and 3' maturation of most stable RNAs because it is the only RNase that can efficiently remove residues near a double-stranded (ds) stem. In the course of study of its specificity and mechanism, we found that RNase T also has single-strand-specific DNase activity. Purified RNase T degrades both single-stranded (ss)RNA and ssDNA in a non-processive manner. However, in contrast to its action on RNA, RNase T binds ssDNA much more tightly and shows less sequence specificity. As with RNA, DNA secondary structure strongly affects its degradation by RNase T. Thus, RNase T action on a dsDNA with a single-stranded 3'-extension efficiently generates blunt-ended DNA. This property of RNase T suggested that it might be a useful enzyme for blunt-ended DNA cloning. We show here that RNase T provides much higher cloning efficiency than the currently used mung bean nuclease.

A ribonuclease specific for inosine-containing RNA: a potential role in antiviral defence?

RNA transcripts in which all guanosine residues are replaced by inosine are degraded at a highly accelerated rate when incubated in extracts from HeLa cells, sheep uterus or pig brain. We report here the partial purification and characterization of a novel ribonuclease, referred to as I-RNase, that is responsible for the degradation of inosine-containing RNA (I-RNA). I-RNase is Mg2+ dependent and specifically degrades single-stranded I-RNA. Comparison of the Km of the enzyme for I-RNA with the Ki for inhibition by normal RNA suggests a approximately 300-fold preferential binding to I-RNA, which can account for the specificity of degradation. The site of cleavage by I-RNase is non-specific; I-RNase acts as a 3'-->5' exonuclease generating 5'-NMPs as products. The presence of alternative unconventional nucleotides in RNA does not result in degradation unless inosine residues are also present. We show that I-RNase is able to degrade RNAs that previously have been modified by the RED-1 double-stranded RNA adenosine deaminase (dsRAD). dsRADs destabilize dsRNA by converting adenosine to inosine, and some of these enzymes are interferon inducible. We therefore speculate that I-RNase in concert with dsRAD may form part of a novel cellular antiviral defence mechanism that acts to degrade dsRNA.