Cap Synthesis Research Articles

Targeting eEF1A reprograms translation and uncovers broad-spectrum antivirals against cap or m6A protein synthesis routes

Plitidepsin is an antitumoral compound safe for treating COVID-19 that targets the translation elongation factor eEF1A. Here we detect that plitidepsin decreases de novo cap-dependent translation of SARS-CoV-2 and non-viral RNAs but affects less than 13% of the host proteome, thus preserving cellular viability. In response to plitidepsin, cells upregulate EIF2AK3 and proteins that reduce translation, but also proteins that support proteostasis via ribosome synthesis and cap-independent translation by eIF4G2 and IGF2BP2. While plitidepsin inhibits cap- or internal ribosome entry sites (IRES)-mediated translation, its impact on N6-methyladenosine (m6A) translation is limited. In agreement, plitidepsin blocks members of Coronaviridae, Flaviviridae, Pneumoviridae and Herpesviridae families. Yet, it fails to inhibit retroviruses that exploit m6A synthesis routes and are blocked by drugs targeting IGF2BP2 m6A reader. By deciphering the molecular fingerprint of cells treated with therapies targeting translation we identify a rational approach to select broad-spectrum antivirals with potential to counteract future pandemic viruses.

Rational Design of Highly Potent SARS-CoV-2 nsp14 Methyltransferase Inhibitors.

The search for new drugs against COVID-19 and its causative agent, SARS-CoV-2, is one of the major trends in the current medicinal chemistry. Targeting capping machinery could be one of the therapeutic concepts based on a unique mechanism of action. Viral RNA cap synthesis involves two methylation steps, the first of which is mediated by the nsp14 protein. Here, we rationally designed and synthesized a series of compounds capable of binding to both the S-adenosyl-l-methionine and the RNA-binding site of SARS-CoV-2 nsp14 N7-methyltransferase. These hybrid molecules showed excellent potency, high selectivity toward various human methyltransferases, nontoxicity, and high cell permeability. Despite the outstanding activity against the enzyme, our compounds showed poor antiviral performance in vitro. This suggests that the activity of this viral methyltransferase has no significant effect on virus transcription and replication at the cellular level. Therefore, our compounds represent unique tools to further explore the role of the SARS-CoV-2 nsp14 methyltransferase in the viral life cycle and the pathogenesis of COVID-19.

Synthesis, in vitro and structural aspects of cap substituted Suberoylanilide hydroxamic acid analogs as potential inducers of apoptosis in Glioblastoma cancer cells via HDAC /microRNA regulation

Glioblastoma multiforme (GBM) is a heterogeneous, aggressive brain cancer characterized by chemo-resistance and cancer stemness. Histone deacetylases (HDACs) are a group of enzymes that regulate chromatin epigenetics which were in turn found to be controlled by microRNAs (miRs). The drug employed in chemotherapy for the treatment of GBM is Temozolomide (TMZ). Unfortunately, many GBM patients exhibit chemo-resistance to this drug. Here we have synthesized various Suberoyl anilide hydroxamic acid (SAHA) analogs with many substitutions at the cap site majority of which not yet studied. These SAHA analogs have exhibited profound cytotoxicity at 2μM, and 4μM concentrations in GBM cancer cell line U87MG, and 1μM, and 2μM concentrations in breast cancer cell line MCF-7. Surprisingly, these analogs have exhibited cytotoxic effects in chronic lymphoid leukemia cells (Raji) at 64μM, and 128μM concentrations due to mutated p53. Among all the synthesized analogs 3-Chloro-SAHA, 3-Chloro-4-fluoro SAHA have exhibited effective cytotoxicity in all cancer cells. These potent analogs inhibited HDAC-8 enzyme activity by 2-folds in U87MG, and MCF-7cell lines and 7-folds decrease in HDAC-8 activity was observed in Raji cell line. These analogs decreased the expression of HDAC-2, HDAC-3 genes and enhanced the expression of p53 tumor suppressor. Interestingly, these compounds decreased the expression of Rictor, the main component of the mTORC2 complex involved cancer cell metabolism. Furthermore, these molecules have decreased oncogenic microRNA expression such as miR-21 and enhanced the expression of tumor suppressor microRNAs such as miR-143. The HDAC binding ability of these molecules was highly significant and have exhibited the ability to cross blood-brain barrier (BBB), and followed the Lipinski rule of five. Thus, these molecules need to be taken up further to clinics for better therapy against GBM either singly or combination therapy.

Nuclear localization of duck Tembusu virus NS5 protein attenuates viral replication in vitro and NS5-NS2B3 interaction

Duck Tembusu virus (TMUV) belongs to the flavivirus genus whose genome replication involved in capping and RNA synthesis dominating by nonstructural protein 5 (NS5). Flaviviral replication has been well documented to occur in the cytoplasm, but the effect of NS5 to gain access to the nucleus remains controversial. Here, TMUV NS5 was observed to localize within the cytoplasm of transfected and infected cells and co-localized with the endoplasmic reticulum. We introduced two arginine mutations into the N390 and Q392 (N390R and Q392R) of the NS5 bipartite nuclear localization sequence (α/βNLS) and designated that mutagenesis as NS5NLSmut, which has shown the ability to access the nucleus and hence attenuates viral replication and production in vitro. Additionally, there was no significant difference between the recovered wild-type TMUV (rTMUV-WT) and engineered mutant (rTMUV-NS5NLSmut) on plaque morphology, survival rate of infected duck embryos or virus copies in tissues. Considering that NS5NLSmut is mainly located in the cytoplasm of rTMUV-NS5NLSmut infected cells at the early stage of infection. We further confirmed that NS5NLSmut attenuated its interaction with nonstructural NS2B-NS3 (NS2B3) following transfection and infection. Meanwhile, the rTMUV-NS5NLSmut tended to stimulate more interferon beta (IFNβ) than rTMUV-WT. However, preliminary study on transient NS5 and NS5NLSmut detected the same levels of IFNβ mRNA mediated by RIG-I detection of NS5 RNA polymerase activity in cell. In summary, these results provide further insights into the relationship between the viral property and subcellular localization of flavivirus NS5 in terms of the NS5-NS2B3 interaction.

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5′ extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

Inhibition of Abl or Src tyrosine kinase decreased porcine circovirus type 2 production in PK15 cells

Porcine circovirus type 2 (PCV2) causes huge economic losses in the global swine industry and has a complex and poorly understood virus-host interaction mechanism. We reported that the C-terminal of the capsid protein of all PCV2 isolates shared a strictly conserved PXXP motif that may interact with SH3 domain-containing tyrosine kinases; however, its roles in PCV2 cell entry and replication remain unknown. In this study, we determined that mRNA levels of two SH3 domain-containing tyrosine kinases family (Abl and Src) had distinct profiles (wild-type and PXXP-mutated) during PCV2 infections of PK15 cells. Therefore, we hypothesized that activities of tyrosine kinases (Abl and Fyn) in PK15 cells may be hijacked by PCV2 via its PXXP motif of the Cap, to favor virus replication. Specific inhibitors PP2 of Lck/Fyn and STI-571 of Abl family kinases decreased viral production through suppression of DNA and Cap synthesis at the replication stage. However, based on indirect immunofluorescence assay (IFA), entry of PCV2 virus-like particles (VLPs) into PK15 cells was not altered. Elucidating mechanisms of PCV2-host interactions should provide new insights for development of new compounds to prevent or reduce PCV2 infections.

Aberrant regulation of the Akt signaling network by human cytomegalovirus allows for targeting of infected monocytes

Primary peripheral blood monocytes are responsible for the hematogenous dissemination of human cytomegalovirus (HCMV) following a primary infection. In order to facilitate viral spread, HCMV extends the naturally short 48-h lifespan of monocytes by stimulating a non-canonical activation of Akt during viral entry, which leads to the increased expression of a specific subset of antiapoptotic proteins. In this study, global analysis of the Akt signaling network showed HCMV induced a more robust activation of the entire network when compared to normal myeloid growth factors. Furthermore, we found a unique interplay between HCMV-activated Akt and the stress response transcription heat shock factor 1 (HSF1) that allowed for the synthesis of both cap- and internal ribosome entry site (IRES)-containing antiapoptotic mRNAs such as myeloid cell leukemia-1 (Mcl-1) and X-linked inhibitor of apoptosis (XIAP), respectively. As generally a switch from cap-dependent to IRES-mediated translation occurs during cellular stress, the ability of HCMV to concurrently drive both types of translation produces a distinct milieu of prosurvival proteins needed for the viability of infected monocytes. Indeed, we found inhibition of XIAP led to death of ∼99% of HCMV-infected monocytes while having minimal effect on the viability of uninfected cells. Taken together, these data indicate that the aberrant activation of the Akt network by HCMV induces the upregulation of a unique subset of antiapoptotic proteins specifically required for the survival of infected monocytes. Consequently, our study highlights the possibility of exploiting these virus-induced changes to prevent viral spread in immunocompromised patients at high-risk for HCMV exposure.

Two-way correspondence between carbon nanotubes and caps: Development of a numerical algorithm and a tool for organic cap synthesis

Abstract A hemispherical or conical carbon aromatic molecule so-called “cap” is considered as a tip of carbon nanotubes (CNTs) that dictates entire CNT structures throughout their production. To design a cap for producing a given CNT, we need to know the correspondence between synthesizable caps and CNTs formed from them, and conversely, the correspondence between target CNTs and possible caps for their formation. We have developed an algorithm and numerical tool that permit the generation of chiral indices from a given cap structure and enable the design of all possible cap structures corresponding to a specified CNT with given chiral indices and also numbers of carbocycles of various sizes, which have been restricted to five and six membered rings so far.

Mutagenesis of Coronavirus nsp14 Reveals Its Potential Role in Modulation of the Innate Immune Response.

Coronavirus (CoV) nonstructural protein 14 (nsp14) is a 60-kDa protein encoded by the replicase gene that is part of the replication-transcription complex. It is a bifunctional enzyme bearing 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single-stranded RNAs and double-stranded RNAs (dsRNAs) and is part of a proofreading system responsible for the high fidelity of CoV replication. nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as "non-self" by the host cell. In this work, a set of point mutants affecting different motifs within the ExoN domain of nsp14 was generated, using transmissible gastroenteritis virus as a model of Alphacoronavirus Mutants lacking ExoN activity were nonviable despite being competent in both viral RNA and protein synthesis. A specific mutation within zinc finger 1 (ZF-C) led to production of a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant recombinant transmissible gastroenteritis virus (TGEV) ZF-C (rTGEV-ZF-C) caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times postinfection. Consequently, the mutant triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of beta interferon (IFN-β), tumor necrosis factor (TNF), and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced compared to the levels seen with the parental virus. Overall, our data revealed a potential role for CoV nsp14 in modulation of the innate immune response. The innate immune response is the first line of antiviral defense that culminates in the synthesis of interferon and proinflammatory cytokines to control viral replication. CoVs have evolved several mechanisms to counteract the innate immune response at different levels, but the role of CoV-encoded ribonucleases in preventing activation of the dsRNA-induced antiviral response has not been described to date. The introduction of a mutation in zinc finger 1 of the ExoN domain of nsp14 led to production of a virus that induced a weak antiviral response, most likely due to the accumulation of lower levels of dsRNA in the late phases of infection. These observations allowed us to propose a novel role for CoV nsp14 ExoN activity in counteracting the antiviral response, which could serve as a novel target for the design of antiviral strategies.

Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses.

RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is N-terminally adjacent to the RdRp and has no apparent homologs elsewhere. Based on its conservation profile, this domain is proposed to have nucleotidylation activity. We used recombinant non-structural protein 9 of the arterivirus equine arteritis virus (EAV) and different biochemical assays, including irreversible labeling with a GTP analog followed by a proteomics analysis, to demonstrate the manganese-dependent covalent binding of guanosine and uridine phosphates to a lysine/histidine residue. Most likely this was the invariant lysine of the newly identified domain, named nidovirus RdRp-associated nucleotidyltransferase (NiRAN), whose substitution with alanine severely diminished the described binding. Furthermore, this mutation crippled EAV and prevented the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in cell culture, indicating that NiRAN is essential for nidoviruses. Potential functions supported by NiRAN may include nucleic acid ligation, mRNA capping and protein-primed RNA synthesis, possibilities that remain to be explored in future studies.

Discovery of m(7)G-cap in eukaryotic mRNAs.

Terminal structure analysis of an insect cytoplasmic polyhedrosis virus (CPV) genome RNA in the early 1970s at the National Institute of Genetics in Japan yielded a 2′-O-methylated nucleotide in the 5′ end of double-stranded RNA genome. This finding prompted me to add S-adenosyl-L-methionine, a natural methylation donor, to the in vitro transcription reaction of viruses that contain RNA polymerase. This effort resulted in unprecedented mRNA synthesis that generates a unique blocked and methylated 5′ terminal structure (referred later to as “cap” or “m7G-cap”) in the transcription of silkworm CPV and human reovirus and vaccinia viruses that contain RNA polymerase in virus particles. Initial studies with viruses paved the way to discover the 5′-cap m7GpppNm structure present generally in cellular mRNAs of eukaryotes. I participated in those studies and was able to explain the pathway of cap synthesis and the significance of the 5′ cap (and capping) in gene expression processes, including transcription and protein synthesis. In this review article I concentrate on the description of these initial studies that eventually led us to a new paradigm of mRNA capping.

RAM function is dependent on Kapβ2-mediated nuclear entry

Eukaryotic gene expression is dependent on the modification of the first transcribed nucleotide of pre-mRNA by the addition of the 7-methylguanosine cap. The cap protects transcripts from exonucleases and recruits complexes which mediate transcription elongation, processing and translation initiation. The cap is synthesized by a series of reactions which link 7-methylguanosine to the first transcribed nucleotide via a 5′ to 5′ triphosphate bridge. In mammals, cap synthesis is catalysed by the sequential action of RNGTT (RNA guanylyltransferase and 5′-phosphatase) and RNMT (RNA guanine-7 methyltransferase), enzymes recruited to RNA pol II (polymerase II) during the early stages of transcription. We recently discovered that the mammalian cap methyltransferase is a heterodimer consisting of RNMT and the RNMT-activating subunit RAM (RNMT-activating mini-protein). RAM activates and stabilizes RNMT and thus is critical for cellular cap methylation and cell viability. In the present study we report that RNMT interacts with the N-terminal 45 amino acids of RAM, a domain necessary and sufficient for maximal RNMT activation. In contrast, smaller components of this RAM domain are sufficient to stabilize RNMT. RAM functions in the nucleus and we report that nuclear import of RAM is dependent on PY nuclear localization signals and Kapβ2 (karyopherin β2) nuclear transport protein.

The D10 Decapping Enzyme of Vaccinia Virus Contributes to Decay of Cellular and Viral mRNAs and to Virulence in Mice

Posttranscriptional mechanisms are important for regulation of cellular and viral gene expression. The presence of the 5' cap structure m(7)G(5')ppp(5')Nm is a general feature of mRNAs that provides protection from exoribonuclease digestion and enhances translation. Vaccinia virus and other poxviruses encode enzymes for both cap synthesis and decapping. Decapping is mediated by two related enzymes, D9 and D10, which are synthesized before and after viral DNA replication, respectively. The timing of D10 synthesis correlates better with the shutdown of host gene expression, and deletion of this gene has been shown to cause persistence of host and viral mRNAs in infected cells. Here, we constructed specific mutant viruses in which translation of D10 was prevented by stop codons or activity of D10 was abrogated by catalytic site mutations, without other genomic alterations. Both mutants formed plaques of normal size and replicated to similar extents as the parental virus in monkey epithelial cells and mouse embryonic fibroblasts. The synthesis of viral proteins was slightly delayed, and cellular and viral mRNAs persisted longer in cells infected with the mutants compared to either the parental virus or clonal revertant. Despite the mild effects in vitro, both mutants were more attenuated than the revertants in intranasal and intraperitoneal mouse models, and less infectious virus was recovered from organs. In addition, there was less lung histopathology following intranasal infection with mutant viruses. These data suggest that the D10 decapping enzyme may help restrict antiviral responses by accelerating host mRNA degradation during poxvirus infection.

Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites

Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II (polymerase II) transcripts, which occurs predominantly during transcription and in mammals is catalysed by RNGTT (RNA guanylyltransferase and 5′ phosphatase) and RNMT (RNA guanine-7 methyltransferase). RNMT has a methyltransferase domain and an N-terminal domain whose function is unclear; it is conserved in mammals, but not required for cap methyltransferase activity. In the present study we report that the N-terminal domain is necessary and sufficient for RNMT recruitment to transcription initiation sites and that recruitment occurs in a DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole)-dependent manner. The RNMT-activating subunit, RAM (RNMT-activating miniprotein), is also recruited to transcription initiation sites via an interaction with RNMT. The RNMT N-terminal domain is required for transcript expression, translation and cell proliferation.

Structure control of silica-supported mono and bimetallic Au–Pt catalysts via mercapto capping synthesis

SiO2-supported monometallic and bimetallic platinum–gold catalysts are prepared by deposition of metal nanoparticles stabilized by mercaptopropyltriethoxysilane (MPTES) after different aging time of the solution containing metal ions and MPTES. The materials are tested in the hydrodesulfurization (HDS) reaction of thiophene and compared with corresponding catalysts prepared by the conventional deposition–precipitation (DP) method. The monometallic Pt and the bimetallic Au–Pt prepared by DP have comparable activity. With respect to the platinum catalyst prepared by DP, the corresponding platinum catalyst prepared by MPTES particle stabilization exhibits a substantial enhancement of the activity regardless the solution aging time. On the contrary, the MPTES-assisted Au–Pt catalysts have different activities, depending on the solution aging time, with the most active being the one obtained with the 5-day-aged solution. In accord with XRD, XPS, and FTIR, the aging time of the solution, through the different interaction of Pt or Au precursors with the mercapto groups, has a crucial effect on the structure and on the surface of the catalysts. The observed differences in the catalytic activity are related to the structural and compositional changes of the bimetallic particles.

Chemical Capping Synthesis of Nickel Oxide Nanoparticles and their Characterizations Studies

This work reports aspect related to chemical capping synthesis of nano-sized particles of nickel oxide. It is a simple, novel and cost effective method. The average particle size, specific surface area, crystallinity index are estimated from XRD analysis. The structural, functional groups and optical characters are analyzed with using of SEM, FTIR and UV- visible techniques. XRD studies confirm the presence of high degree of crystallinity nature of nickel oxide nanoparticles. Their particle size is found to be 12 nm and specific surface area (SSA) is 74m2 g-1. The optical band gap energy value 3.83ev has also been determined from UV-vis spectrum.

Dengue virus nonstructural protein 5 adopts multiple conformations in solution.

Dengue virus (DENV) nonstructural protein 5 (NS5) is composed of two globular domains separated by a 10-residue linker. The N-terminal domain participates in the synthesis of a mRNA cap 1 structure ((7Me)GpppA(2'OMe)) at the 5' end of the viral genome and possesses guanylyltransferase, guanine-N7-methyltransferase, and nucleoside-2'O-methyltransferase activities. The C-terminal domain is an RNA-dependent RNA polymerase responsible for viral RNA synthesis. Although crystal structures of the two isolated domains have been obtained, there are no structural data for full-length NS5. It is also unclear whether the two NS5 domains interact with each other to form a stable structure in which the relative orientation of the two domains is fixed. To investigate the structure and dynamics of DENV type 3 NS5 in solution, we conducted small-angle X-ray scattering experiments with the full-length protein. NS5 was found to be monomeric and well-folded under the conditions tested. The results of these experiments also suggest that NS5 adopts multiple conformations in solution, ranging from compact to more extended forms in which the two domains do not seem to interact with each other. We interpret the multiple conformations of NS5 observed in solution as resulting from weak interactions between the two NS5 domains and flexibility of the linker in the absence of other components of the replication complex.

Stratégies de formation de la structure coiffe chez les virus à ARN

Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential.

ChemInform Abstract: Synthesis and Importance of Bulky Aromatic Cap of Novel SAHA Analogues for HDAC Inhibition and Anticancer Activity.

AbstractCompounds (Ib)—(Id) exhibit a higher inhibitory activity on total HDACs, HDAC1, HDAC2, and HDAC7 and higher anticancer activity against MCF‐7, MDA‐MB‐231, MCF‐7/Dox, MCF‐7/Tam, SK‐OV‐3, LNCaP, and PC3 human cancer cell lines than SAHA.

Synthesis and Importance of Bulky Aromatic Cap of Novel SAHA Analogs for HDAC Inhibition and Anticancer Activity

On the basis of potent HDAC-inhibitory activity and anticancer activity of SAHA, novel SAHA derivatives 3a-d and 7 with a bulky cap such as p-dimethylaminophenyl, 4-phenylaminophenyl, 4-phenyloxyphenyl, 9H-fluorenyl or naphthalenyl ring were synthesized starting from the corresponding aryl amines or naphthalenyl acetic acid using an EDC-mediated amide coupling reaction in the presence of HOBt followed by a nucleophilic addition-elimination reaction with hydroxylamine. Compounds 3b, 3c and 3d showed more potent inhibitory activity on total HDACs (14~27-fold), HDAC1 (8~15-fold), HDAC2 (1.3~25-fold) and HDAC7 (1~3-fold) and more potent anticancer activity (2~22-fold) against MCF-7, MDA-MB-231, MCF-7/Dox, MCF-7/Tam, SK-OV-3, LNCaP and PC3 human cancer cell lines than SAHA.