MTase Domain Research Articles

The complete pathway for co-transcriptional mRNA maturation within a large protein of a non-segmented negative-strand RNA virus.

Non-segmented negative-strand (NNS) RNA viruses, such as rabies, Nipahand Ebola, produce 5'-capped and 3'-polyadenylated mRNAs resembling higher eukaryotic mRNAs. Here, we developed a transcription elongation-coupled pre-mRNA capping system for vesicular stomatitis virus (VSV, a prototypic NNS RNA virus). Using this system, we demonstrate that the single-polypeptide RNA-dependent RNA polymerase (RdRp) large protein (L) catalyzes all pre-mRNA modifications co-transcriptionally in the following order: (i) 5'-capping (polyribonucleotidylation of GDP) to form a GpppA cap core structure, (ii) 2'-O-methylation of GpppA into GpppAm, (iii) guanine-N7-methylation of GpppAm into m7GpppAm (cap 1), (iv) 3'-polyadenylation to yield a poly(A) tail. The GDP polyribonucleotidyltransferase (PRNTase) domain of L generated capped pre-mRNAs of 18 nucleotides or longer via the formation of covalent enzyme-pre-mRNA intermediates. The single methyltransferase domain of L sequentially methylated the cap structure only when pre-mRNAs of 40 nucleotides or longer were associated with elongation complexes. These results suggest that the formation of pre-mRNA closed loop structures in elongation complexes via the RdRp and PRNTase domains followed by the RdRp and MTase domains on the same polypeptide is required for the cap 1 formation during transcription. Taken together, our findings indicate that NNS RNA virus L acts as an all-in-one viral mRNA assembly machinery.

Structural basis of the monkeypox virus mRNA cap N7 methyltransferase complex

ABSTRACT The global outbreak of Mpox, caused by the monkeypox virus (MPXV), has attracted international attention and become another major infectious disease event after COVID-19. The mRNA cap N7 methyltransferase (RNMT) of MPXV methylates the N7 position of the added guanosine to the 5'-cap structure of mRNAs and plays a vital role in evading host antiviral immunity. MPXV RNMT is composed of the large subunit E1 and the small subunit E12. How E1 and E12 of MPXV assembly remains unclear. Here, we report the crystal structures of E12, the MTase domain of E1 with E12 (E1CTD-E12) complex, and the E1CTD-E12-SAM ternary complex, revealing the detailed conformations of critical residues and the structural changes upon E12 binding to E1. Functional studies suggest that E1CTD N-terminal extension (Asp545-Arg562) and the small subunit E12 play an essential role in the binding process of SAM. Structural comparison of the AlphaFold2-predicted E1, E1CTD-E12 complex, and the homologous D1-D12 complex of vaccinia virus (VACV) indicates an allosteric activating effect of E1 in MPXV. Our findings provide the structural basis for the MTase activity stimulation of the E1-E12 complex and suggest a potential interface for screening the anti-poxvirus inhibitors.

Acquiring insights through a sequence-based approach to the critical Zika virus MTase domain

Background: ZIKV is one of the re-emerging arboviruses (viruses carried by arthropods), which is spread through the Aedes mosquito. It is an RNA virus with only one strand that is appropriate to the family Flaviviridae's Flavivirus (genus) & has been linked to other Flaviviruses such as the West Nile virus, chikungunya virus, & dengue (DENV) virus. The envelope, precursor membrane, and capsid are three structural proteins, and seven nonstructural proteins are also encoded by the Zika virus genome.Methods: We conducted an in-silico analysis of the Zika virus' MTase domain protein for this publication. We predicted that methylation would play a significant role in the available Prosite, Pfam, and InterProScan tools to aid in locating the MTase domain. Along with alignment, amino acid composition, charged amino acids, atomic level studies, & molecular weight, we also make predictions for these variables, including theoretical Pi.Results: We also examine the MTase domain's simulated structure (alpha helix, beta sheet, turn) and its specifics, including secondary structure. We also pinpoint the locations where proteins, DNA, and RNA bind. Potential phosphorylation sites can be found on the Ser, Thr, and Tyr residues in the MTase domain.Conclusion: These outcomes imply a complicated interaction between different phosphorylation modifications that modulates the activity of the MTase domain. To fully appreciate the auxiliary and practical perspectives and to clarify the varied roles of PTM in the MTase domain will be a primary goal of future study.Keywords: I-TASSER; Secondary structure; Prosite; α-helix; Pfam; InterProScan; Binding sites; Posttranslational modification; SOPMA; Phyre2

Identification of Novel Non-Nucleoside Inhibitors of Zika Virus NS5 Protein Targeting MTase Activity.

Zika virus (ZIKV) is a positive-sense single-stranded virus member of the Flaviviridae family. Among other arboviruses, ZIKV can cause neurological disorders such as Guillain Barré syndrome, and it can have congenital neurological manifestations and affect fertility. ZIKV nonstructural protein 5 (NS5) is essential for viral replication and limiting host immune detection. Herein, we performed virtual screening to identify novel small-molecule inhibitors of the ZIKV NS5 methyltransferase (MTase) domain. Compounds were tested against the MTases of both ZIKV and DENV, demonstrating good inhibitory activities against ZIKV MTase. Extensive molecular dynamic studies conducted on the series led us to identify other derivatives with improved activity against the MTase and limiting ZIKV infection with an increased selectivity index. Preliminary pharmacokinetic parameters have been determined, revealing excellent stability over time. Preliminary in vivo toxicity studies demonstrated that the hit compound 17 is well tolerated after acute administration. Our results provide the basis for further optimization studies on novel non-nucleoside MTase inhibitors.

A conserved methyltransferase active site residue of Zika virus NS5 is required for the restriction of STING activation and interferon expression.

Zika virus (ZIKV) is a re-emerging RNA virus and causes major public health events due to its link to severe neurological complications in foetuses and neonates. The cGAS-STING signalling pathway regulates innate immunity and plays an important role in the invasion of DNA and RNA viruses. This study reveals a distinct mechanism by which ZIKV restricts the cGAS-STING signalling to repress IFN-β expression. ZIKV attenuates IFN-β expression induced by DNA viruses (herpes simplex virus type 1, HSV-1) or two double-stranded DNAs (dsDNA90 and HSV120) in mouse embryonic fibroblasts (MEFs). Notably, ZIKV NS5, the viral RNA-dependent RNA polymerase, was responsible for the repression of IFN-β. NS5 interacts with STING in the cytoplasm, suppresses IRF3 phosphorylation and nucleus localization and promotes the cleavage of STING K48-linked polyubiquitination. Furthermore, the NS5 methyltransferase (MTase) domain interacts with STING to restrict STING-induced IFN-β expression. Interestingly, point mutation analyses of conserved methyltransferase active site residue D146 indicate that it is critical for repressing IFN-β expression induced by STING stimulation in cGAS-STING signalling.

In Silico Evaluation of 1‐Aminoisoquinoline Derivatives against Dengue Virus: Greener Access via a Sonochemical Method

AbstractThe reported antiviral properties of isoquinolines including that of berberine against dengue virus prompted us exploring a series of 1‐aminoisoquinoline derivatives against RdRp and MTase domain of NS5 protein of dengue virus (DENV) in silico. While MTase is known to assists in viral RNA capping, the RdRp plays a key role in the virus replication. The docking of 1‐aminoisoquinolines into the DENV NS5 protein RdRp domain (PDB: 5I3Q) in silico revealed good interactions of one molecule involving the H‐bond interactions through its NO2 group with GLN802, LEU511 and HIS512 residues. Notably, the docking of these compounds into the DENV NS5 protein DENV3 MTase (PDB: 5EHG) in silico also indicated same molecule as the most encouraging potential entity as the molecule participated in three H‐bond interactions through its NO2 group with GLY86, TRP87, SER56 residues. The synthesis of 1‐aminoisoquinolines was undertaken under sonochemical conditions via the reaction of 1‐chloroisoquinoline with appropriate amines in the presence of Cs2CO3 in DMSO. Among them, three encouraging compounds did not cause any significant cytotoxicity towards Vero cells but showed good to moderate inhibition of DENV2.

A conformational selection mechanism of flavivirus NS5 for species-specific STAT2 inhibition

Flaviviruses, including Zika virus (ZIKV) and Dengue virus (DENV), rely on their non-structural protein 5 (NS5) for both replication of viral genome and suppression of host IFN signaling. DENV and ZIKV NS5s were shown to facilitate proteosome-mediated protein degradation of human STAT2 (hSTAT2). However, how flavivirus NS5s have evolved for species-specific IFN-suppression remains unclear. Here we report structure-function characterization of the DENV serotype 2 (DENV2) NS5−hSTAT2 complex. The MTase and RdRP domains of DENV2 NS5 form an extended conformation to interact with the coiled-coil and N-terminal domains of hSTAT2, thereby promoting hSTAT2 degradation in cells. Disruption of the extended conformation of DENV2/ZIKV NS5, but not the alternative compact state, impaired their hSTAT2 binding. Our comparative structural analysis of flavivirus NS5s further reveals a conserved protein-interaction platform with subtle amino-acid variations likely underpinning diverse IFN-suppression mechanisms. Together, this study uncovers a conformational selection mechanism underlying species-specific hSTAT2 inhibition by flavivirus NS5.

Exploring potential therapeutics: Targeting dengue virus NS5 through molecular docking, ADMET profiling, and DFT analysis

Dengue fever is defined as a mosquito-borne Flaviviridae viral infection. It has become a huge public health issue worldwide. Dengue research has grown in recent years. The structure of the viral genome has been decoded, with the nonstructural protein NS5 acting as a possible target. The NS5 compound has an MTase domain contributing in RNA capping, adding an RdRp domain contributing in viral replication process. On the light of considering the NS5 as a possible medicinal target, this research included molecular docking studies executed using the Schrödinger suite software to examine 1412 compounds that were 80% similar to s-adenosyl-l-homocysteine,Top five compounds were chosen based on their sTable binding energies comparing to the reference. We next used the pkcsm web server to examine the ADMET characteristics of the top five ligands. Four compounds were identified as promising candidates for future therapeutic development. Finally, further analyzed using density functional theory approach.

Docking and MM study of non-structural protein (NS5) of Japanese Encephalitis Virus (JEV) with some derivatives of adenosyl

Introduction: The flavivirus NS5, a non-structural protein of Japanese Encephalitis Virus (JEV), a serious deadly human pathogen responsible for epidemics in South East Asia, consists of N-terminal methyl transferase (MTase) domain and RNA-dependent RNA polymerase (RdRp) is known for unique viral genome replication and cap formation activity. S-adenosyl executes a crucial function in these viral activities. S-adenosyl derivatives are chosen as potential binders with the MTase domain of NS5 based on MM and docking studies.Methods: MM GBSA (Generalized Born Surface Area) simulation were performed to evaluate the binding energy, following the 100 nanosecond (ns) production MD simulation in the periodic boundary condition (PBC) for the selected docked ligands with NS5. Quasi-harmonic entropy of the ligands was also calculated with semi-empirical calculations at the PM3/PM6 level supporting docking and MM-GBSA results.Results and discussion: The residue-wise decomposition energy reveals that the key hydrophobic residues Gly 81, Phe 133, and Ile 147 in the RdRp-MTase interface, indicate the biological relevance. These residues act as the key residue stabilizer, binding vigorously with S-Adenosyl derivatives in the vicinity of the interface between the MTase domain and RdRp. This paves the way for the other potential drug as an inhibitor for the enzymatic activity of the NS5.

Structural basis for the allosteric regulation and dynamic assembly of DNMT3B.

Oligomerization of DNMT3B, a mammalian de novo DNA methyltransferase, critically regulates its chromatin targeting and DNA methylation activities. However, how the N-terminal PWWP and ADD domains interplay with the C-terminal methyltransferase (MTase) domain in regulating the dynamic assembly of DNMT3B remains unclear. Here, we report the cryo-EM structure of DNMT3B under various oligomerization states. The ADD domain of DNMT3B interacts with the MTase domain to form an autoinhibitory conformation, resembling the previously observed DNMT3A autoinhibition. Our combined structural and biochemical study further identifies a role for the PWWP domain and its associated ICF mutation in the allosteric regulation of DNMT3B tetramer, and a differential functional impact on DNMT3B by potential ADD-H3K4me0 and PWWP-H3K36me3 bindings. In addition, our comparative structural analysis reveals a coupling between DNMT3B oligomerization and folding of its substrate-binding sites. Together, this study provides mechanistic insights into the allosteric regulation and dynamic assembly of DNMT3B.

DNA conformational dynamics in the context-dependent non-CG CHH methylation by plant methyltransferase DRM2

DNA methylation provides an important epigenetic mechanism that critically regulates gene expression, genome imprinting, and retrotransposon silencing. In plants, DNA methylation is prevalent not only in a CG dinucleotide context, but also in non-CG contexts, namely CHG and CHH (H = C, T, or A) methylation. It has been established that plant non-CG DNA methylation is highly context dependent, with the +1- and +2-flanking sequences enriched with A/T nucleotides. How DNA sequence, conformation, and dynamics influence non-CG methylation remains elusive. Here, we report structural and biochemical characterizations of the intrinsic substrate preference of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), a plant DNA methyltransferase responsible for establishing all cytosine methylation and maintaining CHH methylation. Among nine CHH motifs, the DRM2 methyltransferase (MTase) domain shows marked substrate preference toward CWW (W = A or T) motifs, correlating well with their relative abundance in planta. Furthermore, we report the crystal structure of DRM2 MTase in complex with a DNA duplex containing a flexible TpA base step at the +1/+2-flanking sites of the target nucleotide. Comparative structural analysis of the DRM2-DNA complexes provides a mechanism by which flanking nucleotide composition impacts DRM2-mediated DNA methylation. Furthermore, the flexibility of the TpA step gives rise to two alternative DNA conformations, resulting in different interactions with DRM2 and consequently temperature-dependent shift of the substrate preference of DRM2. Together, this study provides insights into how the interplay between the conformational dynamics of DNA and temperature as an environmental factor contributes to the context-dependent CHH methylation by DRM2.

Differential Impact of Domain Stratified Non-R882 DNMT3A Mutations in Myeloid Neoplasms

Introduction: DNMT3A mutations are present in approximately 20% of acute myeloid leukemia (AML) patients and other myeloid neoplasms (MN) and are considered the earliest clonal event in leukemogenesis. In AML, 50-60% of DNMT3A mutations are heterozygous missense mutations affecting codon R882, which were shown to be dominant-negative in multiple studies. In contrast, there is still a complete lack of data related to the biological impact of a substantial number of widely dispersed non-R882 mutations and their clinical implications. It is possible that mutations in different DNMT3A domains are unique, leading to specific molecular characteristics and clinical phenotypes. Domain-based characterization and phenotypic effects of non-R882 mutations in more granularity is needed to understand the significance of DNMT3A mutations in MN. Methods: We conducted a large retrospective analysis of 16,565 MN patients by screening publicly available databases of AACR GENIE (v 13.1, n=4167), cBioPortal (Cerami et al., 2012, n=3669) along with Karmanos Cancer Institute (n=800) and a previously published metanalytic cohort (Awada et al., Blood 2021 and Kewan et al., Nature Communications, 2023). Clinical characteristics and genomic data were extracted for DNMT3A MT patients to study associated mutational signatures with respect to various biological characteristics. Chi square test was used for analysis of various parameters and KM curves were used to estimate overall survival (OS). Results: DNMT3A MT was found in 17.5% (2903/16,565) of patients in the whole cohort. Of these, 1635 patients had one or more DNMT3A non-R882 mutations, with pAML in 920 (56.3%), sAML in 355 (21.7%), MDS in 259 (15.8%), MPN in 68 (4.2%) and MDS/MPN in 33(2.0%) patients. Among 2088 non-R882 mutations, the most common type is missense (59%, n=1232), followed by frameshift (16.5%, n=345) and nonsense (12.3%, n=256) mutations. 78 (5%) patients had mutations in the N-terminal domain (NTD), 231 (14.1%) in Pro-Trp-Trp-Pro (PWWP) domain, 311 (19%) in ATRX-DNMT3-DNMT3L (ADD) domain and 777 (47.5%) in the methyltransferase (MTase) domain (p=0.0001). Patients with more than one domain or with splice site mutations were excluded. The median age of patients was 67.6 (59-74.8) ys. and male:female ratio showed no difference across domains (p=0.73 & p=0.64 resp.). Patients with NTD MT were less frequently pAML when compared to the other three domains [38.5% vs. 56%, p=0.002]; however, there was no domain distribution pattern observed for sAML and MDS patients. NTD was enriched with abnormal cytogenetics [58% vs 37%, p=0.0034]. NTD MT patients had only numerically lower BM blast percentage, WBC, and platelet counts. In domain-specific analysis, NTD MT had worse OS than non-NTD MT in pAML [8.2 vs. 15 mo., p=0.002], with no difference in sAML and MDS disease groups. PWWP MT had worse OS than non-PWWP MT in sAML alone [5.2 vs 10.6 mo., p=0.012]. ADD MT status was not related to prognostic outcome in any disease groups. Mtase MT had better OS than non-Mtase MT [11 vs 8 mo., p = 0.003] in sAML alone. In NTD MT pAML patients with poor OS, frameshift MT and abnormal karyotype were enriched than in the non-NTD MT group [31% vs 17.9%, p=0.04 and 57.9% vs 29.1%, p = 0.001, resp.]. PWWP MT sAML patients with worse OS had lower missense [43.2 vs 75.5, p=0.000006], higher nonsense [34.1 vs 9.7, p=0.00006] and abnormal karyotype [68.4% vs 49.1%, p= 0.0086] than in non-PWWP MT. Mtase MT sAML patients with better OS had higher missense [80.2% vs 59.2%, p= 0.002] and lower nonsense mutations [7.3% vs 20.8%, p=0.01]. CEBPA [10% vs 1.7%, p=0.02] and CSF3R [10% vs 2.9%, p=0.03] were mostly co-mutated in NTD MT pAML patients and NRAS [17% vs 7%, p=0.016] in PWWP MT sAML patients. Mtase MT sAML patients had less BCOR [6% vs 13%, p=0.03] and WT1 [1.8% vs 7%, p=0.04] than in non-Mtase MT group. Conclusion: non-R882 DNMT3AMT conveys differential prognosis based on the locus. It conferred poor prognosis in pAML, with more frameshift mutations and abnormal cytogenetics. PWWP MT is associated with worse and MTase MT with better outcomes in the sAML group. This is the first analysis to not only explore the non-R882 mutations at length but also reveal the significance of domain specific non-R882 DNMT3A MT in AML. Our study emphasizes the need to include the domain-based impact of DNMT3A mutational status in the prognostic schema for AML, which may help guide treatment plans and encourage further molecular analysis.

New oxindole carboxamides as inhibitors of DENV NS5 RdRp: Design, synthesis, docking and Biochemical characterization

Dengue is a mosquito-borne disease caused by the dengue virus belonging to family flaviviridae and has grown to be a major global public health issue. Despite decades of effort, the global comeback of dengue is evidence of the inadequacy of present management techniques. Due to the loss of healthy lives and the depletion of scarce medical resources, dengue has a significant negative economic impact in underdeveloped countries. In recent years, research for tackling the incidences of dengue infection has increased. The structure of the viral genome has been deciphered with the non-structural protein, known as NS5 serving as a potential target. NS5 consisting of an MTase domain involved in RNA capping and an RdRp domain involved in viral replication. In the presented work, a series of new Oxindoline Carboxamide derivatives were designed and synthesized for inhibiting the viral RNA dependent RNA-polymerase (RdRp) activity of DENV. The novel compounds were put through tests including molecular docking and surface plasmon resonance (SPR) binding analysis to evaluate their affinity for the viral protein and their potential as novel inhibitors of the virus. From a total of 12 derivative compounds, four compounds OCA-10c, OCA-10f, OCA-10j &OCA-10i, were found to exhibit high affinity for NS5 RdRp, the KD values being 1.376 μM, 1.63 μM, 7.08 μM & 9.32 μM respectively. Overall, we report novel inhibitors of DENV RdRp activity with potential to be utilized against DENV for treating humans after further optimization.

MAb Targeting a Link Between ExoN and MTase of TGEV NSP14.

Porcine transmissible gastroenteritis virus (TGEV) infection results in severe gastrointestinal disease manifesting vomiting, diarrhea in neonatal porcine, with extremely high mortality. Monoclonal antibody (MAb) specific to TGEV nonstructural protein (NSP)14 that contains two functional domains, exonuclease (ExoN) and methyltransferase (MTase) domains, may help elucidate the role of NSP14 in the viral life-cycle. In this study, we developed a murine MAb, designated 12F1, against TGEV NSP14 using traditional cell-fusion technique. It was shown the MAb can exclusively bind to viral NSP14, as evidenced by the results of indirect fluorescent assay and western blotting. Intriguingly, epitope screening assay shown that 12F1 targets a hinge region connecting ExoN and N7-MTase of NSP14.

Development of a sensitive microplate assay for characterizing RNA methyltransferase activity: Implications for epitranscriptomics and drug development

RNA methylation is a ubiquitous post-transcriptional modification found in diverse RNA classes and is a critical regulator of gene expression. In this study, we used Zika virus RNA methyltransferase (MTase) to develop a highly sensitive microplate assay that uses a biotinylated RNA substrate and radiolabeled AdoMet coenzyme. The assay is fast, highly reproducible, exhibits linear progress-curve kinetics under multiple turnover conditions, has high sensitivity in competitive inhibition assays, and significantly lower background levels compared with the currently used method. Using our newly developed microplate assay, we observed no significant difference in the catalytic constants of the full-length nonstructural protein 5 enzyme and the truncated MTase domain. These data suggest that, unlike the Zika virus RNA-dependent RNA polymerase activity, the MTase activity is unaffected by RNA-dependent RNA polymerase-MTase interdomain interaction. Given its quantitative nature and accuracy, this method can be used to characterize various RNA MTases, and, therefore, significantly contribute to the field of epitranscriptomics and drug development against infectious diseases.

Development of Zika Virus Mini-Replicon Based Single-Round Infectious Particles as Gene Delivery Vehicles.

Zika virus (ZIKV) is a type of RNA virus that belongs to the Flaviviridae family. We have reported the construction of a DNA-launched replicon of the Asian-lineage Natal RGN strain and the production of single-round infectious particles (SRIPs) via the combination of prM/E virus-like particles with the replicon. The main objective of the study was to engineer the ZIKV replicon as mammalian expression vectors and evaluate the potential of ZIKV mini-replicon-based SRIPs as delivery vehicles for heterologous gene expression in vitro and in vivo. The mini-replicons contained various genetic elements, including NS4B, an NS5 methyltransferase (MTase) domain, and an NS5 RNA-dependent RNA polymerase (RdRp) domain. Among these mini-replicons, only ZIKV mini-replicons 2 and 3, which contained the full NS5 and NS4B-NS5 genetic elements, respectively, exhibited the expression of reporters (green fluorescent protein (GFP) and cyan fluorescent protein-yellow fluorescent fusion protein (CYP)) and generated self-replicating RNAs. When the mini-replicons were transfected into the cells expressing ZIKV prM/E, this led to the production of ZIKV mini-replicon-based SRIPs. ZIKV mini-replicon 3 SRIPs showed a significantly higher yield titer and a greater abundance of self-replicating replicon RNAs when compared to ZIKV mini-replicon 2 SRIPs. Additionally, there were disparities in the dynamics of CYP expression and cytotoxic effects observed in various infected cell types between ZIKV mini-replicon 2-CYP and 3-CYP SRIPs. In particular, ZIKV mini-replicon 3-CYP SRIPs led to a substantial decrease in the survival rates of infected cells at a MOI of 2. An in vivo gene expression assay indicated that hACE2 expression was detected in the lung and brain tissues of mice following the intravenous administration of ZIKV mini-replicon 3-hACE2 SRIPs. Overall, this study highlights the potential of ZIKV mini-replicon-based SRIPs as promising vehicles for gene delivery applications in vitro and in vivo.

Perspectives on the current antiviral developments towards RNA-dependent RNA polymerase (RdRp) and methyltransferase (MTase) domains of dengue virus non-structural protein 5 (DENV-NS5)

Dengue virus (DENV) infection is one of the most emerging arboviral infections in humans. DENV is a positive-stranded RNA virus in the Flaviviridae family consisting of an 11 kb genome. DENV non-structural protein 5 (DENV-NS5) constitutes the largest among the non-structural proteins, which act as two domains, the RNA-dependent RNA polymerase (RdRp) and RNA methyltransferase enzyme (MTase). The DENV-NS5 RdRp domain contributes to the viral replication stages, whereas the MTase initiates viral RNA capping and facilitates polyprotein translation. Given the functions of both DENV-NS5 domains have made them an important druggable target. Possible therapeutic interventions and drug discoveries against DENV infection were thoroughly reviewed; however, a current update on the therapeutic strategies specific to DENV-NS5 or its active domains was not attempted. Since most potential compounds and drugs targeting the DENV-NS5 were evaluated in both in vitro cultures and animal models, a more detailed evaluation of molecules/drug candidates still requires investigation in randomized controlled clinical trials. This review summarizes current perspectives on the therapeutic strategies adopted to target the DENV-NS5 (RdRp and MTase domains) at the host-pathogen interface and further discusses the directions to identify candidate drugs to combat DENV infection.

AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2′-methyl-2′-fluoro guanosine 5′-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2′-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2′-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC50 ≈ 0.50 μM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.

Inter-domain coordination essential for dengue virus non-structural (5) NS5 interaction with stem loop a (SLA).

Dengue virus is the most prevalent arthropod-borne virus and is a part of the flavivirus family. There are four known dengue serotypes, and humans can be infected with more than one serotype which can lead to severe dengue. There are no clinically approved antiviral drugs for the treatment of dengue infection to date, and vaccines available are limited to seropositive individuals. The non-structural 5 (NS5) protein is the largest protein encoded by flaviviruses and is a major component of the viral replication complex. Dengue NS5 has an N-terminal methyltransferase (MTase) domain responsible for 5’ RNA capping, and a C-terminal RNA-dependent-RNA-polymerase (RdRp) domain responsible for de novo RNA synthesis. Dengue NS5 is also known to interact with RNA elements in the 5’-untranslated regions to promote viral RNA synthesis, in particular the 5’ stem loop A (SLA). However, there is limited structural characterization of the dengue NS5/SLA complex. Here, we characterized the interaction of the full length and individual domains of dengue serotype 2 (DENV2) NS5 with SLA at the 5’-UTR using surface plasmon resonance (SPR) studies, electromobility shift assays (EMSAs) and hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS). HDX-MS studies were used to probe the structural dynamics of DENV2 NS5 and its binding interface with SLA. Results from these studies suggest that coordination is required between the MTase and RdRp domains, which stabilize the interaction between NS5 and SLA. Ultimately, our results will shed more light into how dengue NS5 interacts with SLA to discriminate viral RNA from cellular RNA.

Biochemistry of the Respiratory Syncytial Virus L Protein Embedding RNA Polymerase and Capping Activities.

The human respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus. It is the major cause of severe acute lower respiratory tract infection in infants, the elderly population, and immunocompromised individuals. There is still no approved vaccine or antiviral treatment against RSV disease, but new monoclonal prophylactic antibodies are yet to be commercialized, and clinical trials are in progress. Hence, urgent efforts are needed to develop efficient therapeutic treatments. RSV RNA synthesis comprises viral transcription and replication that are catalyzed by the large protein (L) in coordination with the phosphoprotein polymerase cofactor (P), the nucleoprotein (N), and the M2-1 transcription factor. The replication/transcription is orchestrated by the L protein, which contains three conserved enzymatic domains: the RNA-dependent RNA polymerase (RdRp), the polyribonucleotidyl transferase (PRNTase or capping), and the methyltransferase (MTase) domain. These activities are essential for the RSV replicative cycle and are thus considered as attractive targets for the development of therapeutic agents. In this review, we summarize recent findings about RSV L domains structure that highlight how the enzymatic activities of RSV L domains are interconnected, discuss the most relevant and recent antivirals developments that target the replication/transcription complex, and conclude with a perspective on identified knowledge gaps that enable new research directions.