Structure Of RNA-dependent RNA Polymerase Research Articles

Crystal structures of RNA-dependent RNA polymerases from Jingmen tick virus and Alongshan virus

Jingmenviruses are a group of flavi-like viruses with segmented genome and have been found in various types of hosts, including humans, cattle, monkeys, bats, rodents, sheep, ticks, mosquitoes and nematodes. Jingmenviruses, including the Jingmen tick virus (JMTV) and Alongshan virus (ALSV), have been associated with febrile illness and flu-like symptoms in humans. Viral polymerase plays critical roles in genome replication and transcription and is an ideal target for antiviral drugs. Here, we determined the crystal structures of RNA-dependent RNA polymerase (RdRp) domains of JMTV and ALSV at 2.6 Å and 3.2 Å resolutions, respectively. The overall structures of JMTV and ALSV RdRp domains are similar to those from the typical unsegmented viruses in Flaviviridae family, especially the Flavivirus genus. JMTV and ALSV RdRps can be divided into three subdomains and the catalytical Motif A-G are conserved like the typical flaviviruses, whereas the zinc-binding pockets are absent from the JMTV and ALSV RdRps. The 5′-ends of jingmenvirus genomes are varied in length and sequence, and a highly conserved 8-nucleotide element located on the tip of stem loop A was identified and shown to be required for binding with RdRp and performing de novo replication activity. These findings provide important structural insights into RdRp of segmented flavivirus and reveal the key region of virus genome responsible for replication initiation, which would promote molecular understanding of segmented flavivirus replication and the structure-based design of antiviral drugs against flaviviruses.

Structure of RNA-dependent RNA Polymerase 2 (RDR2) and its implications for double-stranded RNA synthesis in gene silencing

Structure of RNA-dependent RNA Polymerase 2 (RDR2) and its implications for double-stranded RNA synthesis in gene silencing

Histone Deacetylase Inhibitors as Potential COVID-19 Virus RNA-Dependent RNA Polymerase Inhibitors: A Molecular Docking and Dynamics Study

The novel coronavirus disease that initially appeared in 2019, commonly identified as COVID-19 is an acute infectious disease precipitated by the SARSCoV- 2 and has become a severe pandemic health crisis. People stricken with a severe case of COVID-19 are subject to a relatively higher mortality rate, which is brought about predominantly by the difficulty of having potent and specific antiviral drugs for its treatment. In this context, the viral RNA-dependent RNA polymerase (RdRp) is an attractive target for antiviral inhibitors, mainly because of its essential role in processing the polyproteins translated from viral RNA. Moreover, histone deacetylases inhibitors represent one of the most promising antiviral agents. Therefore, in this contribution, the in silico structure-based drug design approach was employed to identify novel structural characteristics for the potential repurposed activity of HDACIs as antivirals against COVID-19. Herein, 12 HDAC inhibitors were screened to explore their potential anti-viral activity against RNA-dependent RNA polymerase (RdRp) (6NUR). Results revealed that large number of the screened HDAC inhibitors are strongly bound into the active binding site of crystallographic structure of RNA-dependent RNA polymerase (RdRp) (6NUR) with lowest CDOCKER energy and CDOCKER interaction energy are very close to those of the control drug remdesivir. Importantly, the virtually screened HDAC inhibitors, particularly, Givinostat, Pracinostat, Panobinostat, Romidepsin (FK228) and its active metabolite (RedF) could be promising candidates for COVID-19 RNA-dependent RNA polymerase (RdRp) inhibitors. Significantly, these inhibitors should be evaluated on their effectivity when treating COVID-19, specifically using the drugs that have been approved for clinical trials with limited toxicity.

Structure‐function relationships in coronavirus and norovirus polymerases explored through the structure and activity of chimeric enzymes

The COVID‐19 pandemic caused by the severe acute respiratory syndrome coronavirus‐2 (SARS CoV‐2) has spurred an unprecedented response from the scientific community to contribute timely solutions to help manage an urgent public health emergency. The RNA‐dependent RNA polymerase (RdRP) plays a critical function in replicating the genome of all RNA viruses and is one of the most intensively studied viral enzyme targets for the development of direct‐acting antiviral therapeutics. Comparisons of recently determined cryo‐EM structures of the SARS CoV‐2 RdRP with crystal structures of calicivirus, picornavirus and flavivirus RdRP's in complex with RNA and nucleoside analogue inhibitors suggest how differences in the sequences and structures of these RdRPs may result in differences in the activity of the enzyme as well as its sensitivity to different inhibitors. Molecular modeling, crystallographic studies and in vitro primer extension assays reveal how chimeric RdRPs bearing different sequence features from the RdRPs of SARS CoV‐2 and human norovirus GII.4 may contribute to differences in polymerase activity and the effects of nucleoside analogue inhibitors. These results suggest how some of the structural features of viral RdRPs may be productively targeted for the design of novel and more potent inhibitors to serve as lead compounds for the development of more effective antiviral therapeutics in the future.

An Analysis of Inhibition of the Severe Acute Respiratory Syndrome Coronavirus 2 RNA-dependent Rna Polymerase By Zinc Ion: an in silico Approach

Background: Coronavirus disease 2019 is caused by exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was reported that Zn2+ is an inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV). We hypothesize that the same applies to the newly discovered SARS-CoV-2. Material & methods: We compared the structure of RNA-dependent RNA polymerase between SARS-CoV and SARS-CoV-2. The RdRp’s binding to Zn2+ was studied by metal ion-binding site prediction and docking server. Results: Several regions containing key residues were detected. The functional aspartic acid residues RdRp, 618D, 760D and 761D were among the predicted Zn2+-binding residues. Conclusion: The most probable mechanism of inhibition of RdRp by Zn2+ is binding to the active aspartic acid triad while other binding sites can further destabilize the enzyme or interfere with the fidelity-check mechanism.

Structural Insights into the Binding Modes of Viral RNA-Dependent RNA Polymerases Using a Function-Site Interaction Fingerprint Method for RNA Virus Drug Discovery.

The coronavirus disease of 2019 (COVID-19) pandemic speaks to the need for drugs that not only are effective but also remain effective given the mutation rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To this end, we describe structural binding-site insights for facilitating COVID-19 drug design when targeting RNA-dependent RNA polymerase (RDRP), a common conserved component of RNA viruses. We combined an RDRP structure data set, including 384 RDRP PDB structures and all corresponding RDRP–ligand interaction fingerprints, thereby revealing the structural characteristics of the active sites for application to RDRP-targeted drug discovery. Specifically, we revealed the intrinsic ligand-binding modes and associated RDRP structural characteristics. Four types of binding modes with corresponding binding pockets were determined, suggesting two major subpockets available for drug discovery. We screened a drug data set of 7894 compounds against these binding pockets and presented the top-10 small molecules as a starting point in further exploring the prevention of virus replication. In summary, the binding characteristics determined here help rationalize RDRP-targeted drug discovery and provide insights into the specific binding mechanisms important for containing the SARS-CoV-2 virus.

COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a newly emerging, highly transmissible, and pathogenic coronavirus in humans that has caused global public health emergencies and economic crises. To date, millions of infections and thousands of deaths have been reported worldwide, and the numbers continue to rise. Currently, there is no specific drug or vaccine against this deadly virus; therefore, there is a pressing need to understand the mechanism(s) through which this virus enters the host cell. Viral entry into the host cell is a multistep process in which SARS-CoV-2 utilizes the receptor-binding domain (RBD) of the spike (S) glycoprotein to recognize angiotensin-converting enzyme 2 (ACE2) receptors on the human cells; this initiates host-cell entry by promoting viral–host cell membrane fusion through large-scale conformational changes in the S protein. Receptor recognition and fusion are critical and essential steps of viral infections and are key determinants of the viral host range and cross-species transmission. In this review, we summarize the current knowledge on the origin and evolution of SARS-CoV-2 and the roles of key viral factors. We discuss the structure of RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 and its significance in drug discovery and explain the receptor recognition mechanisms of coronaviruses. Further, we provide a comparative analysis of the SARS-CoV and SARS-CoV-2 S proteins and their receptor-binding specificity and discuss the differences in their antigenicity based on biophysical and structural characteristics.

Potential RNA-dependent RNA polymerase inhibitors as prospective therapeutics against SARS-CoV-2.

Introduction. The emergence of SARS-CoV-2 has taken humanity off guard. Following an outbreak of SARS-CoV in 2002, and MERS-CoV about 10 years later, SARS-CoV-2 is the third coronavirus in less than 20 years to cross the species barrier and start spreading by human-to-human transmission. It is the most infectious of the three, currently causing the COVID-19 pandemic. No treatment has been approved for COVID-19. We previously proposed targets that can serve as binding sites for antiviral drugs for multiple coronaviruses, and here we set out to find current drugs that can be repurposed as COVID-19 therapeutics.Aim. To identify drugs against COVID-19, we performed an in silico virtual screen with the US Food and Drug Administration (FDA)-approved drugs targeting the RNA-dependent RNA polymerase (RdRP), a critical enzyme for coronavirus replication.Methodology. Initially, no RdRP structure of SARS-CoV-2 was available. We performed basic sequence and structural analysis to determine if RdRP from SARS-CoV was a suitable replacement. We performed molecular dynamics simulations to generate multiple starting conformations that were used for the in silico virtual screen. During this work, a structure of RdRP from SARS-CoV-2 became available and was also included in the in silico virtual screen.Results. The virtual screen identified several drugs predicted to bind in the conserved RNA tunnel of RdRP, where many of the proposed targets were located. Among these candidates, quinupristin is particularly interesting because it is expected to bind across the RNA tunnel, blocking access from both sides and suggesting that it has the potential to arrest viral replication by preventing viral RNA synthesis. Quinupristin is an antibiotic that has been in clinical use for two decades and is known to cause relatively minor side effects.Conclusion. Quinupristin represents a potential anti-SARS-CoV-2 therapeutic. At present, we have no evidence that this drug is effective against SARS-CoV-2 but expect that the biomedical community will expeditiously follow up on our in silico findings.

In situ structures of RNA-dependent RNA polymerase inside bluetongue virus before and after uncoating

Bluetongue virus (BTV), a major threat to livestock, is a multilayered, nonturreted member of the Reoviridae, a family of segmented dsRNA viruses characterized by endogenous RNA transcription through an RNA-dependent RNA polymerase (RdRp). To date, the structure of BTV RdRp has been unknown, limiting our mechanistic understanding of BTV transcription and hindering rational drug design effort targeting this essential enzyme. Here, we report the in situ structures of BTV RdRp VP1 in both the triple-layered virion and double-layered core, as determined by cryo-electron microscopy (cryoEM) and subparticle reconstruction. BTV RdRp has 2 unique motifs not found in other viral RdRps: a fingernail, attached to the conserved fingers subdomain, and a bundle of 3 helices: 1 from the palm subdomain and 2 from the N-terminal domain. BTV RdRp VP1 is anchored to the inner surface of the capsid shell via 5 asymmetrically arranged N termini of the inner capsid shell protein VP3A around the 5-fold axis. The structural changes of RdRp VP1 and associated capsid shell proteins between BTV virions and cores suggest that the detachment of the outer capsid proteins VP2 and VP5 during viral entry induces both global movements of the inner capsid shell and local conformational changes of the N-terminal latch helix (residues 34 to 51) of 1 inner capsid shell protein VP3A, priming RdRp VP1 within the capsid for transcription. Understanding this mechanism in BTV also provides general insights into RdRp activation and regulation during viral entry of other multilayered, nonturreted dsRNA viruses.

Bottlenecks in the transmission of porcine reproductive and respiratory syndrome virus (PRRSV1) to na\xefve pigs and the quasi-species variation of the virus during infection in vaccinated pigs

This paper describes the results of two experiments regarding porcine reproductive and respiratory syndrome virus (PRRSV1): the first one studied the existence of bottlenecks in an experimental one-to-one model of transmission in pigs; while the second analysed the differences between viral quasi-species in vaccinated pigs that developed shorter or longer viraemias after natural challenge. Serum samples, as well as the initial inoculum, were deep-sequenced and a viral quasi-species was constructed per sample. For the first experiment, the results consistently reported a reduction in the quasi-species diversity after a transmission event, pointing to the existence of bottlenecks during PRRSV1 transmission. However, despite the identified preferred and un-preferred transmitted variants not being randomly distributed along the virus genome, it was not possible to identify any variant producing a structural change in any viral protein. In contrast, the mutations identified in GP2, nsp9 and M of the second experiment pointed to changes in the amino acid charges and the viral RNA-dependent RNA polymerase structure. The fact that the affected proteins are known targets of the immunity against PRRSV, plus the differential level of neutralizing antibodies present in pigs developing short or long viraemias, suggests that the immune response selected those changes.

Viral RNA-Dependent RNA Polymerases: A Structural Overview.

Most emerging and re-emerging human and animal viral diseases are associated with RNA viruses. All these pathogens, with the exception of retroviruses, encode a specialized enzyme called RNA-dependent RNA polymerase (RdRP), which catalyze phosphodiester-bond formation between ribonucleotides (NTPs) in an RNA template-dependent manner. These enzymes function either as single polypeptides or in complex with other viral or host components to transcribe and replicate the viral RNA genome. The structures of RdRPs and RdRP catalytic complexes, currently available for several members of (+) ssRNA, (-)ssRNA and dsRNA virus families, have provided high resolution snapshots of the functional steps underlying replication and transcription of viral RNA genomes and their regulatory mechanisms.

Effects of mutations on active site conformation and dynamics of RNA-dependent RNA polymerase from Coxsackievirus B3

Recent crystal structures of RNA-dependent RNA polymerase (3Dpol) from Coxsackievirus B3 (CVB3) revealed that a tyrosine mutation at Phe364 (F364Y) resulted in structures with open active site whereas a hydrophobic mutation at Phe364 (F364A) led to conformations with closed active site. Besides, the crystal structures showed that the F364W mutation had no preference between the open and closed active sites, similar to wild-type. In this paper, we present a molecular dynamics (MD) study on CVB3 3Dpol in order to address some important questions raised by experiments. First, MD simulations of F364Y and F364A were carried out to explore how these mutations at Phe364 influence active site dynamics and conformations. Second, MD simulations of wild-type and mutants were performed to discover the connection between active site dynamics and polymerase function. MD simulations reveal that the effect of mutations on active site dynamics is associated with the interaction between the structural motifs A and D in CVB3 3Dpol. Interestingly, we discover that the active site state is influenced by the formation of a hydrogen bond between backbone atoms of Ala231 (in motif A) and Ala358 (in motif D), which has never been revealed before.

Crystal Structure and Thermostability Characterization of Enterovirus D68 3Dpol.

Enterovirus D68 (EV-D68) is one of the many nonpolio enteroviruses that cause mild to severe respiratory illness. The nonstructural protein 3Dpol is an RNA-dependent RNA polymerase (RdRP) of EV-D68 which plays a critical role in the replication of the viral genome and represents a promising drug target. Here, we report the first three-dimensional crystal structure of the RdRP from EV-D68 in complex with the substrate GTP to 2.3-Å resolution. The RdRP structure is similar to structures of other viral RdRPs, where the three domains, termed the palm, fingers, and thumb, form a structure resembling a cupped right hand. Particularly, an N-terminal fragment (Gly1 to Phe30) bridges the fingers and the thumb domains, which accounts for the enhanced stability of the full-length enzyme over the truncation mutant, as assessed by our thermal shift assays and the dynamic light scattering studies. Additionally, the GTP molecule bound proximal to the active site interacts with both the palm and fingers domains to stabilize the core structure of 3Dpol Interestingly, using limited proteolysis assays, we found that different nucleoside triphosphates (NTPs) stabilize the polymerase structure by various degrees, with GTP and CTP being the most and least stabilizing nucleosides, respectively. Lastly, we derived a model of the core structure of 3Dpol stabilized by GTP, according to our proteolytic studies. The biochemical and biophysical characterizations conducted in this study help us to understand the stability of EV-D68-3Dpol, which may extend to other RdRPs as well.IMPORTANCE Enterovirus D68 (EV-D68) is an emerging viral pathogen, which caused sporadic infections around the world. In recent years, epidemiology studies have reported an increasing number of patients with respiratory diseases globally due to the EV-D68 infection. Moreover, the infection has been associated with acute flaccid paralysis and cranial nerve dysfunction in children. However, there are no vaccines and antiviral treatments specifically targeting the virus to date. In this study, we solved the crystal structure of the RNA-dependent RNA polymerase of EV-D68 and carried out systematic biophysical and biochemical characterizations on the overall and local structural stability of the wild-type (WT) enzyme and several variants, which yields a clear view on the structure-activity relationship of the EV-D68 RNA polymerase.

Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA.

Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3′ terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3′ terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3′ cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3′ termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3′ terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3′ vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.

Significance of the C-terminal amino acid residue in mengovirus RNA-dependent RNA polymerase

Replication of picornavirus genomes is accomplished by the virally encoded RNA-dependent RNA polymerase (RdRP). Although the primary structure of this enzyme exhibits a high level of conservation, there are several significant differences among different picornavirus genera. In particular, a comparative alignment indicates that the C-terminal sequences of cardiovirus RdRP (known also as 3D pol), are 1-amino-acid residue (arginine or tryptophan) longer than that of the enterovirus or rhinovirus enzymes. Here, it is shown that alterations of the last codon of the RdRP-encoding sequence of mengovirus RNA leading to deletion of the C-terminal Trp460 or its replacement by Ala or Phe dramatically impaired viral RNA replication and, in the former case, resulted in a quasi-infectious phenotype (i.e., the mutant RNA might generate a low yield of pseudorevertants acquiring a Tyr residue in place of the deleted Trp460). The replacement of Trp460 by His or Tyr did not appreciably alter the viral growth potential. Homology modeling of three-dimensional structure of mengovirus RdRP suggested that Trp460 may be involved in interaction between the thumb and palm domains of the enzyme. Specifically, Trp460 of the thumb may form a hydrogen bond with Thr219 and hydrophobically interact with Val216 of the palm. The proposed interactions were consistent with the results of in vivo SELEX experiment, which demonstrated that infectious virus could contain Ser or Thr at position 219 and hydrophobic Val, Leu, Ile, as well as Arg (whose side chain has a nonpolar part) at position 216. A similar thumb–palm domain interaction may be a general feature of several RdRPs and its possible functional significance is discussed.

Identification of Residues Critical for the Interferon Antagonist Function of Langat Virus NS5 Reveals a Role for the RNA-Dependent RNA Polymerase Domain

All pathogenic flaviviruses examined thus far inhibit host interferon (IFN) responses by suppressing the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Both Langat virus (LGTV; a member of the tick-borne encephalitis virus serogroup) and Japanese encephalitis virus use the nonstructural protein NS5 to suppress JAK-STAT signaling. However, NS5 is also critical to virus replication, contributing methyltransferase and RNA-dependent RNA polymerase (RdRP) activities. The specific amino acid residues of NS5 involved in IFN antagonism are not known. Here, we demonstrate that the LGTV NS5 JAK-STAT inhibitory domain is contained between amino acids 355 and 735 (of 903), a range which lies within the RdRP domain. Furthermore, we identified two noncontiguous stretches of specific amino acids within the RdRP, 374 to 380 and 624 to 647, as critical for inhibition of JAK-STAT signaling. Despite considerable separation on the linear NS5 sequence, these residues localized adjacent to each other when modeled on the West Nile virus RdRP crystal structure. Due to the general conservation of RdRP structures, these results suggest that the specific residues identified act cooperatively to form a unique functional site on the RdRP responsible for JAK-STAT inhibition. This insight into the mechanism underlying flavivirus IFN evasion strategies will facilitate the design of antiviral therapeutics that potentiate the action of IFN during infection.

Crystal Structures of Active and Inactive Conformations of a Caliciviral RNA-dependent RNA Polymerase

The structure of the RNA-dependent RNA polymerase (RdRP) from the rabbit hemorrhagic disease virus has been determined by x-ray crystallography to a 2.5-A resolution. The overall structure resembles a "right hand," as seen before in other polymerases, including the RdRPs of polio virus and hepatitis C virus. Two copies of the polymerase are present in the asymmetric unit of the crystal, revealing active and inactive conformations within the same crystal form. The fingers and palm domains form a relatively rigid unit, but the thumb domain can adopt either "closed" or "open" conformations differing by a rigid body rotation of approximately 8 degrees. Metal ions bind at different positions in the two conformations and suggest how structural changes may be important to enzymatic function in RdRPs. Comparisons between the structures of the alternate conformational states of rabbit hemorrhagic disease virus RdRP and the structures of RdRPs from hepatitis C virus and polio virus suggest novel structure-function relationships in this medically important class of enzymes.