RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication within the infected cells. RdRP function is critical not only for the virus life cycle but also ...for its adaptive potential. The combination of low fidelity of replication and the absence of proofreading and excision activities within the RdRPs result in high mutation frequencies that allow these viruses a rapid adaptation to changing environments. In this review, we summarize the current knowledge about structural and functional aspects on RdRP catalytic complexes, focused mainly in the Picornaviridae family. The structural data currently available from these viruses provided high-resolution snapshots for a range of conformational states associated to RNA template-primer binding, rNTP recognition, catalysis and chain translocation. As these enzymes are major targets for the development of antiviral compounds, such structural information is essential for the design of new therapies.
Membrane fusion is essential in a myriad of eukaryotic cell biological processes, including the synaptic transmission. Rabphilin-3A is a membrane trafficking protein involved in the calcium-dependent ...regulation of secretory vesicle exocytosis in neurons and neuroendocrine cells, but the underlying mechanism remains poorly understood. Here, we report the crystal structures and biochemical analyses of Rabphilin-3A C2B–SNAP25 and C2B–phosphatidylinositol 4,5-bisphosphate (PIP₂) complexes, revealing how Rabphilin-3A C2 domains operate in cooperation with PIP₂/Ca2+ and SNAP25 to bind the plasma membrane, adopting a conformation compatible to interact with the complete SNARE complex. Comparisons with the synaptotagmin1–SNARE show that both proteins contact the same SNAP25 surface, but Rabphilin-3A uses a unique structural element. Data obtained here suggest a model to explain the Ca2+-dependent fusion process by membrane bending with a myriad of variations depending on the properties of the C2 domain-bearing protein, shedding light to understand the fine-tuning control of the different vesicle fusion events.
Picornavirus RNA replication is initiated by the covalent attachment of a UMP molecule to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction is carried out by the viral ...RNA‐dependent RNA polymerase (3D). Here, we report the X‐ray structure of two complexes between foot‐and‐mouth disease virus 3D, VPg1, the substrate UTP and divalent cations, in the absence and in the presence of an oligoadenylate of 10 residues. In both complexes, VPg fits the RNA binding cleft of the polymerase and projects the key residue Tyr3 into the active site of 3D. This is achieved by multiple interactions with residues of motif F and helix α8 of the fingers domain and helix α13 of the thumb domain of the polymerase. The complex obtained in the presence of the oligoadenylate showed the product of the VPg uridylylation (VPg‐UMP). Two metal ions and the catalytic aspartic acids of the polymerase active site, together with the basic residues of motif F, have been identified as participating in the priming reaction.
Background and Purpose
There is a need for effective anti‐COVID‐19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has ...proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID‐19. The purpose of the present study was to find synergistic antiviral combinations for COVID‐19 based on lethal mutagenesis.
Experimental Approach
The effect of combinations of remdesivir and ribavirin on the infectivity of SARS‐CoV‐2 in cell culture has been tested. Viral populations were monitored by ultra‐deep sequencing, and the decrease of infectivity as a result of the treatment was measured.
Key Results
Remdesivir and ribavirin exerted a synergistic inhibitory activity against SARS‐CoV‐2, quantified both by CompuSyn (Chou‐Talalay method) and Synergy Finder (ZIP‐score model). In serial passage experiments, virus extinction was readily achieved with remdesivir‐ribavirin combinations at concentrations well below their cytotoxic 50 value, but not with the drugs used individually. Deep sequencing of treated viral populations showed that remdesivir, ribavirin, and their combinations evoked significant increases of the number of viral mutations and haplotypes, as well as modification of diversity indices that characterize viral quasi‐species.
Conclusion and Implications
SARS‐CoV‐2 extinction can be achieved by synergistic combination treatments based on lethal mutagenesis. In addition, the results offer prospects of triple drug treatments for effective SARS‐CoV‐2 suppression.
Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme
performs this operation, together with other viral and probably host ...proteins, in the cytoplasm of their host cells. The crystal
structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined
unliganded and bound to a template-primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb
subdomains, with the presence of an NH 2 -terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-primer,
likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication
and the design of antiviral compounds.
RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside ...analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine triphosphate with different RNAs as template-primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3'-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases.
Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance ...to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin -by avoiding the biased repertoire of transition mutations produced by this purine analogue-and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure.
Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the
family that contains three small viral proteins (VPgs), named VPg1, VPg2 and VPg3, linked to the 5'-end of the viral genome. These ...VPg proteins act as primers for RNA replication, which is initiated by the consecutive binding of two UMP molecules to the hydroxyl group of Tyr3 in VPg. This process, termed uridylylation, is catalyzed by the viral RNA-dependent RNA polymerase named 3D
. 5-Fluorouridine triphosphate (FUTP) is a potent competitive inhibitor of VPg uridylylation. Peptide analysis showed FUMP covalently linked to the Tyr3 of VPg. This fluorouridylylation prevents further incorporation of the second UMP residue. The molecular basis of how the incorporated FUMP blocks the incorporation of the second UMP is still unknown. To investigate the mechanism of inhibition of VPg uridylylation by FUMP, we have prepared a simplified 15-mer model of VPg1 containing FUMP and studied its x-ray crystal structure in complex with 3D
. Unfortunately, the fluorouridylylated VPg1 was disordered and not visible in the electron density maps; however, the structure of 3D
in the presence of VPg1-FUMP showed an 8 Å movement of the β9-α11 loop of the polymerase towards the active site cavity relative to the complex of 3D
with VPg1-UMP. The conformational rearrangement of this loop preceding the 3D
B motif seems to block the access of the template nucleotide to the catalytic cavity. This result may be useful in the design of new antivirals against not only FMDV but also other picornaviruses, since all members of this family require the uridylylation of their VPg proteins to initiate the viral RNA synthesis.
Picornavirus genome replication takes place in specialized intracellular membrane compartments that concentrate viral RNA and proteins as well as a number of host factors that also participate in the ...process. The core enzyme in the replication machinery is the viral RNA-dependent RNA polymerase (RdRP) 3D.sup.pol . Replication requires the primer protein 3B (or VPg) attached to two uridine molecules. 3B uridylylation is also catalysed by 3D.sup.pol . Another critical interaction in picornavirus replication is that between 3D.sup.pol and the precursor 3AB, a membrane-binding protein responsible for the localization of 3D.sup.pol to the membranous compartments at which replication occurs. Unlike other picornaviruses, the animal pathogen foot-and-mouth disease virus (FMDV), encodes three non-identical copies of the 3B (3B1, 3B2, and 3B3) that could be specialized in different functions within the replication complex. Here, we have used a combination of biophysics, molecular and structural biology approaches to characterize the functional binding of FMDV 3B1 to the base of the palm of 3D.sup.pol . The 1.7 Å resolution crystal structure of the FMDV 3D.sup.pol -3B1 complex shows that 3B1 simultaneously links two 3D.sup.pol molecules by binding at the bottom of their palm subdomains in an almost symmetric way. The two 3B1 contact surfaces involve a combination of hydrophobic and basic residues at the N- (G5-P6, R9; Region I) and C-terminus (R16, L19-P20; Region II) of this small protein. Enzyme-Linked Immunosorbent Assays (ELISA) show that the two 3B1 binding sites play a role in 3D.sup.pol binding, with region II presenting the highest affinity. ELISA assays show that 3D.sup.pol has higher binding affinity for 3B1 than for 3B2 or 3B3. Membrane-based pull-down assays show that 3B1 region II, and to a lesser extent also region I play essential roles in mediating the interaction of 3AB with the polymerase and its recruitment to intracellular membranes.
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