Point mutations at specific sites of the nsp12-nsp8 interface dramatically affect the RNA polymerization activity of SARS-CoV-2.

In a recent characterization of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variability present in 30 diagnostic samples from patients of the first COVID-19 pandemic wave, 41 amino acid substitutions were documented in the RNA-dependent RNA polymerase (RdRp) nsp12. Eight substitutions were selected in this work to determine whether they had an impact on the RdRp activity of the SARS-CoV-2 nsp12-nsp8-nsp7 replication complex. Three of these substitutions were found around the polymerase central cavity, in the template entry channel (D499G and M668V), and within the motif B (V560A), and they showed polymerization rates similar to the wild type RdRp. The remaining five mutations (P323L, L372F, L372P, V373A, and L527H) were placed near the nsp12-nsp8F contact surface; residues L372, V373, and L527 participated in a large hydrophobic cluster involving contacts between two helices in the nsp12 fingers and the long α-helix of nsp8F. The presence of any of these five amino acid substitutions resulted in important alterations in the RNA polymerization activity. Comparative primer elongation assays showed different behavior depending on the hydrophobicity of their side chains. The substitution of L by the bulkier F side chain at position 372 slightly promoted RdRp activity. However, this activity was dramatically reduced with the L372P, and L527H mutations, and to a lesser extent with V373A, all of which weaken the hydrophobic interactions within the cluster. Additional mutations, specifically designed to disrupt the nsp12-nsp8F interactions (nsp12-V330S, nsp12-V341S, and nsp8-R111A/D112A), also resulted in an impaired RdRp activity, further illustrating the importance of this contact interface in the regulation of RNA synthesis.

Incipient functional SARS-CoV-2 diversification identified through neural network haplotype maps.

Since its introduction in the human population, SARS-CoV-2 has evolved into multiple clades, but the events in its intrahost diversification are not well understood. Here, we compare three-dimensional (3D) self-organized neural haplotype maps (SOMs) of SARS-CoV-2 from thirty individual nasopharyngeal diagnostic samples obtained within a 19-day interval in Madrid (Spain), at the time of transition between clades 19 and 20. SOMs have been trained with the haplotype repertoire present in the mutant spectra of the nsp12- and spike (S)-coding regions. Each SOM consisted of a dominant neuron (displaying the maximum frequency), surrounded by a low-frequency neuron cloud. The sequence of the master (dominant) neuron was either identical to that of the reference Wuhan-Hu-1 genome or differed from it at one nucleotide position. Six different deviant haplotype sequences were identified among the master neurons. Some of the substitutions in the neural clouds affected critical sites of the nsp12-nsp8-nsp7 polymerase complex and resulted in altered kinetics of RNA synthesis in an in vitro primer extension assay. Thus, the analysis has identified mutations that are relevant to modification of viral RNA synthesis, present in the mutant clouds of SARS-CoV-2 quasispecies. These mutations most likely occurred during intrahost diversification in several COVID-19 patients, during an initial stage of the pandemic, and within a brief time period.

Dual role of the foot-and-mouth disease virus 3B1 protein in the replication complex: As protein primer and as an essential component to recruit 3Dpol to membranes.

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) 3Dpol. Replication requires the primer protein 3B (or VPg) attached to two uridine molecules. 3B uridylylation is also catalysed by 3Dpol. Another critical interaction in picornavirus replication is that between 3Dpol and the precursor 3AB, a membrane-binding protein responsible for the localization of 3Dpol 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 3Dpol. The 1.7 Å resolution crystal structure of the FMDV 3Dpol -3B1 complex shows that 3B1 simultaneously links two 3Dpol 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 3Dpol binding, with region II presenting the highest affinity. ELISA assays show that 3Dpol 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.

Atypical Mutational Spectrum of SARS-CoV-2 Replicating in the Presence of Ribavirin.

We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.

Amino Acid Substitutions Associated with Treatment Failure for Hepatitis C Virus Infection.

Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed.

Structural characterization of the Rabphilin-3A-SNAP25 interaction.

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.

Contribution of a Multifunctional Polymerase Region of Foot-and-Mouth Disease Virus to Lethal Mutagenesis.

Viral RNA-dependent RNA polymerases (RdRps) are major determinants of high mutation rates and generation of mutant spectra that mediate RNA virus adaptability. The RdRp of the picornavirus foot-and-mouth disease virus (FMDV), termed 3D, is a multifunctional protein that includes a nuclear localization signal (NLS) in its N-terminal region. Previous studies documented that some amino acid substitutions within the NLS altered nucleotide recognition and enhanced the incorporation of the mutagenic purine analogue ribavirin in viral RNA, but the mutants tested were not viable and their response to lethal mutagenesis could not be studied. Here we demonstrate that NLS amino acid substitution M16A of FMDV serotype C does not affect infectious virus production but accelerates ribavirin-mediated virus extinction. The mutant 3D displays polymerase activity, RNA binding, and copying processivity that are similar to those of the wild-type enzyme but shows increased ribavirin-triphosphate incorporation. Crystal structures of the mutant 3D in the apo and RNA-bound forms reveal an expansion of the template entry channel due to the replacement of the bulky Met by Ala. This is a major difference with other 3D mutants with altered nucleotide analogue recognition. Remarkably, two distinct loop ß9-α11 conformations distinguish 3Ds that exhibit higher or lower ribavirin incorporation than the wild-type enzyme. This difference identifies a specific molecular determinant of ribavirin sensitivity of FMDV. Comparison of several polymerase mutants indicates that different domains of the molecule can modify nucleotide recognition and response to lethal mutagenesis. The connection of this observation with current views on quasispecies adaptability is discussed.IMPORTANCE The nuclear localization signal (NLS) of the foot-and-mouth disease virus (FMDV) polymerase includes residues that modulate the sensitivity to mutagenic agents. Here we have described a viable NLS mutant with an amino acid replacement that facilitates virus extinction by ribavirin. The corresponding polymerase shows increased incorporation of ribavirin triphosphate and local structural modifications that implicate the template entry channel. Specifically, comparison of the structures of ribavirin-sensitive and ribavirin-resistant FMDV polymerases has identified loop ß9-α11 conformation as a determinant of sensitivity to ribavirin mutagenesis.

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.

(F)uridylylated Peptides Linked to VPg1 of Foot-and- Mouth Disease Virus (FMDV): Design, Synthesis and X-Ray Crystallography of the Complexes with FMDV RNA-Dependent RNA Polymerase.

Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the Picornaviridae 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 3Dpol. 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 3Dpol. Unfortunately, the fluorouridylylated VPg1 was disordered and not visible in the electron density maps; however, the structure of 3Dpol in the presence of VPg1-FUMP showed an 8 Å movement of the ß9-α11 loop of the polymerase towards the active site cavity relative to the complex of 3Dpol with VPg1-UMP. The conformational rearrangement of this loop preceding the 3Dpol 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.

Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication.

UNLABELLED: The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic cis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further. IMPORTANCE: Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., in trans) while others must originate from the template (i.e., in cis). Here, we present an analysis of cis and trans activities of the RNA-dependent RNA polymerase 3D. We demonstrate a novel cis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen.

The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.

Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition.

UNLABELLED: The N-terminal region of the foot-and-mouth disease virus (FMDV) 3D polymerase contains the sequence MRKTKLAPT (residues 16 to 24) that acts as a nuclear localization signal. A previous study showed that substitutions K18E and K20E diminished the transport to the nucleus of 3D and 3CD and severely impaired virus infectivity. These residues have also been implicated in template binding, as seen in the crystal structures of different 3D-RNA elongation complexes. Here, we report the biochemical and structural characterization of different mutant polymerases harboring substitutions at residues 18 and 20, in particular, K18E, K18A, K20E, K20A, and the double mutant K18A K20A (KAKA). All mutant enzymes exhibit low RNA binding activity, low processivity, and alterations in nucleotide recognition, including increased incorporation of ribavirin monophosphate (RMP) relative to the incorporation of cognate nucleotides compared with the wild-type enzyme. The structural analysis shows an unprecedented flexibility of the 3D mutant polymerases, including both global rearrangements of the closed-hand architecture and local conformational changes at loop ß9-α11 (within the polymerase motif B) and at the template-binding channel. Specifically, in 3D bound to RNA, both K18E and K20E induced the opening of new pockets in the template channel where the downstream templating nucleotide at position +2 binds. The comparisons of free and RNA-bound enzymes suggest that the structural rearrangements may occur in a concerted mode to regulate RNA replication, processivity, and fidelity. Thus, the N-terminal region of FMDV 3D that acts as a nuclear localization signal (NLS) and in template binding is also involved in nucleotide recognition and can affect the incorporation of nucleotide analogues. IMPORTANCE: The study documents multifunctionality of a nuclear localization signal (NLS) located at the N-terminal region of the foot-and-mouth disease viral polymerase (3D). Amino acid substitutions at this polymerase region can impair the transport of 3D to the nucleus, reduce 3D binding to RNA, and alter the relative incorporation of standard nucleoside monophosphate versus ribavirin monophosphate. Structural data reveal that the conformational changes in this region, forming part of the template channel entry, would be involved in nucleotide discrimination. The results have implications for the understanding of viral polymerase function and for lethal mutagenesis mechanisms.

Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1.

Proteins containing C2 domains are the sensors for Ca(2+) and PI(4,5)P2 in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca(2+) binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P2 and IP3 at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands ß3-ß4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P2/PI(3,4,5)P3, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P2. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca(2+)-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.

The crystal structure of a cardiovirus RNA-dependent RNA polymerase reveals an unusual conformation of the polymerase active site.

UNLABELLED: Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. IMPORTANCE: The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses.

Synergism between remdesivir and ribavirin leads to SARS-CoV-2 extinction in cell culture.

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.

A multi-step process of viral adaptation to a mutagenic nucleoside analogue by modulation of transition types leads to extinction-escape.

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-ß-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.

Structural and mechanistic insights into the association of PKCalpha-C2 domain to PtdIns(4,5)P2.

C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca(2+)-binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCalpha-C2 domain in complex with Ca(2+), 1,2-dihexanoyl-sn-glycero-3-[phospho-L-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P(2)] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns(4,5)P(2) occupies the concave surface of strands beta3 and beta4. Strikingly, the structure reveals a PtdIns(4,5)P(2)-C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of PtdIns(4,5)P(2) severely impaired the ability of PKCalpha to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by PtdIns(4,5)P(2) is presented.

SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations.

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed.

SARS-CoV-2 Point Mutation and Deletion Spectra and Their Association with Different Disease Outcomes.

Mutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultradeep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of 30 nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low-frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype, and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three-dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19. IMPORTANCE The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutation abundance and complexity and that significantly larger values were observed in virus from patients who developed mild COVID-19 symptoms. Mutant spectrum complexity is not a uniform trait among isolates. The nature and location of low-frequency amino acid substitutions present in mutant spectra anticipate great potential for phenotypic diversification of SARS-CoV-2.

Structure of foot-and-mouth disease virus mutant polymerases with reduced sensitivity to ribavirin.

Passage of poliovirus (PV) or foot-and-mouth disease virus (FMDV) in the presence of ribavirin selected for viruses with decreased sensitivity to R, which included different mutations in their polymerase (3D): G64S located in the finger subdomain in the case of PV and M296I located within loop beta9-alpha11 at the active site in the case of FMDV. To investigate why disparate substitutions were selected in two closely related 3Ds, we constructed FMDVs with a 3D that included either G62S (the equivalent replacement in FMDV of PV G64S), M296I, or both substitutions. G62S, but not M296I, inflicts upon FMDV a strong selective disadvantage which is partially compensated for by the substitution M296I. The corresponding mutant polymerases, 3D(G62S), 3D(M296I), and 3D(G62S-M296I), were analyzed functionally and structurally. G62S in 3D impairs RNA-binding, polymerization, and R monophosphate incorporation activities. The X-ray structures of the 3D(G62S)-RNA, 3D(M296I)-RNA, and 3D(G62S-M296I)-RNA complexes show that although the two positions are separated by 13.1 A, the loops where the replacements reside are tightly connected through an extensive network of interactions that reach the polymerase active site. In particular, G62S seems to restrict the flexibility of loop beta9-alpha11 and, as a consequence, the flexibility of the active site and its ability to bind the RNA template. Thus, a localized change in the finger subdomain of 3D may affect the catalytic domain. The results provide a structural interpretation of why different amino acid substitutions were selected to confer R resistance in closely related viruses and reveal a complex network of intra-3D interactions that can affect the recognition of both the RNA template and incoming nucleotide.