Structure of the Respiratory Syncytial Virus Polymerase Complex.

Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). RSV P recruits multiple proteins to the polymerase complex and, with the exception of its oligomerization domain, is thought to be intrinsically disordered. Despite their critical roles in RSV transcription and replication, structures of L and P have remained elusive. Here, we describe the 3.2-Å cryo-EM structure of RSV L bound to tetrameric P. The structure reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation. The structure also rationalizes inhibitor escape mutants and mutations observed in live-attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors.

4'-Fluorouridine mitigates lethal infection with pandemic human and highly pathogenic avian influenza viruses.

Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4'-Fluorouridine (4'-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus (RSV), we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium (HAE) models, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4'-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar to sub-micromolar potency in HAE cells. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4'-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4'-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (pdmCa09) with 4'-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating a broad time window for effective intervention. Therapeutic oral 4'-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pdmCa09. Recoverees were protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4'-FlU and supports 4'-FlU as developmental candidate for the treatment of seasonal and pandemic influenza.

Analysis of SARS-CoV-2 infection dynamic in vivo using reporter-expressing viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

Evaluating Antibody Mediated Protection against Alpha, Beta, and Delta SARS-CoV-2 Variants of Concern in K18-hACE2 Transgenic Mice.

SARS-CoV-2 variants of concern (VoC) are impacting responses to the COVID-19 pandemic. Here, we utilized passive immunization using human convalescent plasma (HCP) obtained from a critically ill COVID-19 patient in the early pandemic to study the efficacy of polyclonal antibodies generated to ancestral SARS-CoV-2 against the Alpha, Beta, and Delta VoC in the K18 human angiotensin converting enzyme 2 (hACE2) transgenic mouse model. HCP protected mice from challenge with the original WA-1 SARS-CoV-2 strain; however, only partially protected mice challenged with the Alpha VoC (60% survival) and failed to save Beta challenged mice from succumbing to disease. HCP treatment groups had elevated receptor binding domain (RBD) and nucleocapsid IgG titers in the serum; however, Beta VoC viral RNA burden in the lung and brain was not decreased due to HCP treatment. While mice could be protected from WA-1 or Alpha challenge with a single dose of HCP, six doses of HCP could not decrease mortality of Delta challenged mice. Overall, these data demonstrate that VoC have enhanced immune evasion and this work underscores the need for in vivo models to evaluate future emerging strains. IMPORTANCE Emerging SARS-CoV-2 VoC are posing new problems regarding vaccine and monoclonal antibody efficacy. To better understand immune evasion tactics of the VoC, we utilized passive immunization to study the effect of early-pandemic SARS-CoV-2 HCP against, Alpha, Beta, and Delta VoC. We observed that HCP from a human infected with the original SARS-CoV-2 was unable to control lethality of Alpha, Beta, or Delta VoC in the K18-hACE2 transgenic mouse model of SARS-CoV-2 infection. Our findings demonstrate that passive immunization can be used as a model to evaluate immune evasion of emerging VoC strains.

Therapeutic targeting of measles virus polymerase with ERDRP-0519 suppresses all RNA synthesis activity.

Morbilliviruses, such as measles virus (MeV) and canine distemper virus (CDV), are highly infectious members of the paramyxovirus family. MeV is responsible for major morbidity and mortality in non-vaccinated populations. ERDRP-0519, a pan-morbillivirus small molecule inhibitor for the treatment of measles, targets the morbillivirus RNA-dependent RNA-polymerase (RdRP) complex and displayed unparalleled oral efficacy against lethal infection of ferrets with CDV, an established surrogate model for human measles. Resistance profiling identified the L subunit of the RdRP, which harbors all enzymatic activity of the polymerase complex, as the molecular target of inhibition. Here, we examined binding characteristics, physical docking site, and the molecular mechanism of action of ERDRP-0519 through label-free biolayer interferometry, photoaffinity cross-linking, and in vitro RdRP assays using purified MeV RdRP complexes and synthetic templates. Results demonstrate that unlike all other mononegavirus small molecule inhibitors identified to date, ERDRP-0519 inhibits all phosphodiester bond formation in both de novo initiation of RNA synthesis at the promoter and RNA elongation by a committed polymerase complex. Photocrosslinking and resistance profiling-informed ligand docking revealed that this unprecedented mechanism of action of ERDRP-0519 is due to simultaneous engagement of the L protein polyribonucleotidyl transferase (PRNTase)-like domain and the flexible intrusion loop by the compound, pharmacologically locking the polymerase in pre-initiation conformation. This study informs selection of ERDRP-0519 as clinical candidate for measles therapy and identifies a previously unrecognized druggable site in mononegavirus L polymerase proteins that can silence all synthesis of viral RNA.

A Bifluorescent-Based Assay for the Identification of Neutralizing Antibodies against SARS-CoV-2 Variants of Concern In Vitro and In Vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the U.S. Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2-neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS-CoV-2 variants of concern (VoC). Here, we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and an rSARS-CoV-2 strain expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta [ß]) VoC in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent-expressing rSARS-CoV-2 strain and the parental wild-type (WT) rSARS-CoV-2 WA-1 strain had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS-CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 is responsible of the COVID-19 pandemic that has warped daily routines and socioeconomics. There is still an urgent need for prophylactics and therapeutics to treat SARS-CoV-2 infections. In this study, we demonstrate the feasibility of using bifluorescent-based assays for the rapid identification of hMAbs with neutralizing activity against SARS-CoV-2, including VoC in vitro and in vivo. Importantly, results obtained with these bifluorescent-based assays recapitulate those observed with individual viruses, demonstrating their feasibility to rapidly advance our understanding of vaccine efficacy and to identify broadly protective human NAbs for the therapeutic treatment of SARS-CoV-2.

Development of an allosteric inhibitor class blocking RNA elongation by the respiratory syncytial virus polymerase complex.

Respiratory syncytial virus (RSV) represents a significant health threat to infants and to elderly or immunocompromised individuals. There are currently no vaccines available to prevent RSV infections, and disease management is largely limited to supportive care, making the identification and development of effective antiviral therapeutics against RSV a priority. To identify effective chemical scaffolds for managing RSV disease, we conducted a high-throughput anti-RSV screen of a 57,000-compound library. We identified a hit compound that specifically blocked activity of the RSV RNA-dependent RNA polymerase (RdRp) complex, initially with moderate low-micromolar potency. Mechanistic characterization in an in vitro RSV RdRp assay indicated that representatives of this compound class block elongation of RSV RNA products after initial extension by up to three nucleotides. Synthetic hit-to-lead exploration yielded an informative 3D quantitative structure-activity relationship (3D-QSAR) model and resulted in analogs with more than 20-fold improved potency and selectivity indices (SIs) of >1,000. However, first-generation leads exhibited limited water solubility and poor metabolic stability. A second optimization strategy informed by the 3D-QSAR model combined with in silico pharmacokinetics (PK) predictions yielded an advanced lead, AVG-233, that demonstrated nanomolar activity against both laboratory-adapted RSV strains and clinical RSV isolates. This anti-RSV activity extended to infection of established cell lines and primary human airway cells. PK profiling in mice revealed 34% oral bioavailability of AVG-233 and sustained high drug levels in the circulation after a single oral dose of 20 mg/kg. This promising first-in-class lead warrants further development as an anti-RSV drug.

The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity.

The paramyxovirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the Morbillivirus genus, a series of measles virus (MeV) and canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design.IMPORTANCE Investigating the role of the paramyxovirus N protein tail domain (Ntail) in virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant viruses in cell culture but result in gradual viral attenuation in a lethal canine distemper virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxovirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxovirus family.

Fine mapping and characterization of the L-polymerase-binding domain of the respiratory syncytial virus phosphoprotein.

UNLABELLED: The minimum requirement for an active RNA-dependent RNA polymerase of respiratory syncytial virus (RSV) is a complex made of two viral proteins, the polymerase large protein (L) and the phosphoprotein (P). Here we have investigated the domain on P that is responsible for this critical P-L interaction. By use of recombinant proteins and serial deletions, an L binding site was mapped in the C-terminal region of P, just upstream of the N-RNA binding site. The role of this molecular recognition element of about 30 amino acid residues in the L-P interaction and RNA polymerase activity was evaluated in cellula using an RSV minigenome system and site-directed mutagenesis. The results highlighted the critical role of hydrophobic residues located in this region. IMPORTANCE: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine and no good antivirals against RSV are available, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. Like all negative-strand RNA viruses, RSV codes for its own machinery to replicate and transcribe its genome. The core of this machinery is composed of two proteins, the phosphoprotein (P) and the large protein (L). Here, using recombinant proteins, we have mapped and characterized the P domain responsible for this L-P interaction and the formation of an active L-P complex. These findings extend our understanding of the mechanism of action of RSV RNA polymerase and allow us to define a new target for the development of drugs against RSV.

Identification and characterization of the binding site of the respiratory syncytial virus phosphoprotein to RNA-free nucleoprotein.

UNLABELLED: The RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy with Paramyxoviridae and Rhabdoviridae, it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N(0)-P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N(0) monomer. We used this N mutant, designated N(mono), as a substitute for N(0) in order to characterize the P regions involved in the N(0)-P complex formation. Using a series of P fragments, we determined by glutathione S-transferase (GST) pulldown assays that the N and C termini of P are able to interact with N(mono). We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and N(mono) in vitro. Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N(0)-P interaction could constitute a potential antiviral strategy. IMPORTANCE: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment is available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase cofactor, is believed to function as a chaperon protein, maintaining N as a nonassembled, RNA-free protein (N(0)) competent for RNA encapsidation. In this paper, we provide the first evidence, to our knowledge, that the N terminus of P contains a domain that binds specifically to this RNA-free form of N. We further show that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis. These findings extend our understanding of the function of RSV RNA polymerase and point to a new target for the development of drugs against this virus.

Orally efficacious lead of the AVG inhibitor series targeting a dynamic interface in the respiratory syncytial virus polymerase.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory infections in infants and the immunocompromised, yet no efficient therapeutic exists. We have identified the AVG class of allosteric inhibitors of RSV RNA synthesis. Here, we demonstrate through biolayer interferometry and in vitro RNA-dependent RNA polymerase (RdRP) assays that AVG compounds bind to the viral polymerase, stalling the polymerase in initiation conformation. Resistance profiling revealed a unique escape pattern, suggesting a discrete docking pose. Affinity mapping using photoreactive AVG analogs identified the interface of polymerase core, capping, and connector domains as a molecular target site. A first-generation lead showed nanomolar potency against RSV in human airway epithelium organoids but lacked in vivo efficacy. Docking pose-informed synthetic optimization generated orally efficacious AVG-388, which showed potent efficacy in the RSV mouse model when administered therapeutically. This study maps a druggable target in the RSV RdRP and establishes clinical potential of the AVG chemotype against RSV disease.

SARS-CoV-2 variant of concern type and biological sex affect efficacy of molnupiravir in dwarf hamster model of severe COVID-19.

SARS-CoV-2 variants of concern (VOC) have triggered distinct infection waves in the coronavirus disease 2019 (COVID-19) pandemic, culminating in currently all-time high incidence rates of VOC omicron. Orally available direct-acting antivirals such as molnupiravir promise to improve disease management and limit SARS-CoV-2 spread. However, molnupiravir efficacy against VOC delta was questioned based on clinical trial results and its potency against omicron is unknown. This study evaluates molnupiravir against a panel of relevant VOC in three efficacy models: primary human airway epithelium organoids, the ferret model of upper respiratory disease, and a lethal Roborovski dwarf hamster efficacy model of severe COVID-19-like acute lung injury. All VOC were equally efficiently inhibited by molnupiravir in cultured cells and organoids. Treatment consistently reduced upper respiratory VOC shedding in ferrets and prevented viral transmission. Pathogenicity in the dwarf hamsters was VOC-dependent and highest for gamma, omicron, and delta with fulminant lung histopathology. Oral molnupiravir started 12 hours after infection resulted in complete survival of treated dwarf hamsters independent of challenge VOC. However, reduction in lung virus differed VOC-dependently, ranging from one (delta) to four (gamma) orders of magnitude compared to vehicle-treated animals. Dwarf hamsters infected with VOC omicron showed significant individual variation in response to treatment. Virus load reduction was significant in treated males, but not females. The dwarf hamster model recapitulates mixed efficacy of molnupiravir seen in human trials and alerts that therapeutic benefit of approved antivirals must be continuously reassessed in vivo as new VOC emerge.

4'-Fluorouridine is an oral antiviral that blocks respiratory syncytial virus and SARS-CoV-2 replication.

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and older adults. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with high selectivity index in cells and human airway epithelia organoids. Polymerase inhibition within in vitro RNA-dependent RNA polymerase assays established for RSV and SARS-CoV-2 revealed transcriptional stalling after incorporation. Once-daily oral treatment was highly efficacious at 5 milligrams per kilogram (mg/kg) in RSV-infected mice or 20 mg/kg in ferrets infected with different SARS-CoV-2 variants of concern, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2, and related RNA virus infections.

SARS-CoV-2 VOC type and biological sex affect molnupiravir efficacy in severe COVID-19 dwarf hamster model.

SARS-CoV-2 variants of concern (VOC) have triggered infection waves. Oral antivirals such as molnupiravir promise to improve disease management, but efficacy against VOC delta was questioned and potency against omicron is unknown. This study evaluates molnupiravir against VOC in human airway epithelium organoids, ferrets, and a lethal Roborovski dwarf hamster model of severe COVID-19-like lung injury. VOC were equally inhibited by molnupiravir in cells and organoids. Treatment reduced shedding in ferrets and prevented transmission. Pathogenicity in dwarf hamsters was VOC-dependent and highest for delta, gamma, and omicron. All molnupiravir-treated dwarf hamsters survived, showing reduction in lung virus load from one (delta) to four (gamma) orders of magnitude. Treatment effect size varied in individual dwarf hamsters infected with omicron and was significant in males, but not females. The dwarf hamster model recapitulates mixed efficacy of molnupiravir in human trials and alerts that benefit must be reassessed in vivo as VOC evolve.

Progress and pitfalls of a year of drug repurposing screens against COVID-19.

Near the end of 2019, a new betacoronavirus started to efficiently transmit between humans, resulting in the current COVID-19 pandemic. Unprecedented worldwide efforts were made to identify and repurpose antiviral therapeutics from collections of approved drugs and known bioactive compounds. Typical pitfalls of this approach (promiscuous/cytotoxic compounds leading to false positives), combined with bypassing antiviral drug development parameters due to urgency have resulted in often disappointing outcomes. A flood of publications, press-releases, and media posts, created confusion in the general public and sometime mobilized precious resources for clinical trials with minimal prospect of success. Breakthroughs have been made, not in the laboratory but in the clinic, resulting from the empiric identification of mitigators of clinical signs such as the discovery of improved disease management through immunomodulators. This opinion piece will aim to capture some of the lessons that we believe the COVID-19 pandemic has taught about drug repurposing screens.

4'-Fluorouridine is a broad-spectrum orally efficacious antiviral blocking respiratory syncytial virus and SARS-CoV-2 replication.

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and the elderly. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and SARS-CoV-2 with high selectivity index in cells and well-differentiated human airway epithelia. Polymerase inhibition in in vitro RdRP assays established for RSV and SARS-CoV-2 revealed transcriptional pauses at positions i or i +3/4 post-incorporation. Once-daily oral treatment was highly efficacious at 5 mg/kg in RSV-infected mice or 20 mg/kg in ferrets infected with SARS-CoV-2 WA1/2020 or variant-of-concern (VoC) isolate CA/2020, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2 and related RNA virus infections. ONE-SENTENCE SUMMARY: 4'-Fluorouridine is an orally available ribonucleoside analog that efficiently treats RSV and SARS-CoV-2 infections in vivo .

SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern induce lethal disease in K18-hACE2 transgenic mice despite convalescent plasma therapy.

SARS-CoV-2 variants of concern (VoCs) are impacting responses to the COVID-19 pandemic. Here we present a comparison of the SARS-CoV-2 USA-WA1/2020 (WA-1) strain with B.1.1.7 and B.1.351 VoCs and identify significant differences in viral propagation in vitro and pathogenicity in vivo using K18-hACE2 transgenic mice. Passive immunization with plasma from an early pandemic SARS-CoV-2 patient resulted in significant differences in the outcome of VoC-infected mice. WA-1-infected mice were protected by plasma, B.1.1.7-infected mice were partially protected, and B.1.351-infected mice were not protected. Serological correlates of disease were different between VoC-infected mice, with B.1.351 triggering significantly altered cytokine profiles than other strains. In this study, we defined infectivity and immune responses triggered by VoCs and observed that early 2020 SARS-CoV-2 human immune plasma was insufficient to protect against challenge with B.1.1.7 and B.1.351 in the mouse model.

Oral prodrug of remdesivir parent GS-441524 is efficacious against SARS-CoV-2 in ferrets.

Remdesivir is an antiviral approved for COVID-19 treatment, but its wider use is limited by intravenous delivery. An orally bioavailable remdesivir analog may boost therapeutic benefit by facilitating early administration to non-hospitalized patients. This study characterizes the anti-SARS-CoV-2 efficacy of GS-621763, an oral prodrug of remdesivir parent nucleoside GS-441524. Both GS-621763 and GS-441524 inhibit SARS-CoV-2, including variants of concern (VOC) in cell culture and human airway epithelium organoids. Oral GS-621763 is efficiently converted to plasma metabolite GS-441524, and in lungs to the triphosphate metabolite identical to that generated by remdesivir, demonstrating a consistent mechanism of activity. Twice-daily oral administration of 10 mg/kg GS-621763 reduces SARS-CoV-2 burden to near-undetectable levels in ferrets. When dosed therapeutically against VOC P.1 gamma γ, oral GS-621763 blocks virus replication and prevents transmission to untreated contact animals. These results demonstrate therapeutic efficacy of a much-needed orally bioavailable analog of remdesivir in a relevant animal model of SARS-CoV-2 infection.

Viral evolution identifies a regulatory interface between paramyxovirus polymerase complex and nucleocapsid that controls replication dynamics.

Paramyxoviruses are negative-polarity RNA viruses of major clinical importance. The dynamic interaction of the RNA-dependent RNA polymerase (RdRP) complex with the encapsidated RNA genome is mechanistically and structurally poorly understood. Having generated recombinant measles (MeV) and canine distemper (CDV) viruses with truncated nucleocapsid (N) protein showing defects in replication kinetics, we have applied a viral evolution approach to the problem. Passaging of recombinants resulted in long-range compensatory mutations that restored RdRP bioactivity in minigenome assays and efficient replication of engineered viruses. Compensatory mutations clustered at an electronically compatible acidic loop in N-core and a basic face of the phosphoprotein X domain (P-XD). Co-affinity precipitations, biolayer interferometry, and molecular docking revealed an electrostatic-driven transiently forming interface between these domains. The compensatory mutations reduced electrostatic compatibility of these microdomains and lowered coprecipitation efficiency, consistent with a molecular checkpoint function that regulates paramyxovirus polymerase mobility through modulation of conformational stability of the P-XD assembly.

High-Throughput Screening Assay to Identify Small Molecule Inhibitors of Marburg Virus VP40 Protein.

Marburg virus (MARV) causes sporadic outbreaks of severe disease with high case fatality rates in humans. To date, neither therapeutics nor prophylactic approaches have been approved for MARV disease. The MARV matrix protein VP40 (mVP40) plays central roles in virus assembly and budding. mVP40 also inhibits interferon signaling by inhibiting the function of Janus kinase 1. This suppression of host antiviral defenses likely contributes to MARV virulence and therefore is a potential therapeutic target. We developed and optimized a cell-based high-throughput screening (HTS) assay in 384-well format to measure mVP40 interferon (IFN) antagonist function such that inhibitors could be identified. We performed a pilot screen of 1280 bioactive compounds and identified 3 hits, azaguanine-8, tosufloxacin hydrochloride, and linezolid, with Z scores > 3 and no significant cytotoxicity. Of these, azaguanine-8 inhibited MARV growth at noncytotoxic concentrations. These data demonstrate the suitability of the HTS mVP40 assay for drug discovery and suggest potential directions for anti-MARV therapeutic development.