Influenza Polymerase Research Articles

Identification of novel influenza polymerase PB2 inhibitors using virtual screening approach and molecular dynamics simulation analysis of active compounds

Hierarchical virtual screening combined with ADME prediction and cluster analysis methods were used to identify influenza virus PB2 inhibitors with high activity, good druggability properties, and diverse structures. The 200,000 molecules in the ChemDiv core library were narrowed down to a final set of 97 molecules, of which six compounds were found to rescue cells from both H1N1 and H3N2 virus-induced CPE with EC50 values ranging from 5.81 μM to 42.77 μM, and could bind to the PB2 CBD of H1N1, with Kd values of 0.11 μM to 6.4 μM. The six compounds have novel structures and low molecular weight and are, thus, suitable serve as lead compounds for development as PB2 inhibitors. A receptor-based pharmacophore model was successfully constructed using key amino acid residues for the binding of inhibitors to PB2, provided by the MD simulations. This pharmacophore model suggested that to improve the activity of our active compounds, we should mainly focus on optimizing their existing structures with the aim of increasing their adaptability to the binding site, rather than adding chemical fragments to increase their binding to adjacent sites. This pharmacophore construction method facilitates the creation of a reasonable pharmacophore model without the need to fully understand the structure-activity relationships, and our descriptions provide a useful reference for similar research.

Rare variant MX1 alleles increase human susceptibility to zoonotic H7N9 influenza virus.

Zoonotic avian influenza A virus (IAV) infections are rare. Sustained transmission of these IAVs between humans has not been observed, suggesting a role for host genes. We used whole-genome sequencing to compare avian IAV H7N9 patients with healthy controls and observed a strong association between H7N9 infection and rare, heterozygous single-nucleotide variants in the MX1 gene. MX1 codes for myxovirus resistance protein A (MxA), an interferon-induced antiviral guanosine triphosphatase known to control IAV infections in transgenic mice. Most of the MxA variants identified lost the ability to inhibit avian IAVs, including H7N9, in transfected human cell lines. Nearly all of the inactive MxA variants exerted a dominant-negative effect on the antiviral function of wild-type MxA, suggesting an MxA null phenotype in heterozygous carriers. Our study provides genetic evidence for a crucial role of the MX1-based antiviral defense in controlling zoonotic IAV infections in humans.

Poultry passport to pandemic

Influenza What conditions are required to nurture the seeds of a pandemic? The avian influenza virus H7N9 rarely spills over into humans, but when it does, mortality exceeds 30%, far in excess of that of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Chen et al. used whole-genome sequencing to investigate the contribution of rare mutations among poultry workers, who can be exposed to high levels of H7N9. Multiple defective single-nucleotide variants in the myxovirus resistance Mx1 locus were prevalent in H7N9 patients. In vitro infection experiments and influenza polymerase activity assays showed that 14 of the 17 MxA protein variants had no antiviral activity. Thus, when exposed to high virus loads, individuals with such genetic vulnerabilities may act as crucibles for transmission of virulent new influenza subtypes. Science , abg5953, this issue p. [918][1] [1]: /lookup/doi/10.1126/science.abg5953

Ultrasound and Influenza: The Spectrum of Lung and Cardiac Ultrasound Findings in Patients with Suspected Influenza A and B

In patients with influenza, cardiac and lung ultrasound may help determine the severity of illness and predict clinical outcomes. To determine the ultrasound characteristics of influenza and define the spectrum of lung and cardiac findings in patients with suspected influenza A or B, we conducted a prospective observational study in patients presenting to the emergency department at a tertiary care academic institution. An ultrasound protocol consisting of cardiac, lung and inferior vena cava scans was performed within 6 h of admission. We compared the ultrasound findings in cases with positive and negative influenza polymerase chain reaction, while controlling for comorbidities. We enrolled 117 patients, 41.9% of whom (49/117) tested positive for influenza. In those with influenza, ultrasound confirmed preserved left ventricular and right ventricular (RV) function in 81.3% of patients. The most common cardiac pathology was RV dilation (10.4%), followed by left ventricular systolic dysfunction (8.3%). Patients with negative influenza polymerase chain reaction with RV dysfunction demonstrated higher hospital admission than those those with normal RV function (45.1%, 23/51, vs. 17.9%, 5/28; p = 0.016). B-lines were prevalent in both influenza and non-influenza groups (40.8% and 69.1%, respectively; p = 0.013). Lung consolidation was identified in only 8.25% of patients with influenza. In conclusion, in patients with influenza we were unable to define distinct ultrasound features specific to influenza A or B, suggesting that ultrasound may not be beneficial in diagnosing influenza nor in evaluating its severity.

Synthesis and In Vitro Evaluation of C-7 and C-8 Luteolin Derivatives as Influenza Endonuclease Inhibitors.

The part of the influenza polymerase PA subunit featuring endonuclease activity is a target for anti-influenza therapies, including the FDA-approved drug Xofluza. A general feature of endonuclease inhibitors is their ability to chelate Mg2+ or Mn2+ ions located in the enzyme’s catalytic site. Previously, we screened a panel of flavonoids for PA inhibition and found luteolin and its C-glucoside orientin to be potent inhibitors. Through structural analysis, we identified the presence of a 3′,4′-dihydroxyphenyl moiety as a crucial feature for sub-micromolar inhibitory activity. Here, we report results from a subsequent investigation exploring structural changes at the C-7 and C-8 positions of luteolin. Experimental IC50 values were determined by AlphaScreen technology. The most potent inhibitors were C-8 derivatives with inhibitory potencies comparable to that of luteolin. Bio-isosteric replacement of the C-7 hydroxyl moiety of luteolin led to a series of compounds with one-order-of-magnitude-lower inhibitory potencies. Using X-ray crystallography, we solved structures of the wild-type PA-N-terminal domain and its I38T mutant in complex with orientin at 1.9 Å and 2.2 Å resolution, respectively.

1,2,4-Triazolo[1,5-a]pyrimidines: Efficient one-step synthesis and functionalization as influenza polymerase PA-PB1 interaction disruptors.

In the search for new anti-influenza virus (IV) compounds, we have identified the 1,2,4-triazolo[1,5-a]pyrimidine (TZP) as a very suitable scaffold to obtain compounds able to disrupt IV RNA-dependent RNA polymerase (RdRP) PA-PB1 subunits heterodimerization. In this work, in order to acquire further SAR insights for this class of compounds and identify more potent derivatives, we designed and synthesized additional series of analogues to investigate the role of the substituents around the TZP core. To this aim, we developed four facile and efficient one-step procedures for the synthesis of 5-phenyl-, 6-phenyl- and 7-phenyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines, and 2-amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol. Two analogues having the ethyl carboxylate moiety at the C-2 position of the TZP were also prepared in good yields. Then, the scaffolds herein synthesized and two previous scaffolds were functionalized and evaluated for their anti-IAV activity, leading to the identification of compound 22 that showed both anti-PA-PB1 (IC50=19.5μM) and anti-IAV activity (EC50=16μM) at non-toxic concentrations, thus resulting among the most active TZP derivatives reported to date by us. A selection of the synthesized compounds, along with a set of in-house available analogues, was also tested against SARS-CoV-2. The most promising compound 49 from this series displayed an EC50 value of 34.47μM, highlighting the potential of the TPZ scaffold in the search for anti-CoV agents.

The argument for rapid influenza polymerase chain reaction (PCR) during the COVID-19 pandemic: Quicker turnaround times correlated with decreased antimicrobial use, reduced admission rates, and shorter length of stay.

The innovation of rapid influenza polymerase chain reaction (XT-PCR) has allowed quick, highly sensitive test results. Consequently, physicians can differentiate influenza from other respiratory illnesses and rapidly initiate treatment. We examined the effect of implementing XT-PCR on antimicrobial use, admission rates, and length of stay at a tertiary healthcare system.

Identification of Novel Influenza Polymerase PB2 Inhibitors Using a Cascade Docking Virtual Screening Approach.

To discover novel inhibitors that target the influenza polymerase basic protein 2 (PB2) cap-binding domain (CBD), commercial ChemBridge compound libraries containing 384,796 compounds were screened using a cascade docking of LibDock–LigandFit–GOLD, and 60 compounds were selected for testing with cytopathic effect (CPE) inhibition assays and surface plasmon resonance (SPR) assay. Ten compounds were identified to rescue cells from H1N1 virus-mediated death at non-cytotoxic concentrations with EC50 values ranging from 0.30 to 67.65 μM and could bind to the PB2 CBD of H1N1 with Kd values ranging from 0.21 to 6.77 μM. Among these, four compounds (11D4, 12C5, 21A5, and 21B1) showed inhibition of a broad spectrum of influenza virus strains, including oseltamivir-resistant ones, the PR/8-R292K mutant (H1N1, recombinant oseltamivir-resistant strain), the PR/8-I38T mutant (H1N1, recombinant baloxavir-resistant strain), and the influenza B/Lee/40 virus strain. These compounds have novel chemical scaffolds and relatively small molecular weights and are suitable for optimization as lead compounds. Based on sequence and structure comparisons of PB2 CBDs of various influenza virus subtypes, we propose that the Phe323/Gln325, Asn429/Ser431, and Arg355/Gly357 mutations, particularly the Arg355/Gly357 mutation, have a marked impact on the selectivities of PB2 CBD-targeted inhibitors of influenza A and influenza B.

Structural characterization of the interaction between the C-terminal domain of the influenza polymerase PA subunit and an optimized small peptide inhibitor

Influenza viruses can cause severe respiratory infections in humans, leading to nearly half a million deaths worldwide each year. Improved antiviral drugs are needed to address the threat of development of novel pandemic strains. Current therapeutic interventions target three key proteins in the viral life cycle: neuraminidase, the M2 channel and RNA-dependent-RNA polymerase. Protein-protein interactions between influenza polymerase subunits are potential new targets for drug development. Using a newly developed assay based on AlphaScreen technology, we screened a peptide panel for protein-protein interaction inhibitors to identify a minimal PB1 subunit-derived peptide that retains high inhibition potential and can be further modified. Here, we present an X-ray structure of the resulting decapeptide bound to the C-terminal domain of PA polymerase subunit from pandemic isolate A/California/07/2009 H1N1 at 1.6 Å resolution and discuss its implications for the design of specific, potent influenza polymerase inhibitors.

Molecular basis of host-adaptation interactions between influenza virus polymerase PB2 subunit and ANP32A

Avian influenza polymerase undergoes host adaptation in order to efficiently replicate in human cells. Adaptive mutants are localised on the C-terminal (627-NLS) domains of the PB2 subunit. In particular, mutation of PB2 residue 627 from E to K rescues polymerase activity in mammalian cells. A host transcription regulator ANP32A, comprising a long C-terminal intrinsically disordered domain (IDD), is responsible for this adaptation. Human ANP32A IDD lacks a 33 residue insertion compared to avian ANP32A, and this deletion restricts avian influenza polymerase activity. We used NMR to determine conformational ensembles of E627 and K627 forms of 627-NLS of PB2 in complex with avian and human ANP32A. Human ANP32A IDD transiently binds to the 627 domain, exploiting multivalency to maximise affinity. E627 interrupts the polyvalency of the interaction, an effect compensated by an avian-unique motif in the IDD. The observed binding mode is maintained in the context of heterotrimeric influenza polymerase, placing ANP32A in the immediate vicinity of known host-adaptive PB2 mutants.

Dihydromyricetin is a new inhibitor of influenza polymerase PB2 subunit and influenza-induced inflammation

Development of new and effective anti-influenza drugs is critical for the treatment of influenza virus infection. The polymerase basic 2 (PB2) subunit as a core subunit of influenza A virus RNA polymerase complex is considered to be an attractive drug target for anti-influenza drug discovery. Dihydromyricetin, as a natural flavonoid, has a wide range of biological activities, but its anti-influenza A virus activity is ambiguous. Here, we found dihydromyricetin could inhibit the replication of a variety of influenza A virus strains. Mechanism studies demonstrated that dihydromyricetin reduced viral polymerase activity via selective inhibition of viral PB2 subunit, and decreased relative amounts of viral mRNA and genomic RNA during influenza A virus infection. The binding affinity and molecular docking analyses revealed that dihydromyricetin interacted with the PB2 cap-binding pocket, functioned as a cap-binding competitor. Interestingly, dihydromyricetin also reduced cellular immune injury by inhibiting TLR3 signaling pathway. Additionally, combination treatment of dihydromyricetin with zanamivir exerted a synergistic anti-influenza effect. Altogether, our experiments reveal the antiviral and anti-inflammatory activities of dihydromyricetin in vitro against influenza virus infection, which provides a new insight into the development of novel anti-influenza drugs.

Baloxavir for the treatment of Influenza in allogeneic hematopoietic stem cell transplant recipients previously treated with oseltamivir

Seasonal influenza causes significant morbidity and mortality in allogeneic stem cell transplant (SCT) recipients. In this population, influenza virus can replicate for prolonged periods, despite neuraminidase inhibitor treatment, leading to resistance and treatment failure. Baloxavir targets the influenza polymerase and may be an effective treatment option in these patients. We used baloxavir to treat five allogeneic SCT recipients that were still symptomatic and shedding influenza virus after completing one or more treatment courses of oseltamivir and characterized the viral isolates before and during treatment. Two patients were infected with influenza A/H1pdm09 carrying a neuraminidase variant (H275Y) linked to oseltamivir resistance. Both these two patients were successfully treated with baloxavir. Of the three patients infected with wild-type influenza virus, two cleared the virus after baloxavir treatment, while the third patient developed the polymerase I38T variant linked to baloxavir resistance. Our data suggest that baloxavir treatment can be effective in treating neuraminidase inhibitor-resistant influenza in profoundly immunocompromised patients. Randomized clinical trials are needed to define the role of baloxavir alone and combined with oseltamivir for the treatment of influenza in SCT recipients and other immunocompromised populations.

Acute-onset smell and taste disorders in the context of COVID-19: a pilot multicentre polymerase chain reaction based case-control study.

Background and purposeSpecific respiratory tract infections, including COVID‐19, may cause smell and/or taste disorders (STDs) with increased frequency. The aim was to determine whether new‐onset STDs are more frequent amongst COVID‐19 patients than influenza patients.MethodThis was a case–control study including hospitalized patients of two tertiary care centres. Consecutive patients positive for COVID‐19 polymerase chain reaction (cases) and patients positive for influenza polymerase chain reaction (historical control sample) were assessed during specific periods, employing a self‐reported STD questionnaire.ResultsSeventy‐nine cases and 40 controls were included. No significant differences were found in basal features between the two groups. New‐onset STDs were significantly more frequent amongst cases (31, 39.2%) than in the control group (5, 12.5 %) [adjusted odds ratio 21.4 (2.77–165.4, P = 0.003)]. COVID‐19 patients with new‐onset STDs were significantly younger than COVID‐19 patients without STDs (52.6 ± 17.2 vs. 67.4 ± 15.1, P < 0.001). Amongst COVID‐19 patients who presented STDs, 22 (70.9%) recalled an acute onset and it was an initial manifestation in 11 (35.5%). Twenty‐five (80.6%) presented smell disorders (mostly anosmia, 14, 45.2%) and 28 (90.3%) taste disorders (mostly ageusia, 14, 45.2%). Only four (12.9 %) reported concomitant nasal obstruction. The mean duration of STD was 7.5 ± 3.2 days and 12 patients (40%) manifested complete recovery after 7.4 ± 2.3 days of onset.ConclusionNew‐onset STDs were significantly more frequent amongst COVID‐19 patients than influenza patients; they usually had an acute onset and were commonly an initial manifestation. The use of STD assessment in anamnesis as a hint for COVID‐19 and to support individuals’ self‐isolation in the current epidemic context is suggested.

Structure and Function of Influenza Polymerase.

Influenza polymerase (FluPol) plays a key role in the viral infection cycle by transcribing and replicating the viral genome. FluPol is a multifunctional, heterotrimeric enzyme with cap-binding, endonuclease, RNA-dependent RNA polymerase and polyadenylation activities. It performs its functions in the context of the viral ribonucleoprotein particle (RNP), wherein the template viral RNA is coated by multiple copies of viral nucleoprotein. Moreover, it interacts with a number of host proteins that are essential cofactors and, consequently, adaptive mutations in the polymerase are required for crossing the avian-human species barrier. In this review, we show how mechanistic understanding of how FluPol performs its multiple functions has greatly advanced over the last decade through determination of high-resolution structures by X-ray crystallography and cryo-electron microscopy. These have revealed not only the detailed architecture of FluPol but highlighted the remarkably conformational flexibility that is inherent to its functioning as a dynamic RNA synthesis machine. Structural studies are also underpinning current attempts to develop next-generation anti-influenza drugs that directly target FluPol.

A Structure-Based Model for the Complete Transcription Cycle of Influenza Polymerase

Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). Here, we visualize by cryoelectron microscopy the conformational dynamics of the polymerase during the complete transcription cycle from pre-initiation to termination, focusing on the template trajectory. After exiting the active site cavity, the template 3' extremity rebinds into a specific site on the polymerase surface. Here, it remains sequestered during all subsequent transcription steps, forcing the template to loop out as it further translocates. At termination, the strained connection between thebound template 5' end and the active site results in polyadenylation by stuttering at uridine 17. Upon product dissociation, further conformational changes release the trapped template, allowing recycling back into the pre-initiation state. Influenza polymerase thus performs transcription while tightly binding to and protecting both template ends, allowing efficient production of multiple mRNAs from a single vRNP.

Influenza Polymerase Inhibitors: Mechanisms of Action and Resistance.

The influenza virus RNA-dependent RNA polymerase is highly conserved among influenza A, B, C, and D viruses. It comprises three subunits: polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA) in influenza A and B viruses or polymerase 3 protein (P3) in influenza C and D viruses. Because this polymerase is essential for influenza virus replication, it has been considered as a target for antiviral agents. Recently, several polymerase inhibitors that target each subunit have been developed. This review discusses the mechanism of action, antiviral activity, and emergence of resistance to three inhibitors approved for the treatment of influenza or in late-phase clinical trials: the PB1 inhibitor favipiravir, the PB2 inhibitor pimodivir, and the PA inhibitor baloxavir marboxil.

Noninterruptive Clinical Decision Support Decreases Ordering of Respiratory Viral Panels during Influenza Season

A growing body of evidence suggests that testing for influenza virus alone is more appropriate than multiplex respiratory viral panel (RVP) testing for general populations of patients with respiratory tract infections. We aimed to decrease the proportion of RVPs out of total respiratory viral testing ordered during influenza season. We implemented two consecutive interventions: reflex testing for RVPs only after a negative influenza test, and noninterruptive clinical decision support (CDS) including modifications of the computerized physician order entry search behavior and cost display. We conducted an interrupted time series of RVPs and influenza polymerase chain reaction tests pre- and postintervention, and performed a mixed-effects logistic regression analysis with a primary outcome of proportion of RVPs out of total respiratory viral tests. The primary predictor was the intervention period, and covariates included the provider, clinical setting, associated diagnoses, and influenza incidence. From March 2013 to April 2019, there were 24,294 RVPs and 26,012 influenza tests (n = 50,306). Odds of ordering an RVP decreased during the reflex testing period (odds ratio: 0.432, 95% confidence interval: 0.397-0.469), and decreased more dramatically during the noninterruptive CDS period (odds ratio: 0.291, 95% confidence interval: 0.259-0.327). The odds of ordering an RVP were 71% less with the noninterruptive CDS intervention, which projected 4,773 fewer RVPs compared with baseline. Assuming a cost equal to Medicare reimbursement rates for RVPs and influenza tests, this would generate an estimated averted cost of $1,259,474 per year. Noninterruptive CDS interventions are effective in reducing unnecessary and expensive testing, and avoid typical pitfalls such as alert fatigue.

A unique feature of swine ANP32A provides susceptibility to avian influenza virus infection in pigs.

Both the replication and transcription of the influenza virus are catalyzed by the viral polymerase complex. The polymerases of most avian influenza A viruses have poor performance in mammalian cells, which is considered to be one of the important species barriers. Pigs have been long considered as important intermediate hosts for interspecies transmission of the avian influenza virus, because of their susceptibility to infection with both avian and mammalian influenza viruses. However, the molecular basis of influenza polymerase adaptation in pigs remains largely unknown. ANP32A and ANP32B proteins have been identified as playing fundamental roles in influenza virus replication and host range determination. In this study, we found that swine ANP32A (swANP32A), unlike swine ANP32B or other mammalian ANP32A or B, shows stronger supporting activity to avian viral polymerase. Knockout of ANP32A in pig cells PK15 dramatically reduced avian influenza polymerase activity and viral infectivity, suggesting a unique feature of swANP32A in supporting avian influenza viral polymerase. This species-specific activity is mapped to two key sites, 106V and 156S, in swANP32A. Interestingly, the amino acid 106V is unique to pigs among all the vertebrate species studied, and when combined with 156S, exhibits positive epistasis in pigs. Mutation of 106V and 156S to the signature found in ANP32As from other mammalian species weakened the interaction between swANP32A and chicken viral polymerase, and reduced polymerase activity. Understanding the molecular basis of ANP32 proteins may help to discover new antiviral targets and design avian influenza resistant genome edited pigs.

Rapid detection of an I38T amino acid substitution in influenza polymerase acidic subunit associated with reduced susceptibility to baloxavir marboxil.

BackgroundThe novel cap‐dependent endonuclease inhibitor baloxavir marboxil was approved in February 2018 for the treatment of influenza virus infection in Japan. In vitro studies have revealed that an I38T substitution in the polymerase acidic subunit (PA) is associated with reduced susceptibility of influenza viruses to baloxavir.ObjectivesDevelopment of a rapid and simple method for monitoring influenza A(H1N1)pdm09, A(H3N2), and B viruses possessing the I38T substitution in PA.MethodsThree assays were developed based on RNase H2‐dependent PCR (rhPCR) and named A/H1pdm PA_I38T rhPCR, A/H3 PA_I38T rhPCR, and B PA_I38T rhPCR. The assays were evaluated using cDNAs synthesized from in vitro‐transcribed PA gene RNA controls, RNAs purified from viruses isolated in the 2017/2018 and 2018/2019 influenza seasons, and RNAs purified from clinical specimens collected in the 2018/2019 influenza season.ResultsThe assays developed in this study accurately discriminated PA I38 and PA T38 with high sensitivity.ConclusionsOur assays should be considered a powerful tool for monitoring the emergence of baloxavir‐resistant influenza viruses.

Postpandemic Influenza Mortality of Venovenous Extracorporeal Membrane Oxygenation–Treated Patients in Reunion Island: A Retrospective Single Center Study

The aim of the present study was to assess the post-pandemic mortality of influenza in patients receiving venovenous extracorporeal membrane oxygenation (VV-ECMO) in Reunion Island, France, by comparing the incidence with other patients undergoing VV-ECMO. Retrospective, descriptive, and single-center cohort study. The primary outcome was the standardized mortality ratio for influenza based on the quartiles of the Respiratory Extracorporeal Membrane Oxygenation Survival Prediction score (RESP Score) in the reference population of patients undergoing VV-ECMO. Intensive care unit (ICU), Felix Guyon Hospital, University Teaching Hospital of La Réunion, La Réunion, France. Consecutive patients on ECMO with positive polymerase chain reaction for influenza. None. One hundred twenty-seven consecutive patients were hospitalized in the ICU with a positive influenza polymerase chain reaction from January 2013 to December 2017. Twenty-four influenza patients underwent ECMO including 18 patients with VV-ECMO. During this period, 72 patients requiring VV-ECMO were hospitalized in the ICU. The overall mortality rate of influenza patients on VV-ECMO was 61% versus 46% for non-influenza patients. The standardized mortality ratio per quartile of RESP Score was 1.28 (95% confidence interval 0.61-2.35). In Reunion, the mortality of patients undergoing VV-ECMO for severe influenza is not lower than the expected mortality of all patients undergoing VV-ECMO.