Influenza Virus Polymerase Complex Research Articles

The mechanism of genome replication and transcription in bunyaviruses.

Bunyaviruses are negative sense, single-strand RNA viruses that infect a wide range of vertebrate, invertebrate and plant hosts. WHO lists three bunyavirus diseases as priority diseases requiring urgent development of medical countermeasures highlighting their high epidemic potential. While the viral large (L) protein containing the RNA-dependent RNA polymerase is a key enzyme in the viral replication cycle and therefore a suitable drug target, our knowledge on the structure and activities of this multifunctional protein has, until recently, been very limited. However, in the last few years, facilitated by the technical advances in the field of cryogenic electron microscopy, many structures of bunyavirus L proteins have been solved. These structures significantly enhance our mechanistic understanding of bunyavirus genome replication and transcription processes and highlight differences and commonalities between the L proteins of different bunyavirus families. Here, we provide a review of our current understanding of genome replication and transcription in bunyaviruses with a focus on the viral L protein. Further, we compare within bunyaviruses and with the related influenza virus polymerase complex and highlight open questions.

A natural variant in ANP32B impairs influenza virus replication in human cells.

Viruses require host factors to support their replication, and genetic variation in such factors can affect susceptibility to infectious disease. Influenza virus replication in human cells relies on ANP32 proteins, which are involved in assembly of replication-competent dimeric influenza virus polymerase (FluPol) complexes. Here, we investigate naturally occurring single nucleotide variants (SNV) in the human Anp32A and Anp32B genes. We note that variant rs182096718 in Anp32B is found at a higher frequency than other variants in either gene. This SNV results in a D130A substitution in ANP32B, which is less able to support FluPol activity than wild-type ANP32B and binds FluPol with lower affinity. Interestingly, ANP32B-D130A exerts a dominant negative effect over wild-type ANP32B and interferes with the functionally redundant paralogue ANP32A. FluPol activity and virus replication are attenuated in CRISPR-edited cells expressing wild-type ANP32A and mutant ANP32B-D130A. We propose a model in which the D130A mutation impairs FluPol dimer formation, thus resulting in compromised replication. We suggest that both homozygous and heterozygous carriers of rs182096718 may have some genetic protection against influenza viruses.

The active form of the influenza cap-snatching endonuclease inhibitor baloxavir marboxil is a tight binding inhibitor

Baloxavir marboxil (BXM) is an FDA-approved antiviral prodrug for the treatment of influenza A and B infection and postexposure prophylaxis. The active form, baloxavir acid (BXA), targets the cap-snatching endonuclease (PA) of the influenza virus polymerase complex. The nuclease activity delivers the primer for transcription, and previous reports have shown that BXA blocks the nuclease activity with high potency. However, biochemical studies on the mechanism of action are lacking. Structural data have shown that BXA chelates the two divalent metal ions at the active site, like inhibitors of the human immunodeficiency virus type 1 (HIV-1) integrase or ribonuclease (RNase) H. Here we studied the mechanisms underlying the high potency of BXA and how the I38T mutation confers resistance to the drug. Enzyme kinetics with the recombinant heterotrimeric enzyme (FluB-ht) revealed characteristics of a tight binding inhibitor. The apparent inhibitor constant (Kiapp) is 12 nM, while the I38T mutation increased Kiapp by ∼18-fold. Order-of-addition experiments show that a preformed complex of FluB-ht, Mg2+ ions and BXA is required to observe inhibition, which is consistent with active site binding. Conversely, a preformed complex of FluB-ht and RNA substrate prevents BXA from accessing the active site. Unlike integrase inhibitors that interact with the DNA substrate, BXA behaves like RNase H inhibitors that compete with the nucleic acid at the active site. The collective data support the conclusion that BXA is a tight binding inhibitor and the I38T mutation diminishes these properties.

Synthesis, inhibitory activity and oral dosing formulation of AV5124, the structural analogue of influenza virus endonuclease inhibitor baloxavir.

The development and clinical implementation of the cap-dependent endonuclease (CEN) inhibitor baloxavir marboxil was a breakthrough in influenza therapy, but it was associated with the emergence of drug-resistant variants. To design and synthesize structural analogues of CEN inhibitors and evaluate their safety, pharmacokinetics and antiviral potency in vitro and in vivo. The drug candidate AV5124 and its active metabolite AV5116 were synthesized based on pharmacophore modelling. Stability in plasma and microsomes, plasma protein binding, cytotoxicity and antiviral activities were assessed in vitro. Pharmacokinetics after IV or oral administration were analysed in CD-1 mice. Acute toxicity and protective efficacy against lethal A(H1N1)pdm09 influenza virus challenge were examined in BALB/c mice. Pharmacophore model-assisted, 3D molecular docking predicted key supramolecular interactions of the metal-binding group and bulky hydrophobic group of AV5116 with the CEN binding site (Protein Data Bank code: 6FS6) that are essential for high antiviral activity. AV5116 inhibited influenza virus polymerase complexes in cell-free assays and replication of oseltamivir-susceptible and -resistant influenza A and B viruses at nanomolar concentrations. Notably, AV5116 was equipotent or more potent than baloxavir acid (BXA) against WT (I38-WT) viruses and viruses with reduced BXA susceptibility carrying an I38T polymerase acidic (PA) substitution. AV5116 exhibited low cytotoxicity in Madin-Darby canine kidney cells and lacked mitochondrial toxicity, resulting in favourable selective indices. Treatment with 20 or 50 mg/kg AV5124 prevented death in 60% and 100% of animals, respectively. Overall, AV5124 and A5116 are promising inhibitors of the influenza virus CEN and warrant further development as potent anti-influenza agents.

Baloxavir Marboxil: A Review in Acute Uncomplicated Influenza.

Baloxavir marboxil (Xofluza®; hereafter referred to as baloxavir), the prodrug of baloxavir acid, is a first-in-class, small molecule inhibitor of the polymerase acidic (PA) protein subunit of the influenza virus polymerase complex. Baloxavir (after conversion to baloxavir acid) acts to block influenza virus replication by inhibiting the cap-dependent endonuclease activity of the PA protein. Taken orally as a single dose, baloxavir is approved in the USA for the treatment of acute uncomplicated influenza in patients ≥12years of age who have been symptomatic for ≤48h. Data from randomized, double-blind, placebo- and oseltamivir-controlled phaseIII trials have shown that baloxavir is efficacious in improving influenza symptoms both in otherwise healthy adolescents and adults and in those at high risk of influenza complications, displaying similar efficacy to that of oseltamivir. Furthermore, there is evidence that baloxavir can reduce influenza viral load more rapidly than oseltamivir. Baloxavir has activity against influenzaA and B viruses (including strains resistant to neuraminidase inhibitors) and is well tolerated. Evidence of the emergence and likely human-to-human transmission of variant viruses with reduced susceptibility to baloxavir highlights the importance of monitoring and surveillance for changes in influenza virus drug susceptibility patterns. However, currently available evidence suggests that baloxavir, with the benefits of a single oral dose regimen, provides a useful alternative to neuraminidase inhibitors for the treatment of acute uncomplicated influenza in adolescents and adults.

Focusing on the Influenza Virus Polymerase Complex: Recent Progress in Drug Discovery and Assay Development.

Influenza viruses are severe human pathogens that pose persistent threat to public health. Each year more people die of influenza virus infection than that of breast cancer. Due to the limited efficacy associated with current influenza vaccines, as well as emerging drug resistance from small molecule antiviral drugs, there is a clear need to develop new antivirals with novel mechanisms of action. The influenza virus polymerase complex has become a promising target for the development of the next-generation of antivirals for several reasons. Firstly, the influenza virus polymerase, which forms a heterotrimeric complex that consists of PA, PB1, and PB2 subunits, is highly conserved. Secondly, both individual polymerase subunit (PA, PB1, and PB2) and inter-subunit interactions (PA-PB1, PB1- PB2) represent promising drug targets. Lastly, growing insight into the structure and function of the polymerase complex has spearheaded the structure-guided design of new polymerase inhibitors. In this review, we highlight recent progress in drug discovery and assay development targeting the influenza virus polymerase complex and discuss their therapeutic potentials.

Structure of a functional cap-binding domain in Rift Valley fever virus L protein.

Rift Valley fever virus (RVFV) belongs to the family of Phenuiviridae within the order of Bunyavirales. The virus may cause fatal disease both in livestock and humans, and therefore, is of great economical and public health relevance. In analogy to the influenza virus polymerase complex, the bunyavirus L protein is assumed to bind to and cleave off cap structures of cellular mRNAs to prime viral transcription. However, even though the presence of an endonuclease in the N-terminal domain of the L protein has been demonstrated for several bunyaviruses, there is no evidence for a cap-binding site within the L protein. We solved the structure of a C-terminal 117 amino acid-long domain of the RVFV L protein by X-ray crystallography. The overall fold of the domain shows high similarity to influenza virus PB2 cap-binding domain and the putative non-functional cap-binding domain of reptarenaviruses. Upon co-crystallization with m7GTP, we detected the cap-analogue bound between two aromatic side chains as it has been described for other cap-binding proteins. We observed weak but specific interaction with m7GTP rather than GTP in vitro using isothermal titration calorimetry. The importance of m7GTP-binding residues for viral transcription was validated using a RVFV minigenome system. In summary, we provide structural and functional evidence for a cap-binding site located within the L protein of a virus from the Bunyavirales order.

Multiple polymerase gene mutations for human adaptation occurring in Asian H5N1 influenza virus clinical isolates

The role of the influenza virus polymerase complex in host range restriction has been well-studied and several host range determinants, such as the polymerase PB2-E627K and PB2-D701N mutations, have been identified. However, there may be additional, currently unknown, human adaptation polymerase mutations. Here, we used a database search of influenza virus H5N1 clade 1.1, clade 2.3.2.1 and clade 2.3.4 strains isolated from 2008–2012 in Southern China, Vietnam and Cambodia to identify polymerase adaptation mutations that had been selected in infected patients. Several of these mutations acted either alone or together to increase viral polymerase activity in human airway cells to levels similar to the PB2-D701N and PB2-E627K single mutations and to increase progeny virus yields in infected mouse lungs to levels similar to the PB2-D701N single mutation. In particular, specific mutations acted synergistically with the PB2-D701N mutation and showed synergistic effects on viral replication both in human airway cells and mice compared with the corresponding single mutations. Thus, H5N1 viruses in infected patients were able to acquire multiple polymerase mutations that acted cooperatively for human adaptation. Our findings give new insight into the human adaptation of AI viruses and help in avian influenza virus risk assessment.

The amyloidogenicity of the influenza virus PB1-derived peptide sheds light on its antiviral activity

The influenza virus polymerase complex is a promising target for new antiviral drug development. It is known that, within the influenza virus polymerase complex, the PB1 subunit region from the 1st to the 25th amino acid residues has to be is in an alpha-helical conformation for proper interaction with the PA subunit. We have previously shown that PB1(6–13) peptide at low concentrations is able to interact with the PB1 subunit N-terminal region in a peptide model which shows aggregate formation and antiviral activity in cell cultures.In this paper, it was shown that PB1(6–13) peptide is prone to form the amyloid-like fibrillar aggregates. The peptide homo-oligomerization kinetics were examined, and the affinity and characteristic interaction time of PB1(6–13) peptide monomers and the influenza virus polymerase complex PB1 subunit N-terminal region were evaluated by the SPR and TR-SAXS methods. Based on the data obtained, a hypothesis about the PB1(6–13) peptide mechanism of action was proposed: the peptide in its monomeric form is capable of altering the conformation of the PB1 subunit N-terminal region, causing a change from an alpha helix to a beta structure. This conformational change disrupts PB1 and PA subunit interaction and, by that mechanism, the peptide displays antiviral activity.

Role of the PB1 protein in the fidelity of the influenza virus polymerase complex

Role of the PB1 protein in the fidelity of the influenza virus polymerase complex

Role of the PB1 protein in the influenza virus polymerase complex fidelity

Role of the PB1 protein in the influenza virus polymerase complex fidelity

Screening for Novel Small-Molecule Inhibitors Targeting the Assembly of Influenza Virus Polymerase Complex by a Bimolecular Luminescence Complementation-Based Reporter System.

Influenza virus RNA-dependent RNA polymerase consists of three viral protein subunits: PA, PB1, and PB2. Protein-protein interactions (PPIs) of these subunits play pivotal roles in assembling the functional polymerase complex, which is essential for the replication and transcription of influenza virus RNA. Here we developed a highly specific and robust bimolecular luminescence complementation (BiLC) reporter system to facilitate the investigation of influenza virus polymerase complex formation. Furthermore, by combining computational modeling and the BiLC reporter assay, we identified several novel small-molecule compounds that selectively inhibited PB1-PB2 interaction. Function of one such lead compound was confirmed by its activity in suppressing influenza virus replication. In addition, our studies also revealed that PA plays a critical role in enhancing interactions between PB1 and PB2, which could be important in targeting sites for anti-influenza intervention. Collectively, these findings not only aid the development of novel inhibitors targeting the formation of influenza virus polymerase complex but also present a new tool to investigate the exquisite mechanism of PPIs. IMPORTANCE Formation of the functional influenza virus polymerase involves complex protein-protein interactions (PPIs) of PA, PB1, and PB2 subunits. In this work, we developed a novel BiLC assay system which is sensitive and specific to quantify both strong and weak PPIs between influenza virus polymerase subunits. More importantly, by combining in silico modeling and our BiLC assay, we identified a small molecule that can suppress influenza virus replication by disrupting the polymerase assembly. Thus, we developed an innovative method to investigate PPIs of multisubunit complexes effectively and to identify new molecules inhibiting influenza virus polymerase assembly.

The Influenza Virus Polymerase Complex: An Update on Its Structure, Functions, and Significance for Antiviral Drug Design.

Influenza viruses cause seasonal epidemics and pandemic outbreaks associated with significant morbidity and mortality, and a huge cost. Since resistance to the existing anti‐influenza drugs is rising, innovative inhibitors with a different mode of action are urgently needed. The influenza polymerase complex is widely recognized as a key drug target, given its critical role in virus replication and high degree of conservation among influenza A (of human or zoonotic origin) and B viruses. We here review the major progress that has been made in recent years in unravelling the structure and functions of this protein complex, enabling structure‐aided drug design toward the core regions of the PA endonuclease, PB1 polymerase, or cap‐binding PB2 subunit. Alternatively, inhibitors may target a protein–protein interaction site, a cellular factor involved in viral RNA synthesis, the viral RNA itself, or the nucleoprotein component of the viral ribonucleoprotein. The latest advances made for these diverse pharmacological targets have yielded agents in advanced (i.e., favipiravir and VX‐787) or early clinical testing, besides several experimental inhibitors in various stages of development, which are all covered here.

Influenza Virus and Chromatin: Role of the CHD1 Chromatin Remodeler in the Virus Life Cycle.

Influenza A virus requires ongoing cellular transcription to carry out the cap-snatching process. Chromatin remodelers modify chromatin structure to produce an active or inactive conformation, which enables or prevents the recruitment of transcriptional complexes to specific genes; viral transcription thus depends on chromatin dynamics. Influenza virus polymerase associates with chromatin components of the infected cell, such as RNA polymerase II (RNAP II) or the CHD6 chromatin remodeler. Here we show that another CHD family member, CHD1 protein, also interacts with the influenza virus polymerase complex. CHD1 recognizes the H3K4me3 (histone 3 with a trimethyl group in lysine 4) histone modification, a hallmark of active chromatin. Downregulation of CHD1 causes a reduction in viral polymerase activity, viral RNA transcription, and the production of infectious particles. Despite the dependence of influenza virus on cellular transcription, RNAP II is degraded when viral transcription is complete, and recombinant viruses unable to degrade RNAP II show decreased pathogenicity in the murine model. We describe the CHD1-RNAP II association, as well as the parallel degradation of both proteins during infection with viruses showing full or reduced induction of degradation. The H3K4me3 histone mark also decreased during influenza virus infection, whereas a histone mark of inactive chromatin, H3K27me3, remained unchanged. Our results indicate that CHD1 is a positive regulator of influenza virus multiplication and suggest a role for chromatin remodeling in the control of the influenza virus life cycle. Although influenza virus is not integrated into the genome of the infected cell, it needs continuous cellular transcription to synthesize viral mRNA. This mechanism implies functional association with host genome expression and thus depends on chromatin dynamics. Influenza virus polymerase associates with transcription-related factors, such as RNA polymerase II, and with chromatin remodelers, such as CHD6. We identified the association of viral polymerase with another chromatin remodeler, the CHD1 protein, which positively modulated viral polymerase activity, viral RNA transcription, and virus multiplication. Once viral transcription is complete, RNAP II is degraded in infected cells, probably as a virus-induced mechanism to reduce the antiviral response. CHD1 associated with RNAP II and paralleled its degradation during infection with viruses that induce full or reduced degradation. These findings suggest that RNAP II degradation and CHD1 degradation cooperate to reduce the antiviral response.

Identification of a small-molecule inhibitor of influenza virus via disrupting the subunits interaction of the viral polymerase

Assembly of the heterotrimeric influenza virus polymerase complex from the individual subunits PB1, PA, and PB2 is a prerequisite for viral replication, in which the interaction between the C terminal of PA (PAC) and the N-terminal of PB1 (PB1N) may be a desired target for antiviral development. In this study, we compared the feasibility of high throughput screening by enzyme-linked immunosorbent assay (ELISA) and fluorescence polarization assay. Among the two, ELISA was demonstrated to own broader dynamic range so that it was used for screening inhibitors that blocked PAC and PB1N interaction. Several binding inhibitors of PAC-PB1N were identified and subsequently tested for the antiviral efficacy. Apparently, 3-(2-chlorophenyl)-6-ethyl-7-methyl[1,2,4]triazolo[4,3-a]pyrimidin-5-ol, designated ANA-1, was found to be a strong inhibitor of viral polymerase activity and act as a potent antiviral agent against the infections of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 subtypes, in cell cultures. Intranasal administration of ANA-1 protected mice from lethal challenge and reduced lung viral loads in H1N1 virus infected BALB/c mice. Docking analyses predicted that ANA-1 bound to an allosteric site of PAC, which might cause conformational changes thereby disrupting the PAC-PB1N interaction. Overall, our study has identified a novel compound with potential to be developed as an anti-influenza drug.

Identification of mammalian-adapting mutations in the polymerase complex of an avian H5N1 influenza virus.

Avian influenza viruses of the H5N1 subtype pose a serious global health threat due to the high mortality (>60%) associated with the disease caused by these viruses and the lack of protective antibodies to these viruses in the general population. The factors that enable avian H5N1 influenza viruses to replicate in humans are not completely understood. Here we use a high-throughput screening approach to identify novel mutations in the polymerase genes of an avian H5N1 virus that confer efficient polymerase activity in mammalian cells. Several of the identified mutations (which have previously been found in natural isolates) increase viral replication in mammalian cells and virulence in infected mice compared with the wild-type virus. The identification of amino-acid mutations in avian H5N1 influenza virus polymerase complexes that confer increased replication and virulence in mammals is important for the identification of circulating H5N1 viruses with an increased potential to infect humans.Supplementary informationThe online version of this article (doi:10.1038/ncomms8491) contains supplementary material, which is available to authorized users.

Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity

The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.

Molecular determinants of pathogenicity in the polymerase complex.

Viral pathogenesis involves numerous interactions between viral and cellular factors. In recent years, the influenza virus polymerase complex has emerged as a major determinant of interspecies transmission and pathogenicity. The viral RNA-dependent RNA polymerase, in concert with the nucleoprotein, mediates transcription and replication of the viral RNA genome in the nucleus of the infected cell. The activity by which the viral polymerase complex performs these processes in mammalian cells is considered to be a major contributor to viral pathogenicity in mammals. In this chapter, we summarise our current understanding on the pathogenicity determinants in the viral polymerase complex and highlight some of its cellular interaction partners. We particularly discuss the role of importin-α isoforms in host adaptation and pathogenesis as well as the role of the viral polymerase in regulating cellular responses to viral infection.

CHD6, a Cellular Repressor of Influenza Virus Replication, Is Degraded in Human Alveolar Epithelial Cells and Mice Lungs during Infection

The influenza virus polymerase associates to an important number of transcription-related proteins, including the largest subunit of the RNA polymerase II complex (RNAP II). Despite this association, degradation of the RNAP II takes place in the infected cells once viral transcription is completed. We have previously shown that the chromatin remodeler CHD6 protein interacts with the influenza virus polymerase complex, represses viral replication, and relocalizes to inactive chromatin during influenza virus infection. In this paper, we report that CHD6 acts as a negative modulator of the influenza virus polymerase activity and is also subjected to degradation through a process that includes the following characteristics: (i) the cellular proteasome is not implicated, (ii) the sole expression of the three viral polymerase subunits from its cloned cDNAs is sufficient to induce proteolysis, and (iii) degradation is also observed in vivo in lungs of infected mice and correlates with the increase of viral titers in the lungs. Collectively, the data indicate that CHD6 degradation is a general effect exerted by influenza A viruses and suggest that this viral repressor may play an important inhibitory role since degradation and accumulation into inactive chromatin occur during the infection.

Effect of Envelope Proteins on the Mechanical Properties of Influenza Virus

The envelope of the influenza virus undergoes extensive structural change during the viral life cycle. However, it is unknown how lipid and protein components of the viral envelope contribute to its mechanical properties. Using atomic force microscopy, here we show that the lipid envelope of spherical influenza virions is ∼10 times softer (∼0.05 nanonewton nm(-1)) than a viral protein-capsid coat and sustains deformations of one-third of the virion's diameter. Compared with phosphatidylcholine liposomes, it is twice as stiff, due to membrane-attached protein components. We found that virus indentation resulted in a biphasic force-indentation response. We propose that the first phase, including a stepwise reduction in stiffness at ∼10-nm indentation and ∼100 piconewtons of force, is due to mobilization of membrane proteins by the indenting atomic force microscope tip, consistent with the glycoprotein ectodomains protruding ∼13 nm from the bilayer surface. This phase was obliterated for bromelain-treated virions with the ectodomains removed. Following pH 5 treatment, virions were as soft as pure liposomes, consistent with reinforcing proteins detaching from the lipid bilayer. We propose that the soft, pH-dependent mechanical properties of the envelope are critical for the pH-regulated life cycle and support the persistence of the virus inside and outside the host.