Anti-influenza Drugs Research Articles

Interaction of Influenza A Nucleoprotein with Host hnRNP-C Is Implicated in Viral Replication.

The host interactome of influenza viral proteins is ever-expanding. In this work, we report the identification of host heterogeneous nuclear ribonucleoprotein C (hnRNP-C) as an interacting partner of influenza A virus nucleoprotein (NP). We confirmed that this interaction exists across different influenza A subtypes and strains. Using biochemical methods, we determined that hnRNP-C interacts with NP via its C-terminal auxiliary domain. Further, we determined that the hnRNP-C is a negative regulator of influenza viral growth. Its interaction with NP is implicated in the promotion of host cell apoptosis during viral infection. It is the first time that the interaction between influenza nucleoprotein and host heterogeneous nuclear ribonucleoprotein C is characterized in detail. Overall, these findings not only characterize the interaction between NP and its host interacting partner hnRNP-C but also clarify the functional significance of this interaction. This work may lead to a new therapeutic target for the development of anti-influenza drugs.

Antiviral Potential of Natural Resources against Influenza Virus Infections.

Influenza is a severe contagious disease caused by influenza A and B viruses. The WHO estimates that annual outbreaks lead to 3-5 million severe infections of which approximately 10% lead to the death of the patient. While vaccination is the cornerstone of prevention, antiviral drugs represent the most important treatment option of acute infections. Only two classes of drugs are currently approved for the treatment of influenza in numerous countries: M2 channel blockers and neuraminidase inhibitors. In some countries, additional compounds such as the recently developed cap-dependent endonuclease inhibitor baloxavir marboxil or the polymerase inhibitor favipiravir are available. However, many of these compounds suffer from poor efficacy, if not applied early after infection. Furthermore, many influenza strains have developed resistances and lost susceptibility to these compounds. As a result, there is an urgent need to develop new anti-influenza drugs against a broad spectrum of subtypes. Natural products have made an important contribution to the development of new lead structures, particularly in the field of infectious diseases. Therefore, this article aims to review the research on the identification of novel lead structures isolated from natural resources suitable to treat influenza infections.

In Silico Targeting of influenza virus haemagglutinin receptor protein using Diosmetin, Tangeritin, and Anthocyanidins as potential drugs

Influenza viruses cause acute respiratory illnesses in birds, humans, and other mammals, and are a major public health concern around the world. Pandemic flu could be caused by an unforeseen human adaptation of an influenza subtype or strain rather than currently circulating influenza viruses. The need for plant metabolites-based new anti-influenza drugs appears to be urgent. Blocking Haemeagglutinin (HA) protein is one of the most appealing drug targets to halt the growth of the virus. The influenza virus can acquire resistance to currently existing therapies, therefore necessitating the development of new medications. The plant's bioactive metabolites, flavanoids are having potential medicinal efficacy. The current study aimed to identify certain flavonoids (Diosmetin, Tangeritin, and Anthocyanidins) that might interact with the HA protein of the influenza virus and help in inhibiting its growth. We used PyRx v0.8 for virtual screening and docking studies. The highest binding affinity docked structures were analyzed using PyMOL and Discovery Studio Visualizer. The present study revealed that these naturally occurring compounds interacted with HA protein, resulting in the minimization of energy in the range of -5.2 to -7.0 kcal/mol. Diosmetin showed the best binding affinity of -7.0Kcal/mol. The molecular binding studies revealed that Diosmetin, Tangeritin, and Anthocyanidins are potential compounds to test against HA protein and can be used to develop effective anti-influenza agents.

A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

ABSTRACT The quadrilateral reassortant IAV A/(H1N1) pdm09 is the pathogen responsible for the first influenza pandemic of the 21st century. The virus spread rapidly among hosts causing high mortality within human population. Efficient accumulation of virions is known to be important for the rapid transmission of virus. However, the mechanism by which A/(H1N1) pdm09 promotes its rapid replication has not been fully studied. Here, we found the NS1 of A/(H1N1) pdm09 mediated complete macroautophagy/autophagy, and then facilitated self-replication, which may be associated with the more rapid spread of this virus compared with H1N1WSN and H3N8JL89. We found that the promotion of self-replication could be mainly attributed to NS1pdm09 strongly antagonizing the inhibitory effect of LRPPRC on autophagy. The interaction between NS1pdm09 and LRPPRC competitively blocked the interaction of LRPPRC with BECN1/Beclin1, resulting in increased recruitment of BECN1 for PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3) and induction of the initiation of autophagy. In conclusion, we uncover the unique molecular mechanism by which A/(H1N1) pdm09 utilizes autophagy to promote self-replication, and we provide theoretical basics for the analysis of the etiological characteristics of the A/(H1N1) pdm09 pandemic and the development of anti-influenza drugs and vaccines. Abbreviations: 293T: human embryonic kidney 293 cells; 293T_LRPPRC: stable LRPPRC expression 293T cells; 3-MA: 3-methyladenine; A549 cells: human non-small cell lung cancer cells; AA: amino acid; ACTB: actin beta; BECN1: beclin 1; BECN1 KO: BECN1 knockout 293T cells; Cal: calyculin A; Co-IP: co-immunoprecipitation; CQ: chloroquine; DC: dendritic cell; Eug: eugenol; GFP: green fluorescent protein; HA: hemagglutinin; HIV: human immunodeficiency virus; IAVs: Influenza A viruses; IFN: interferon; JL89: A/equine/Jilin/1/1989 (H3N8); LAMP2: lysosomal associated membrane protein 2; LRPPRC: leucine rich pentatriicopeptide repeat containing; LRPPRC KO: LRPPRC knockout 293T cells; M2: matrix 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MDCK: Madin-Darby canine kidney cells; MOI: multiplicity of infection; MS: mass spectrometry; NP: nucleoprotein; NS1: non-structural protein 1; NS1JL89: non-structural protein 1 of A/equine/Jilin/1/1989 (H3N8); NS1pdm09: non-structural protein 1 of A/(H1N1) pdm09; NS1SC09: non-structural protein 1 of A/Sichuan/2009 (H1N1); NS1WSN: non-structural protein 1 of A/WSN/1933 (H1N1); PB1: polymerase basic protein 1; PB1-F2: alternate reading frame discovered in PB1 gene segment; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PR8: A/PR/8/34 (H1N1); Rapa: rapamycin; RFP: red fluorescent protein; SC09: A/Sichuan/2009 (H1N1); SQSTM1/p62: sequestosome 1; STK4/MST1: serine/threonine kinase 4; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20; WHO: World Health Organization; WSN: A/WSN/1933 (H1N1); WSN-NS1JL89: WSN recombinant strain in which NS1 was replaced with that of JL89; WSN-NS1SC09: WSN recombinant strain in which NS1 was replaced with that of SC09.

Sialidase Inhibitors with Different Mechanisms.

Sialidases, or neuraminidases, are enzymes that catalyze the hydrolysis of sialic acid (Sia)-containing molecules, mostly removal of the terminal Sia (desialylation). By desialylation, sialidase can modulate the functionality of the target compound and is thus often involved in biological pathways. Inhibition of sialidases with inhibitors is an important approach for understanding sialidase function and the underlying mechanisms and could serve as a therapeutic approach as well. Transition-state analogues, such as anti-influenza drugs oseltamivir and zanamivir, are major sialidase inhibitors. In addition, difluoro-sialic acids were developed as mechanism-based sialidase inhibitors. Further, fluorinated quinone methide-based suicide substrates were reported. Sialidase product analogue inhibitors were also explored. Finally, natural products have shown competitive inhibiton against viral, bacterial, and human sialidases. This Perspective describes sialidase inhibitors with different mechanisms and their activities and future potential, which include transition-state analogue inhibitors, mechanism-based inhibitors, suicide substrate inhibitors, product analogue inhibitors, and natural product inhibitors.

Physiochemical characterisation of drugs and related leads for swine flu

Influenza viruses have acquired resistance to approved neuraminidase-targeting drugs, increasing the need for new drug targets for the development of novel anti-influenza drugs. The control of influenza virus infection is a major public health concern due to the significant morbidity and mortality it causes through seasonal epidemics and pandemics. I try to find those drugs which are based on Lipinski rule for this. we used data for the collect information in the study and abstracted the following databases, which is PubChem and drug bank. I retrieved 20 drug of swine flu from drug bank and retrieve the similar drug from Pubchem database and perform clustering of drug to group than on the basis of chemical properties and further observe that which drugs are follow the Lipinski rule which is based on in-silico method. Further, the selected drug structure was docked with chemical compounds using AutoDock1.5.6. This study also highlights stronger binding energy having good ADMET property with H1N1 drugs.

Evolution of Influenza Viruses-Drug Resistance, Treatment Options, and Prospects.

Viral evolution refers to the genetic changes that a virus accumulates during its lifetime which can arise from adaptations in response to environmental changes or the immune response of the host. Influenza A virus is one of the most rapidly evolving microorganisms. Its genetic instability may lead to large changes in its biological properties, including changes in virulence, adaptation to new hosts, and even the emergence of infectious diseases with a previously unknown clinical course. Genetic variability makes it difficult to implement effective prophylactic programs, such as vaccinations, and may be responsible for resistance to antiviral drugs. The aim of the review was to describe the consequences of the variability of influenza viruses, mutations, and recombination, which allow viruses to overcome species barriers, causing epidemics and pandemics. Another consequence of influenza virus evolution is the risk of the resistance to antiviral drugs. Thus far, one class of drugs, M2 protein inhibitors, has been excluded from use because of mutations in strains isolated in many regions of the world from humans and animals. Therefore, the effectiveness of anti-influenza drugs should be continuously monitored in reference centers representing particular regions of the world as a part of epidemiological surveillance.

Antiviral activity of soybean GL 2626/96 (Glycine max) ethanolic extract against influenza A virus in vitro and in vivo

Influenza viruses cause respiratory infections in humans with high morbidity and mortality rates. Neuraminidase inhibitors such as oseltamivir and peramivir are the most commonly used drugs for influenza virus infections. However, the emergence of resistant viruses necessitates the urgent need to develop next-generation anti-influenza drugs. Soybean (Glycine max L. Merr.) is widely cultivated and used as food worldwide. In addition, soybean has long been used as a nutritional supplement and herbal medicine. However, the potential anti-influenza properties of the soybean cultivar “GL 2626/96″ (SG2626) are yet to be investigated. Herein, we determined whether the ethanolic extract of SG2626 (SG2626E) has anti-viral activity through performing SG2626E pre-, co-, and post-treatment assays, using the influenza green fluorescent protein (GFP)-tagged influenza A/PR/8/34 (A/PR/8/34-GFP) virus. SG2626E showed anti-influenza virus activity in pre- and co-treated cells in a dose-dependent manner, but not in post-treated cells. SG2626E imparted a considerable inhibitory effect on influenza A virus (IAV) infection through blocking viral attachment. SG2626E inhibited the activity of viral hemagglutinin, but not viral neuraminidase of the IAV. SG2626E inhibited IAV infection by reducing intracellular calcium levels in infected human lung epithelial A549 cells. Additionally, SG2626E reduced body weight loss, decreased mortality, and increased the survival rate through reducing viral replication in the lungs of IAV-infected mice. Overall, these results suggest that SG2626E inhibits IAV infection and is a potential novel anti-influenza agent.

Identification of HNH endonuclease domain in the basic protein 2 subunit of the polymerase of human influenza viruses

RNA polymerases of human influenza viruses A, B and C do not have a capping enzyme, as other RNA viruses, to cap their mRNAs for translation in the host cells. So, they employ a unique ‘cap-snatching’ mechanism, where the mRNA cap structures are snatched from the host cell mRNAs and used as a primer to initiate its own mRNA synthesis. One of the RNA polymerase subunits, the polymerase basic protein subunit 2 (PB2), is shown to involve in the ‘cap-snatching’ mechanism. The active sites for the ‘cap-snatching’ and endonuclease by the PB2 were analyzed by multiple sequence alignment (MSA) analysis and corroborated with the results available from biochemical, site-directed mutagenesis (SDM) and X-ray crystallographic techniques. It is found that the PB2 subunit in all three human influenza viruses habours both the cap-binding motif (CBM) and a HNH/N type endonuclease domain. The CBM is aromatic amino acid rich and the HNH/N is a –DH- based endonuclease in influenza viruses A and C and a –DQ- based one in influenza virus B. The invariant H is proposed to act as a general base to initiate catalysis and the invariant first N is implicated in nucleotide binding. In addition, the nuclear localization signals were also identified in all three human influenza viruses. By sequence similarity, similar HNH/N domain are found in the RNA cleaving CRISPR-Cas13a/13b and CRISPR-Cas12a endoribonucleases. The identification of HNH/N domain in all three human influenza viruses suggests that the PB2 subunit itself could cleave the cap structures from the host cell mRNAs, which are subsequently used as primers to initiate viral mRNA synthesis. These results will facilitate the optimization of endonuclease inhibitors as potential new anti-influenza drugs, and could also help in developing new drugs for flu treatments in the future.

Genetic Evolution of Avian Influenza A (H9N2) Viruses Isolated from Domestic Poultry in Uganda Reveals Evidence of Mammalian Host Adaptation, Increased Virulence and Reduced Sensitivity to Baloxavir.

A (H9N2) avian influenza A viruses were first detected in Uganda in 2017 and have since established themselves in live bird markets. The aim of this study was to establish the subsequent genetic evolution of H9N2 viruses in Uganda. Cloacal samples collected from live bird market stalls in Kampala from 2017 to 2019 were screened by RT-PCR for influenza A virus and H9N2 viruses were isolated in embryonated eggs. One hundred and fifty H9N2 isolates were subjected to whole genome sequencing on the Illumina MiSeq platform. The sequence data analysis and comparison with contemporary isolates revealed that the virus was first introduced into Uganda in 2014 from ancestors in the Middle East. There has since been an increase in nucleotide substitutions and reassortments among the viruses within and between live bird markets, leading to variations in phylogeny of the different segments, although overall diversity remained low. The isolates had several mutations such as HA-Q226L and NS-I106M that enable mammalian host adaptation, NP-M105V, PB1-D3V, and M1-T215A known for increased virulence/pathogenicity and replication, and PA-E199D, NS-P42S, and M2-S31N that promote drug resistance. The PA-E199D substitution in particular confers resistance to the endonuclease inhibitor Baloxavir acid, which is one of the new anti-influenza drugs. Higher EC50 was observed in isolates with a double F105L+E199D substitution that may suggest a possible synergistic effect. These H9N2 viruses have established an endemic situation in live bird markets in Uganda because of poor biosecurity practices and therefore pose a zoonotic threat. Regular surveillance is necessary to further generate the needed evidence for effective control strategies and to minimize the threats.

Low prevalence of influenza A strains with resistance markers in Brazil during 2017-2019 seasons.

The influenza A virus (IAV) is of a major public health concern as it causes annual epidemics and has the potential to cause pandemics. At present, the neuraminidase inhibitors (NAIs) are the most widely used anti-influenza drugs, but, more recently, the drug baloxavir marboxil (BXM), a polymerase inhibitor, has also been licensed in some countries. Mutations in the viral genes that encode the antiviral targets can lead to treatment resistance. Worldwide, a low prevalence of antiviral resistant strains has been reported. Despite that, this situation can change rapidly, and resistant strain surveillance is a priority. Thus, the aim of this was to evaluate Brazilian IAVs antiviral resistance from 2017 to 2019 through the identification of viral mutations associated with reduced inhibition of the drugs and by testing the susceptibility of IAV isolates to oseltamivir (OST), the most widely used NAI drug in the country. Initially, we analyzed 282 influenza A(H1N1)pdm09 and 455 A(H3N2) genetic sequences available on GISAID. The amino acid substitution (AAS) NA:S247N was detected in one A(H1N1)pdm09 strain. We also identified NA:I222V (n = 6) and NA:N329K (n = 1) in A(H3N2) strains. In addition, we performed a molecular screening for NA:H275Y in 437 A(H1N1)pdm09 samples, by pyrosequencing, which revealed a single virus harboring this mutation. Furthermore, the determination of OST IC50 values for 222 A(H1N1)pdm09 and 83 A(H3N2) isolates revealed that all isolates presented a normal susceptibility profile to the drug. Interestingly, we detected one A(H3N2) virus presenting with PA:E119D AAS. Moreover, the majority of the IAV sequences had the M2:S31N adamantanes resistant marker. In conclusion, we show a low prevalence of Brazilian IAV strains with NAI resistance markers, in accordance with what is reported worldwide, indicating that NAIs still remain an option for the treatment of influenza infections in Brazil. However, surveillance of influenza resistance should be strengthened in the country for improving the representativeness of investigated viruses and the robustness of the analysis.

Broadly neutralizing antibodies recognizing different antigenic epitopes act synergistically against the influenza B virus.

The discovery of broadly neutralizing monoclonal antibodies against influenza viruses has raised hope for the successful development of new antiviral drugs. However, due to the speed and variety of mutations in influenza viruses, single-component antibodies that recognize specific epitopes are susceptible to viral escape and have limited efficacy when administration is delayed. Hence, it is necessary to develop alternative strategies with better antiviral activity. Influenza B virus infection can cause severe illness in children and the elderly. Commonly used anti-influenza drugs have low clinical efficacy against influenza B virus. In this study, we investigated the antiviral efficacy of combinations of representative monoclonal antibodies targeting different antigenic epitopes against the influenza B virus. We found that combinations of antibodies recognizing the hemagglutinin (HA) head and stem regions showed a stronger neutralizing activity than single antibodies and other antibody combinations in vitro. In addition, we found that pair-wise combinations of antibodies recognizing the HA head region, HA stem region, and neuraminidase enzyme-activated region showed superior antiviral activity than single antibodies in both mouse and ferret in vivo protection assays. Notably, these antibody combinations still displayed good antiviral efficacy when treatment was delayed. Mechanistic studies further revealed that combining antibodies recognizing different epitope regions resulted in extremely strong antibody-dependent cell-mediated cytotoxicity, which may partly explain their superior antiviral effects. Together, the findings of this study provide new avenues for the development of better antiviral drugs and vaccines against influenza viruses.

The Influence of Influenza Virus Infections in Patients with Chronic Obstructive Pulmonary Disease.

IntroductionChronic obstructive pulmonary disease (COPD) is a common disease and is preventable and treatable. A previous study showed that influenza virus infections were also associated with the risk of acute exacerbation in patients with COPD, and other studies showed that the influenza virus might increase the risk of stroke. However, studies on the influence of influenza infection among COPD patients are limited. In this study, we review the role of influenza infection in contributing to mortality, pneumonia, respiratory failure, COPD acute exacerbation, and ischemic stroke among COPD patients.Materials and MethodsWe performed a population-based cohort study of COPD patients using data from Taiwan between January 1, 2011, and December 31, 2019. We excluded patients with lung cancer, lung transplantation and asthma. We also excluded patients who lacked COPD medication prescriptions and those treated with anti-influenza drugs without flu diagnosis records. Patients with missing or incomplete data were also excluded from the study cohort.ResultsAfter 1:1 matching by age, sex, COPD duration, diagnosed years and comorbidities, we enrolled 10,855 cases and controls for further analysis. The risks of pneumonia, respiratory failure, COPD acute exacerbation, and ischemic stroke were 1.770 (95% CI=1.638–1.860; P<0.0001), 1.097 (95% CI=1.008–1.194; P=0.0319), 1.338 (95% CI=1.248–1.435; P<0.0001), and 1.134 (95% CI=1.039–1.239, P=0.0051), respectively, in the influenza infection group compared with COPD patients without influenza infection.ConclusionInfluenza infections are linked to an increased risk of ischemic stroke, pneumonia, respiratory failure, and COPD acute exacerbation among COPD patients. In conclusion, patients with COPD need to be closely monitored after having an influenza infection.

Enhancing neutralizing activity against influenza H1N1/PR8 by engineering a single-domain VL-M2 specific into a bivalent form.

Flu disease, with high mortality and morbidity, is caused by the influenza virus. Influenza infections are most effectively prevented through vaccination, but it requires annual reformulation due to the antigenic shift or drift of hemagglutinin and neuraminidase proteins. Increasing resistance to available anti-influenza drugs was also recently reported. The M2 surface protein of the influenza virus is an attractive target for universal vaccine development as it is highly conserved and multifunctional throughout the viral life cycle. This study aimed to discover a single-chain variable fragment (scFv) targeting the M2 protein of influenza A H1N1/PR8, showing neutralizing activity through plaque inhibition in virus replication. Several candidates were isolated using bio-panning, including scFv and single-domain VL target M2 protein, which was displayed on the yeast surface. The scFv/VL proteins were obtained with high yield and high purity through soluble expression in E. coli BL21 (DE3) pLysE strains. A single-domain VL-M2-specific antibody, NVLM10, exhibited the highest binding affinity to influenza virions and was engineered into a bivalent format (NVL2M10) to improve antigen binding. Both antibodies inhibited virus replication in a dose-dependent manner, determined using plaque reduction- and immunocytochemistry assays. Furthermore, bivalent anti-M2 single-domain VL antibodies significantly reduced the plaque number and viral HA protein intensity as well as viral genome (HA and NP) compared to the monovalent single-domain VL antibodies. This suggests that mono- or bivalent single-domain VL antibodies can exhibit neutralizing activity against influenza virus A, as determined through binding to virus particle activity.

Insights into binding molecular mechanism of hemagglutinin H3N2 of influenza virus complexed with arbidol and its derivative: A molecular dynamics simulation perspective

Recently, the H3N2 influenza outbreak has caused serious global public health concern for future control of the next influenza pandemic. Since using current anti-influenza drugs targeting neuraminidase (oseltamivir and zanamivir) and the proton M2 channel (amantadine and rimantadine) leads to drug resistance, it is essential to seek new anti-viral agents that act on additional viral targets. Hemagglutinin (HA), a glycoprotein embedded in the viral surface and playing a critical role in influenza the viral replication cycle has become an attractive target. This work investigates the molecular binding mechanism of HA H3N2 of influenza virus complexed with the fusion inhibitor, arbidol and its derivative (der-arbidol), by means of molecular dynamics simulation. The result showed that the arbidol derivative could form many and strong hydrogen bonds with the HA surrounding amino acids comprising GLU1032(1), LYS3071(1) and LYS3102(1) while arbidol makes this type of interaction with only LYS582(1). The introduction of hydroxyl group at the meta-position of the thiophenol ring was detected to replace the nearby water molecule, thus allowing the direct hydrogen bond formation between der-arbidol and GLU1032(1) of HA residue. Furthermore, the salt bridge networks established among residues GLU572(1)···ARG542(1)···GLU972(2) were considerably more stable in HA-Der-arbidol than that found in HA-Arbidol. The predicted protein-ligand binding free energies were in agreement with experimental data indicating that der-arbidol exhibits higher inhibitory potency against HA H3N2 of influenza virus. Detailed information could be useful for further designing and optimizing HA fusion inhibitors with improved efficiency.

Neuraminidases-Key Players in the Inflammatory Response after Pathophysiological Cardiac Stress and Potential New Therapeutic Targets in Cardiac Disease.

Glycoproteins and glycolipids on the cell surfaces of vertebrates and higher invertebrates contain α-keto acid sugars called sialic acids, terminally attached to their glycan structures. The actual level of sialylation, regulated through enzymatic removal of the latter ones by NEU enzymes, highly affects protein-protein, cell-matrix and cell-cell interactions. Thus, their regulatory features affect a large number of different cell types, including those of the immune system. Research regarding NEUs within heart and vessels provides new insights of their involvement in the development of cardiovascular pathologies and identifies mechanisms on how inhibiting NEU enzymes can have a beneficial effect on cardiac remodelling and on a number of different cardiac diseases including CMs and atherosclerosis. In this regard, a multitude of clinical studies demonstrated the potential of N-acetylneuraminic acid (Neu5Ac) to serve as a biomarker following cardiac diseases. Anti-influenza drugs i.e., zanamivir and oseltamivir are viral NEU inhibitors, thus, they block the enzymatic activity of NEUs. When considering the improvement in cardiac function in several different cardiac disease animal models, which results from NEU reduction, the inhibition of NEU enzymes provides a new potential therapeutic treatment strategy to treat cardiac inflammatory pathologies, and thus, administrate cardioprotection.

Binding mechanism of oseltamivir and influenza neuraminidase suggests perspectives for the design of new anti-influenza drugs.

Oseltamivir is a widely used influenza virus neuraminidase (NA) inhibitor that prevents the release of new virus particles from host cells. However, oseltamivir-resistant strains have emerged, but effective drugs against them have not yet been developed. Elucidating the binding mechanisms between NA and oseltamivir may provide valuable information for the design of new drugs against NA mutants resistant to oseltamivir. Here, we conducted large-scale (353.4 μs) free-binding molecular dynamics simulations, together with a Markov State Model and an importance-sampling algorithm, to reveal the binding process of oseltamivir and NA. Ten metastable states and five major binding pathways were identified that validated and complemented previously discovered binding pathways, including the hypothesis that oseltamivir can be transferred from the secondary sialic acid binding site to the catalytic site. The discovery of multiple new metastable states, especially the stable bound state containing a water-mediated hydrogen bond between Arg118 and oseltamivir, may provide new insights into the improvement of NA inhibitors. We anticipated the findings presented here will facilitate the development of drugs capable of combating NA mutations.

Inhibition of H1N1 Influenza Virus-induced Apoptosis by Ebselen Through ROS-mediated ATM/ATR Signaling Pathways.

Influenza A viruses can cause global outbreaks and seasonal pandemics. However, the use of conventional anti-influenza drugs leads to an increase in drug-resistant mutations in influenza viruses worldwide. Therefore, numerous studies have focused on developing effective anti-influenza drugs. It is feasible to treat influenza by targeting influenza-mediated oxidative damage. Ebselen is a synthetic organoselenium compound which provides glutathione peroxidase-like activity. It has been shown to play a role in anti-influenza therapy, but the mechanism remains to be further explored. This experiment verified the anti-influenza effect of ebselen. CCK-8 and PCR showed that ebselen had a significant inhibitory effect on virus replication compared with the virus group. In addition, the mechanistic investigations revealed that ebselen could inhibit influenza-mediated apoptosis, mitochondrial damage, accumulation of reactive oxygen species, and DNA breakage. At the same time, ebselen significantly inhibited the phosphorylation of ATM and ATR and promoted the activation of PARP and Caspase-3. Ebselen, on the other hand, reduced the inflammatory response caused by influenza. These results suggest that ebselen is a promising inhibitor for H1N1.

PSMD12-Mediated M1 Ubiquitination of Influenza A Virus at K102 Regulates Viral Replication.

ABSTRACTThe M1 of influenza A virus (IAV) is important for the virus life cycle, especially for the assembly and budding of viruses, which is a multistep process that requires host factors. Identifying novel host proteins that interact with M1 and understanding their functions in IAV replication are of great interest in antiviral drug development. In this study, we identified 19 host proteins in DF1 cells suspected to interact with the M1 protein of an H5N6 virus through immunoprecipitation (IP)/mass spectrometry. Among them, PSMD12, a 26S proteasome regulatory subunit, was shown to interact with influenza M1, acting as a positive host factor in IAV replication in avian and human cells. The data showed that PSMD12 promoted K63-linked ubiquitination of M1 at the K102 site. H5N6 and PR8 with an M1-K102 site mutant displayed a significantly weaker replication ability than the wild-type viruses. Mechanistically, PSMD12 promoted M1-M2 virus-like particle (VLP) release, and an M1-K102 mutation disrupted the formation of supernatant M1-M2 VLPs. An H5N6 M1-K102 site mutation or knockdown PSMD12 disrupted the budding release of the virus in chicken embryo fibroblast (CEF) cells, which was confirmed by transmission electron microscopy. Further study confirmed that M1-K102 site mutation significantly affected the virulence of H5N6 and PR8 viruses in mice. In conclusion, we report the novel host factor PSMD12 which affects the replication of influenza virus by mediating K63-linked ubiquitination of M1 at K102. These findings provide novel insight into the interactions between IAV and host cells, while suggesting an important target for anti-influenza virus drug research.IMPORTANCE M1 is proposed to play multiple biologically important roles in the life cycle of IAV, which relies largely on host factors. This study is the first one to identify that PSMD12 interacts with M1, mediates K63-linked ubiquitination of M1 at the K102 site, and thus positively regulates influenza virus proliferation. PSMD12 promoted M1-M2 VLP egress, and an M1-K102 mutation affected the M1-M2 VLP formation. Furthermore, we demonstrate the importance of this site to the morphology and budding of influenza viruses by obtaining mutant viruses, and the M1 ubiquitination regulator PSMD12 has a similar function to the M1 K102 mutation in regulating virus release and virus morphology. Additionally, we confirm the reduced virulence of H5N6 and PR8 (H1N1) viruses carrying the M1-K102 site mutation in mice. These findings provide novel insights into IAV interactions with host cells and suggest a valid and highly conserved candidate target for antiviral drug development.

Identification of cytochrome c oxidase subunit 4 isoform 1 as a positive regulator of influenza virus replication.

Human infection with highly pathogenic H5N1 influenza virus causes severe respiratory diseases. Currently, the drugs against H5N1 are limited to virus-targeted inhibitors. However, drug resistance caused by these inhibitors is becoming a serious threat to global public health. An alternative strategy to reduce the resistance risk is to develop antiviral drugs targeting host cell proteins. In this study, we demonstrated that cytochrome c oxidase subunit 4 isoform 1 (COX41) of host cell plays an important role in H5N1 infection. Overexpression of COX41 promoted viral replication, which was inhibited by silencing or knockout the expression of COX41 in the host cell. The ribonucleoproteins (RNPs) of H5N1 were retained in the cell nucleus after knockout cellular COX41. Strikingly, inhibition of cellular COX41 by lycorine, a small-molecule compound isolated from Amaryllidaceae plants, reduced the levels of COX41-induced ROS and phosphorylation of extracellular signal-regulated kinase (ERK) in cells, thus resulting in the blockage of nuclear export of vRNP and inhibition of viral replication. In H5N1-infected mice that were treated with lycorine, we observed a reduction of viral titers and inhibition of pathological changes in the lung and trachea tissues. Importantly, no resistant virus was generated after culturing the virus with the continuous treatment of lycorine. Collectively, these findings suggest that COX41 is a positive regulator of H5N1 replication and might serve as an alternative target for anti-influenza drug development.