Drug repositioning of Clopidogrel or Triamterene to inhibit influenza virus replication in vitro

Influenza viruses cause respiratory tract infections and substantial health concerns. Infection may result in mild to severe respiratory disease associated with morbidity and some mortality. Several anti-influenza drugs are available, but these agents target viral components and are susceptible to drug resistance. There is a need for new antiviral drug strategies that include repurposing of clinically approved drugs. Drugs that target cellular machinery necessary for influenza virus replication can provide a means for inhibiting influenza virus replication. We used RNA interference screening to identify key host cell genes required for influenza replication, and then FDA-approved drugs that could be repurposed for targeting host genes. We examined the effects of Clopidogrel and Triamterene to inhibit A/WSN/33 (EC50 5.84 uM and 31.48 uM, respectively), A/CA/04/09 (EC50 6.432 uM and 3.32 uM, respectively), and B/Yamagata/16/1988 (EC50 0.28 uM and 0.11 uM, respectively) replication. Clopidogrel and Triamterene provide a druggable approach to influenza treatment across multiple strains and subtypes.

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Network-Guided Discovery of Influenza Virus Replication Host Factors

mBio ◽

10.1128/mbio.02002-18 ◽

2018 ◽

Vol 9 (6) ◽

Cited By ~ 14

Author(s):

Emily E. Ackerman ◽

Eiryo Kawakami ◽

Manami Katoh ◽

Tokiko Watanabe ◽

Shinji Watanabe ◽

...

Keyword(s):

Influenza Virus ◽

Viral Replication ◽

Virus Replication ◽

Drug Targets ◽

Antiviral Drug ◽

Viral Proteins ◽

Host Factors ◽

Ppi Network ◽

Host Proteins ◽

Influenza Virus Replication

ABSTRACTThe positions of host factors required for viral replication within a human protein-protein interaction (PPI) network can be exploited to identify drug targets that are robust to drug-mediated selective pressure. Host factors can physically interact with viral proteins, be a component of virus-regulated pathways (where proteins do not interact with viral proteins), or be required for viral replication but unregulated by viruses. Here, we demonstrate a method of combining human PPI networks with virus-host PPI data to improve antiviral drug discovery for influenza viruses by identifying target host proteins. Analysis shows that influenza virus proteins physically interact with host proteins in network positions significant for information flow, even after the removal of known abundance-degree bias within PPI data. We have isolated a subnetwork of the human PPI network that connects virus-interacting host proteins to host factors that are important for influenza virus replication without physically interacting with viral proteins. The subnetwork is enriched for signaling and immune processes distinct from those associated with virus-interacting proteins. Selecting proteins based on subnetwork topology, we performed an siRNA screen to determine whether the subnetwork was enriched for virus replication host factors and whether network position within the subnetwork offers an advantage in prioritization of drug targets to control influenza virus replication. We found that the subnetwork is highly enriched for target host proteins—more so than the set of host factors that physically interact with viral proteins. Our findings demonstrate that network positions are a powerful predictor to guide antiviral drug candidate prioritization.IMPORTANCEIntegrating virus-host interactions with host protein-protein interactions, we have created a method using these established network practices to identify host factors (i.e., proteins) that are likely candidates for antiviral drug targeting. We demonstrate that interaction cascades between host proteins that directly interact with viral proteins and host factors that are important to influenza virus replication are enriched for signaling and immune processes. Additionally, we show that host proteins that interact with viral proteins are in network locations of power. Finally, we demonstrate a new network methodology to predict novel host factors and validate predictions with an siRNA screen. Our results show that integrating virus-host proteins interactions is useful in the identification of antiviral drug target candidates.

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Inhibitory effect of hirsutine on influenza virus replication in vitro

Antiviral Research ◽

10.1016/s0166-3542(97)83306-2 ◽

1997 ◽

Vol 34 (2) ◽

pp. A88 ◽

Cited By ~ 2

Author(s):

K. Konno ◽

H. Inoue ◽

M. Fujiwara ◽

T. Mizuta ◽

H. Takayama ◽

...

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Inhibitory Effect ◽

Influenza Virus Replication

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Role of Initiating Nucleoside Triphosphate Concentrations in the Regulation of Influenza Virus Replication and Transcription

Journal of Virology ◽

10.1128/jvi.00627-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 6902-6910 ◽

Cited By ~ 27

Author(s):

Frank T. Vreede ◽

Hugh Gifford ◽

George G. Brownlee

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Influenza A ◽

De Novo ◽

Nucleoside Triphosphate ◽

Ribonucleoprotein Complexes ◽

Influenza Virus Replication ◽

High Concentrations

ABSTRACT The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Previous studies in our laboratory have shown that virion-derived viral ribonucleoprotein complexes synthesize both mRNA and cRNA in vitro and early in the infection cycle in vivo. Our continued studies showed that de novo synthesis of cRNA in vitro is more sensitive to the concentrations of ATP, CTP, and GTP than capped-primer-dependent synthesis of mRNA. Using rescued recombinant influenza A/WSN/33 viruses, we now demonstrate that the 3′-terminal sequence of the vRNA promoter dictates the requirement for a high nucleoside triphosphate (NTP) concentration during de novo-initiated replication to cRNA, whereas this is not the case for the extension of capped primers during transcription to mRNA. In contrast to some other viral polymerases, for which only the initiating NTP is required at high concentrations, influenza virus polymerase requires high concentrations of the first three NTPs. In addition, we show that base pair mutations in the vRNA promoter can lead to nontemplated dead-end mutations during replication to cRNA in vivo. Based on our observations, we propose a new model for the de novo initiation of influenza virus replication.

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Cryptoporic acid E from Cryptoporus volvatus inhibits influenza virus replication in vitro

Antiviral Research ◽

10.1016/j.antiviral.2017.02.010 ◽

2017 ◽

Vol 143 ◽

pp. 106-112 ◽

Cited By ~ 5

Author(s):

Li Gao ◽

Jiayuan Han ◽

Jianyong Si ◽

Junchi Wang ◽

Hexiang Wang ◽

...

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Influenza Virus Replication

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N-linked glycosylation plays an important role in budding of NA protein and virulence of influenza viruses

Journal of Virology ◽

10.1128/jvi.02042-20 ◽

2020 ◽

Author(s):

Danqi Bao ◽

Ruixue Xue ◽

Min Zhang ◽

Chenyang Lu ◽

Tianxin Ma ◽

...

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Immune Escape ◽

H1n1 Influenza ◽

Influenza Viruses ◽

H1n1 Influenza Virus ◽

Protein Loss ◽

Knock Out

Neuraminidase (NA) has multiple functions in the life cycle of influenza virus, especially in the late stage of virus replication. Both of Hemagglutinin (HA) and NA are highly glycosylated proteins. N-linked glycosylation (NLG) of HA has been reported to contribute to immune escape and virulence of influenza viruses. However, the function of NLG of NA remains largely unclear. In this study, we found that NLG is critical for budding ability of NA. Tunicamycin treatment or NLG knock-out significantly inhibited the budding of NA. Further studies showed that the NLG knock-out caused attenuation of virus in vitro and in vivo. Notably the NLG at 219 position plays an important role in budding, replication, and virulence of H1N1 influenza virus. To explore the underlying mechanism, unfolded protein response (UPR) was determined in NLG knock-out NA overexpressed cells, which showed that the mutant NA was mainly located in ER, and the UPR markers BIP and p-eIF2α were upregulated, and XBP1 was downregulated. All the results indicated that NLG knock-out NA was stacked in ER and triggered UPR, which might shut down the budding process of NA. Overall, the study shed light on the function of NLG of NA in virus replication and budding. IMPORTANCE NA is a highly glycosylated protein. Nevertheless, how the NLG affects the function of NA protein remains largely unclear. In this study, we found that NLG plays important roles in budding and Neuraminidase activity of NA protein. Loss of NLG attenuated viral budding and replication. Especially the 219 NLG site mutation significantly attenuated the replication and virulence of H1N1 influenza virus in vitro and in vivo, which suggested that NLG of NA protein is a novel virulence marker for influenza viruses.

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Replication Dynamics, Pathogenicity, and Evolution of Influenza Viruses in Intestinal Caco-2 Cells

University of the Future: Re-Imagining Research and Higher Education ◽

10.29117/quarfe.2020.0166 ◽

2020 ◽

Author(s):

Israa Elbashir ◽

Heba Al Khatib ◽

Hadi Yassine

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Deep Sequencing ◽

Virus Evolution ◽

Influenza Viruses ◽

Intestinal Cells ◽

Sequencing Analysis ◽

Influenza Virus Replication ◽

Ha Protein ◽

Deep Sequencing Analysis

Background: Influenza virus is a major cause of respiratory infections worldwide. Besides the common respiratory symptoms, namouras cases with gastrointestinal symptoms have been reported. Moreover, influenza virus has been detected in feces of up to 20.6 % of influenza-infected patients. Therefore, direct infection of intestinal cells with influenza virus is suspected; however, the mechanism of this infection has not been explored. AIM: To investigate influenza virus replication, cellular responses to infection, and virus evolution following serial infection in human Caucasian colon adenocarcinoma cells (Caco-2 cells). Method: Two influenza A subtypes (A/H3N2 and A/H1N1pdm 09) and one influenza B virus (B/Yamagata) were serially passaged in Caco-2. Quantitative PCR was used to study hormones and cytokines expression following infection. Deep sequencing analysis of viral genome was used to assess the virus evolution. Results: The replication capacity of the three viruses was maintained throughout 12 passages, with H3N2 virus being the fastest in adaptation. The expression of hormone and cytokines in Caco-2 cells was considerably different between the viruses and among the passages, however, a pattern of induction was observed at the late phase of infection. Deep sequencing analysis revealed a few amino acid substitutions in the HA protein of H3N2 and H1N1 viruses, mostly in the antigenic site. Moreover, virus evolution at the quasispecies level based on HA protein revealed that H3N2 and H1N1 harbored more diverse virus populations when compared to IBV, indicating their higher evolution within Caco-2 cells. Conclusion: The findings of this study indicate the possibility of influenza virus replication in intestinal cells. To further explain the gastrointestinal complications of influenza infections in-vivo experiments with different influenza viruses are needed.

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Interferon Inducible Transmembrane Protein 3 Inhibits Influenza Virus Replication and Inflammation by Interacting With ABHD16A

10.21203/rs.3.rs-106351/v1 ◽

2020 ◽

Author(s):

Chen Liang ◽

Limei Zhu ◽

Jun Chen

Keyword(s):

Influenza Virus ◽

Confocal Microscopy ◽

Virus Replication ◽

Influenza Viruses ◽

Expression Level ◽

Hek293 Cells ◽

Yeast Two Hybrid ◽

Related Factors ◽

Influenza Virus Replication ◽

Two Hybrid

Abstract Background: Studies have shown that human interferon inducible transmembrane proteins (IFITM) family proteins have broad-spectrum antiviral capabilities. Preliminary studies in our laboratory have preliminarily proved that IFITMs have the effect of inhibiting influenza viruses. In order to further study its mechanism and role in the occurrence and development of influenza, relevant studies have been carried out.Methods: Fluorescence quantitative polymerase chain reaction (PCR) detection, yeast two-hybrid test and optical confocal microscopy were used to investigate the effect of hIFITM3 on influenza virus replication, the interaction with human abhydrolase domain containing 16A (hABHD16A) and the expression of inflammation-related factors.Results: In HEK293 cells, overexpression of hIFITM3 protein significantly inhibited the replication of influenza virus at 24h, 48h, and 72h; yeast two-hybrid experiment proved that IFITM3 interacts with ABHD16A; laser confocal microscopy observations showed that IFITM3 and ABHD16A co-localized in cell membrane area; the expression level of inflammation-related factors in cells overexpressing hIFITM3 or hABHD16A was detected by fluorescence quantitative PCR, and the results showed that the mRNA levels of interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-a and cyclooxygenase 2 (COX2) were significantly increased . But when IFITM3/ABHD16A was co-expressed, the mRNA expression levels of these cytokines were significantly reduced except for COX2. When influenza virus infected cells co-expressing IFITM3/ABHD16A, the expression level of inflammatory factors decreased compared with the control group, indicating that IFITM3 can play an important role in regulating inflammation balance.Conclusions: This study confirmed that hIFITM3 has an effect of inhibiting influenza virus replication. Furthermore, it was found that hIFITM3 interacts with hABHD16A, following which it can better inhibit the replication of influenza virus and the inflammatory response caused by the disease process.

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Network-Guided Discovery of Influenza Virus Replication Host Factors

10.1101/344861 ◽

2018 ◽

Cited By ~ 1

Author(s):

Emily E. Ackerman ◽

Eiryo Kawakami ◽

Manami Katoh ◽

Tokiko Watanabe ◽

Shinji Watanabe ◽

...

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Drug Targets ◽

Antiviral Drug ◽

Viral Proteins ◽

Host Factors ◽

Host Protein ◽

Ppi Network ◽

Host Proteins ◽

Influenza Virus Replication

ABSTRACTThe position of host factors required for viral replication within a human protein-protein interaction (PPI) network can be exploited to identify drug targets that are robust to drug-mediated selective pressure. Host factors can physically interact with viral proteins, be a component of pathways regulated by viruses (where proteins themselves do not interact with viral proteins) or be required for viral replication but unregulated by viruses. Here, we demonstrate a method of combining a human PPI network with virus-host protein interaction data to improve antiviral drug discovery for influenza viruses by identifying target host proteins. Network analysis shows that influenza virus proteins physically interact with host proteins in network positions significant for information flow. We have isolated a subnetwork of the human PPI network which connects virus-interacting host proteins to host factors that are important for influenza virus replication without physically interacting with viral proteins. The subnetwork is enriched for signaling and immune processes. Selecting proteins based on network topology within the subnetwork, we performed an siRNA screen to determine if the subnetwork was enriched for virus replication host factors and if network position within the subnetwork offers an advantage in prioritization of drug targets to control influenza virus replication. We found that the subnetwork is highly enriched for target host proteins – more so than the set of host factors that physically interact with viral proteins. Our findings demonstrate that network positions are a powerful predictor to guide antiviral drug candidate prioritization.IMPORTANCEIntegrating virus-host interactions with host protein-protein interactions, we have created a method using these established network practices to identify host factors (i.e. proteins) that are likely candidates for antiviral drug targeting. We demonstrate that interaction cascades between host proteins that directly interact with viral proteins and host factors that are important to influenza replication are enriched for signaling and immune processes. Additionally, we show that host proteins that interact with viral proteins are in network locations of power. Finally, we demonstrate a new network methodology to predict novel host factors and validate predictions with an siRNA screen. Our results show that integrating virus-host proteins interactions is useful in the identification of antiviral drug target candidates.

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MicroRNAs affect GPCR and Ion channel genes needed for influenza replication

Journal of General Virology ◽

10.1099/jgv.0.001691 ◽

2021 ◽

Vol 102 (11) ◽

Author(s):

Nichole Orr-Burks ◽

Jackelyn Murray ◽

Kyle V. Todd ◽

Abhijeet Bakre ◽

Ralph A. Tripp

Keyword(s):

Influenza Virus ◽

Ion Channel ◽

Virus Replication ◽

Target Genes ◽

A549 Cells ◽

Influenza Viruses ◽

Economic Losses ◽

Alveolar Epithelial ◽

Influenza Virus Replication ◽

Host Genes

Influenza virus causes seasonal epidemics and sporadic pandemics resulting in morbidity, mortality, and economic losses worldwide. Understanding how to regulate influenza virus replication is important for developing vaccine and therapeutic strategies. Identifying microRNAs (miRs) that affect host genes used by influenza virus for replication can support an antiviral strategy. In this study, G-protein coupled receptor (GPCR) and ion channel (IC) host genes in human alveolar epithelial (A549) cells used by influenza virus for replication (Orr-Burks et al., 2021) were examined as miR target genes following A/CA/04/09- or B/Yamagata/16/1988 replication. Thirty-three miRs were predicted to target GPCR or IC genes and their miR mimics were evaluated for their ability to decrease influenza virus replication. Paired miR inhibitors were used as an ancillary measure to confirm or not the antiviral effects of a miR mimic. Fifteen miRs lowered influenza virus replication and four miRs were found to reduce replication irrespective of virus strain and type differences. These findings provide evidence for novel miR disease intervention strategies for influenza viruses.

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