An A14U Substitution in the 3′ Noncoding Region of the M Segment of Viral RNA Supports Replication of Influenza Virus with an NS1 Deletion by Modulating Alternative Splicing of M Segment mRNAs

ABSTRACTThe NS1 protein of influenza virus has multiple functions and is a determinant of virulence. Influenza viruses with NS1 deletions (DelNS1 influenza viruses) are a useful tool for studying virus replication and can serve as effective live attenuated vaccines, but deletion of NS1 severely diminishes virus replication, hampering functional studies and vaccine production. We found that WSN-DelNS1 viruses passaged in cells consistently adapted to gain an A14U substitution in the 3′ noncoding region of the M segment of viral RNA (vRNA) which restored replicative ability. DelNS1-M-A14U viruses cannot inhibit interferon expression in virus infected-cells, providing an essential model for studying virus replication in the absence of the NS1 protein. Characterization of DelNS1-M-A14U virus showed that the lack of NS1 has no apparent effect on expression of other viral proteins, with the exception of M mRNAs. Expression of the M transcripts, M1, M2, mRNA3, and mRNA4, is regulated by alternative splicing. The A14U substitution changes the splicing donor site consensus sequence of mRNA3, altering expression of M transcripts, with M2 expression significantly increased and mRNA3 markedly suppressed in DelNS1-M-A14U, but not DelNS1-M-WT, virus-infected cells. Further analysis revealed that the A14U substitution also affects promoter function during replication of the viral genome. The M-A14U mutation increases M vRNA synthesis in DelNS1 virus infection and enhances alternative splicing of M2 mRNA in the absence of other viral proteins. The findings demonstrate that NS1 is directly involved in influenza virus replication through modulation of alternative splicing of M transcripts and provide strategic information important to construction of vaccine strains with NS1 deletions.IMPORTANCENonstructural protein (NS1) of influenza virus has multiple functions. Besides its role in antagonizing host antiviral activity, NS1 is also believed to be involved in regulating virus replication, but mechanistic details are not clear. The NS1 protein is a virulence determinant which inhibits both innate and adaptive immunity and live attenuated viruses with NS1 deletions show promise as effective vaccines. However, deletion of NS1 causes severe attenuation of virus replication during infection, impeding functional studies and vaccine development. We characterized a replication-competent DelNS1 virus which carries an A14U substitution in the 3′ noncoding region of the vRNA M segment. We found that M-A14U mutation supports virus replication through modulation of alternative splicing of mRNAs transcribed from the M segment. Our findings give insight into the role of NS1 in influenza virus replication and provide an approach for constructing replication-competent strains with NS1 deletions for use in functional and vaccine studies.

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Dynamic phospho-modification of viral proteins as a crucial regulatory layer of influenza A virus replication and innate immune responses

Biological Chemistry ◽

10.1515/hsz-2021-0241 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Yvonne Boergeling ◽

Linda Brunotte ◽

Stephan Ludwig

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Influenza A ◽

Current Knowledge ◽

Viral Proteins ◽

Transport Processes ◽

Influenza Viruses ◽

Innate Immune ◽

A Genome ◽

Influenza Virus Replication

Abstract Influenza viruses are small RNA viruses with a genome of about 13 kb. Because of this limited coding capacity, viral proteins have evolved to fulfil multiple functions in the infected cell. This implies that there must be mechanisms allowing to dynamically direct protein action to a distinct activity in a spatio-temporal manner. Furthermore, viruses exploit many cellular processes, which also have to be dynamically regulated during the viral replication cycle. Phosphorylation and dephosphorylation of proteins are fundamental for the control of many cellular responses. There is accumulating evidence that this mechanism represents a so far underestimated level of regulation in influenza virus replication. Here, we focus on the current knowledge of dynamics of phospho-modifications in influenza virus replication and show recent examples of findings underlining the crucial role of phosphorylation in viral transport processes as well as activation and counteraction of the innate immune response.

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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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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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Safflomin A inhibits neuraminidase activity and influenza virus replication

RSC Advances ◽

10.1039/c5ra17336a ◽

2015 ◽

Vol 5 (114) ◽

pp. 94053-94066 ◽

Cited By ~ 10

Author(s):

Miao Yu ◽

Ye Wang ◽

Li Tian ◽

Yanyan Wang ◽

Xizhu Wang ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Virus Replication ◽

Neuraminidase Activity ◽

Viral Release ◽

Infected Cells ◽

Influenza Virus Replication

Neuraminidase (NA) is a glycoprotein on the surface of the influenza virus that plays an important role in the early processes of virus infection and viral release from the infected cells.

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The Microbiota Contributes to the Control of Highly Pathogenic H5N9 Influenza Virus Replication in Ducks

Journal of Virology ◽

10.1128/jvi.00289-20 ◽

2020 ◽

Vol 94 (10) ◽

Cited By ~ 1

Author(s):

Thomas Figueroa ◽

Pierre Bessière ◽

Amelia Coggon ◽

Kim M. Bouwman ◽

Roosmarijn van der Woude ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Virus Replication ◽

Clinical Signs ◽

Influenza Viruses ◽

Highly Pathogenic ◽

Antiviral Immune Response ◽

Influenza Virus Replication

ABSTRACT Ducks usually show little or no clinical signs following highly pathogenic avian influenza virus infection. In order to analyze whether the microbiota could contribute to the control of influenza virus replication in ducks, we used a broad-spectrum oral antibiotic treatment to deplete the microbiota before infection with a highly pathogenic H5N9 avian influenza virus. Antibiotic-treated ducks and nontreated control ducks did not show any clinical signs following H5N9 virus infection. We did not detect any significant difference in virus titers neither in the respiratory tract nor in the brain nor spleen. However, we found that antibiotic-treated H5N9 virus-infected ducks had significantly increased intestinal virus excretion at days 3 and 5 postinfection. This was associated with a significantly decreased antiviral immune response in the intestine of antibiotic-treated ducks. Our findings highlight the importance of an intact microbiota for an efficient control of avian influenza virus replication in ducks. IMPORTANCE Ducks are frequently infected with avian influenza viruses belonging to multiple subtypes. They represent an important reservoir species of avian influenza viruses, which can occasionally be transmitted to other bird species or mammals, including humans. Ducks thus have a central role in the epidemiology of influenza virus infection. Importantly, ducks usually show little or no clinical signs even following infection with a highly pathogenic avian influenza virus. We provide evidence that the microbiota contributes to the control of influenza virus replication in ducks by modulating the antiviral immune response. Ducks are able to control influenza virus replication more efficiently when they have an intact intestinal microbiota. Therefore, maintaining a healthy microbiota by limiting perturbations to its composition should contribute to the prevention of avian influenza virus spread from the duck reservoir.

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

PLoS ONE ◽

10.1371/journal.pone.0259129 ◽

2021 ◽

Vol 16 (10) ◽

pp. e0259129

Author(s):

Nichole Orr-Burks ◽

Jackelyn Murray ◽

Kyle V. Todd ◽

Abhijeet Bakre ◽

Ralph A. Tripp

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Drug Repositioning ◽

Antiviral Drug ◽

Influenza Viruses ◽

Influenza Virus Replication ◽

Approved Drugs ◽

Multiple Strains ◽

Tract Infections

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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Inhibition of Influenza Virus Replication by Nitric Oxide

Journal of Virology ◽

10.1128/jvi.73.10.8880-8883.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8880-8883 ◽

Cited By ~ 72

Author(s):

Guus F. Rimmelzwaan ◽

Marianne M. J. W. Baars ◽

Pim de Lijster ◽

Ron A. M. Fouchier ◽

Albert D. M. E. Osterhaus

Keyword(s):

Nitric Oxide ◽

Influenza Virus ◽

Influenza A ◽

Rna Synthesis ◽

Virus Production ◽

Influenza Viruses ◽

No Donor ◽

Infected Cells ◽

Influenza Virus Replication ◽

Darby Canine Kidney

ABSTRACT Nitric oxide (NO) has been shown to contribute to the pathogenesis of influenza virus-induced pneumonia in mouse models. Here we show that replication of influenza A and B viruses in Mabin Darby canine kidney cells is severely impaired by the NO donor,S-nitroso-N-acetylpenicillamine. Reduction of productively infected cells and virus production proved to correlate with inhibition of viral RNA synthesis, indicating that NO affects an early step in the replication cycle of influenza viruses.

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