Annexin II Incorporated into Influenza Virus Particles Supports Virus Replication by Converting Plasminogen into Plasmin

ABSTRACT For influenza viruses to become infectious, the proteolytic cleavage of hemagglutinin (HA) is essential. This usually is mediated by trypsin-like proteases in the respiratory tract. The binding of plasminogen to influenza virus A/WSN/33 leads to the cleavage of HA, a feature determining its pathogenicity and neurotropism in mice. Here, we demonstrate that plasminogen also promotes the replication of other influenza virus strains. The inhibition of the conversion of plasminogen into plasmin blocked influenza virus replication. Evidence is provided that the activation of plasminogen is mediated by the host cellular protein annexin II, which is incorporated into the virus particles. Indeed, the inhibition of plasminogen binding to annexin II by using a competitive inhibitor inhibits plasminogen activation into plasmin. Collectively, these results indicate that the annexin II-mediated activation of plasminogen supports the replication of influenza viruses, which may contribute to their pathogenicity.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Application of Super-Resolution and Advanced Quantitative Microscopy to the Spatio-Temporal Analysis of Influenza Virus Replication

Viruses ◽

10.3390/v13020233 ◽

2021 ◽

Vol 13 (2) ◽

pp. 233 ◽

Cited By ~ 1

Author(s):

Emma Touizer ◽

Christian Sieben ◽

Ricardo Henriques ◽

Mark Marsh ◽

Romain F. Laine

Keyword(s):

Influenza Virus ◽

Host Cell ◽

Virus Replication ◽

Large Scale ◽

Super Resolution ◽

Living Cells ◽

Influenza Viruses ◽

Animal Populations ◽

Influenza Virus Replication ◽

Super Resolution Microscopy

With an estimated three to five million human cases annually and the potential to infect domestic and wild animal populations, influenza viruses are one of the greatest health and economic burdens to our society, and pose an ongoing threat of large-scale pandemics. Despite our knowledge of many important aspects of influenza virus biology, there is still much to learn about how influenza viruses replicate in infected cells, for instance, how they use entry receptors or exploit host cell trafficking pathways. These gaps in our knowledge are due, in part, to the difficulty of directly observing viruses in living cells. In recent years, advances in light microscopy, including super-resolution microscopy and single-molecule imaging, have enabled many viral replication steps to be visualised dynamically in living cells. In particular, the ability to track single virions and their components, in real time, now allows specific pathways to be interrogated, providing new insights to various aspects of the virus-host cell interaction. In this review, we discuss how state-of-the-art imaging technologies, notably quantitative live-cell and super-resolution microscopy, are providing new nanoscale and molecular insights into influenza virus replication and revealing new opportunities for developing antiviral strategies.

Download Full-text

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

10.1101/778258 ◽

2019 ◽

Author(s):

Thomas Figueroa ◽

Pierre Bessière ◽

Amelia Coggon ◽

Roosmarijn van der Woude ◽

Maxence Delverdier ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Virus Replication ◽

Intestinal Microbiota ◽

Clinical Signs ◽

Influenza Viruses ◽

Highly Pathogenic ◽

Influenza Virus Replication

ABSTRACTDucks usually show little or no clinical signs following highly pathogenic avian influenza virus infection. In order to analyze if the gut microbiota could contribute to the control of influenza virus replication in ducks, we used a broad-spectrum oral antibiotic treatment to deplete the gut microbiota before infection with a highly pathogenic H5N9 avian influenza virus. Antibiotic-treated ducks and non-treated control ducks did not show any clinical signs following H5N9 virus infection. We did not detect any difference in virus titers neither in the respiratory tract, nor in the brain and spleen. However, we found that antibiotic-treated H5N9 virus infected ducks had significantly increased intestinal virus excretion at day 3 and 5 post-infection. 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.IMPORTANCEDucks 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 intestinal 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 prevention of avian influenza virus spread from the duck reservoir.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.00919-15 ◽

2015 ◽

Vol 89 (20) ◽

pp. 10273-10285 ◽

Cited By ~ 7

Author(s):

Min Zheng ◽

Pui Wang ◽

Wenjun Song ◽

Siu-Ying Lau ◽

Siwen Liu ◽

...

Keyword(s):

Influenza Virus ◽

Alternative Splicing ◽

Virus Replication ◽

Viral Proteins ◽

Influenza Viruses ◽

Noncoding Region ◽

Ns1 Protein ◽

Functional Studies ◽

Infected Cells ◽

Influenza Virus Replication

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.

Download Full-text

Oseltamivir Inhibits H7 Influenza Virus Replication in Mice Inoculated by the Ocular Route

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06101-11 ◽

2011 ◽

Vol 56 (3) ◽

pp. 1616-1618 ◽

Cited By ~ 9

Author(s):

Jessica A. Belser ◽

Katrina Sleeman ◽

Melissa B. Pearce ◽

Jacqueline M. Katz ◽

Larisa V. Gubareva ◽

...

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Influenza Viruses ◽

Ocular Tissue ◽

Neuraminidase Inhibitors ◽

Virus Challenge ◽

Influenza Virus Replication ◽

Virus Exposure ◽

Human Infections ◽

H7n3 Virus

ABSTRACTThe majority of human infections associated with H7 influenza viruses have resulted in ocular and not respiratory disease. While oseltamivir has been prescribed to individuals presenting with conjunctivitis following H7 virus exposure, it is unknown if oseltamivir inhibits virus replication in ocular tissue. We demonstrate that H7 viruses possess sensitivity to neuraminidase inhibitors and that administration of oseltamivir before ocular virus challenge in mice inhibits H7N7 and H7N3 virus replication in ocular and respiratory tissues.

Download Full-text