Library-based analysis reveals segment and length dependent characteristics of defective influenza genomes

Found in a diverse set of viral populations, defective interfering particles are parasitic variants that are unable to replicate on their own yet rise to relatively high frequencies. Their presence is associated with a loss of population fitness, both through the depletion of key cellular resources and the stimulation of innate immunity. For influenza A virus, these particles contain large internal deletions in the genomic segments which encode components of the heterotrimeric polymerase. Using a library-based approach, we comprehensively profile the growth and replication of defective influenza species, demonstrating that they possess an advantage during genome replication, and that exclusion during population expansion reshapes population composition in a manner consistent with their final, observed, distribution in natural populations. We find that an innate immune response is not linked to the size of a deletion; however, replication of defective segments can enhance their immunostimulatory properties. Overall, our results address several key questions in defective influenza A virus biology, and the methods we have developed to answer those questions may be broadly applied to other defective viruses.

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Library-based analysis reveals segment and length dependent characteristics of defective influenza genomes

10.1101/2021.09.06.459165 ◽

2021 ◽

Author(s):

Marisa Mendes ◽

Alistair B Russell

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Natural Populations ◽

Innate Immune ◽

Viral Population ◽

Genome Replication ◽

Defective Interfering Particles ◽

High Frequencies ◽

Stimulation Of ◽

Key Questions

Parasitic elements of the viral population which are unable to replicate on their own yet rise to high frequencies, defective interfering particles are found in a variety of different viruses. Their presence is associated with a loss of population fitness, both through the depletion of key cellular resources and the stimulation of innate immunity. For influenza A virus, these particles contain large internal deletions in the genomic segments which encode components of the heterotrimeric polymerase. Using a library-based approach, we comprehensively profile the growth and replication of defective influenza species, demonstrating that they possess an advantage during genome replication, and that exclusion during packaging reshapes population composition in a manner consistent with their final, observed, distribution in natural populations. We find that an innate immune response is not linked to the size of a deletion; however, replication of defective segments can enhance their immunostimulatory properties. Overall, our results address several key questions in defective influenza A virus biology, and the methods we have developed to answer those questions may be broadly applied to other defective viruses.

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Antiviral activity of influenza A virus defective interfering particles against SARS-CoV-2 replication in vitro through stimulation of innate immunity

10.1101/2021.02.19.431972 ◽

2021 ◽

Author(s):

U. Rand ◽

S.Y. Kupke ◽

H. Shkarlet ◽

M.D. Hein ◽

T. Hirsch ◽

...

Keyword(s):

Innate Immunity ◽

Influenza A Virus ◽

Influenza A ◽

Immune Escape ◽

Antiviral Treatment ◽

Specific Treatment ◽

Janus Kinase ◽

Defective Interfering Particles ◽

Stimulation Of

AbstractSARS-CoV-2 causing COVID-19 emerged in late 2019 and resulted in a devastating pandemic. Although the first approved vaccines were already administered by the end of 2020, vaccine availability is still limited. Moreover, immune escape variants of the virus are emerging against which the current vaccines may confer only limited protection. Further, existing antivirals and treatment options against COVID-19 only show limited efficacy. Influenza A virus (IAV) defective interfering particles (DIPs) were previously proposed not only for antiviral treatment of the influenza disease but also for pan-specific treatment of interferon (IFN)-sensitive respiratory virus infections. To investigate the applicability of IAV DIPs as an antiviral for the treatment of COVID-19, we conducted in vitro co-infection experiments with produced, cell culture-derived DIPs and the IFN-sensitive SARS-CoV-2. We show that treatment with IAV DIPs leads to complete abrogation of SARS-CoV-2 replication. Moreover, this inhibitory effect was dependent on janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. These results suggest an unspecific stimulation of the innate immunity by IAV DIPs as a major contributor in suppressing SARS-CoV-2 replication. Thus, we propose IAV DIPs as an effective antiviral agent for treatment of COVID-19, and potentially also for suppressing the replication of new variants of SARS-CoV-2.

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238 Innate immune response triggered by influenza A virus is negatively regulated by suppressor of cytokine signaling (SOCS)1 and SOCS3 through a RIG-I/IFNAR1-dependent pathway

Cytokine ◽

10.1016/j.cyto.2008.07.303 ◽

2008 ◽

Vol 43 (3) ◽

pp. 296

Author(s):

Julien Pothlichet ◽

Michel Chignard ◽

Mustapha Si-Tahar

Keyword(s):

Immune Response ◽

Influenza A Virus ◽

Innate Immune Response ◽

Influenza A ◽

Innate Immune ◽

Cytokine Signaling ◽

Suppressor Of Cytokine Signaling ◽

Dependent Pathway

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Influenza A virus PB1‐F2 protein: An ambivalent innate immune modulator and virulence factor

Journal of Leukocyte Biology ◽

10.1002/jlb.4mr0320-206r ◽

2020 ◽

Vol 107 (5) ◽

pp. 763-771 ◽

Cited By ~ 2

Author(s):

Pak‐Hin Hinson Cheung ◽

Tak‐Wang Terence Lee ◽

Chi‐Ping Chan ◽

Dong‐Yan Jin

Keyword(s):

Influenza A Virus ◽

Virulence Factor ◽

Influenza A ◽

Innate Immune ◽

Immune Modulator

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Production of Defective Interfering Particles of Influenza A Virus in Parallel Continuous Cultures at Two Residence Times—Insights From qPCR Measurements and Viral Dynamics Modeling

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2019.00275 ◽

2019 ◽

Vol 7 ◽

Cited By ~ 2

Author(s):

Felipe Tapia ◽

Tanja Laske ◽

Milena A. Wasik ◽

Markus Rammhold ◽

Yvonne Genzel ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Residence Times ◽

Viral Dynamics ◽

Dynamics Modeling ◽

Continuous Cultures ◽

Defective Interfering Particles

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The Zα2 domain of ZBP1 is a molecular switch regulating influenza-induced PANoptosis and perinatal lethality during development

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013752 ◽

2020 ◽

Vol 295 (24) ◽

pp. 8325-8330 ◽

Cited By ~ 14

Author(s):

Sannula Kesavardhana ◽

R. K. Subbarao Malireddi ◽

Amanda R. Burton ◽

Shaina N. Porter ◽

Peter Vogel ◽

...

Keyword(s):

Cell Death ◽

Nucleic Acids ◽

Influenza A Virus ◽

Nlrp3 Inflammasome ◽

Influenza A ◽

Defense Responses ◽

Innate Immune ◽

Inflammasome Activation ◽

Perinatal Lethality

Z-DNA-binding protein 1 (ZBP1) is an innate immune sensor of nucleic acids that regulates host defense responses and development. ZBP1 activation triggers inflammation and pyroptosis, necroptosis, and apoptosis (PANoptosis) by activating receptor-interacting Ser/Thr kinase 3 (RIPK3), caspase-8, and the NLRP3 inflammasome. ZBP1 is unique among innate immune sensors because of its N-terminal Zα1 and Zα2 domains, which bind to nucleic acids in the Z-conformation. However, the specific role of these Zα domains in orchestrating ZBP1 activation and subsequent inflammation and cell death is not clear. Here we generated Zbp1ΔZα2/ΔZα2 mice that express ZBP1 lacking the Zα2 domain and demonstrate that this domain is critical for influenza A virus–induced PANoptosis and underlies perinatal lethality in mice in which the RIP homotypic interaction motif domain of RIPK1 has been mutated (Ripk1mRHIM/mRHIM). Deletion of the Zα2 domain in ZBP1 abolished influenza A virus–induced PANoptosis and NLRP3 inflammasome activation. Furthermore, deletion of the Zα2 domain of ZBP1 was sufficient to rescue Ripk1mRHIM/mRHIM mice from perinatal lethality caused by ZBP1-driven cell death and inflammation. Our findings identify the essential role of the Zα2 domain of ZBP1 in several physiological functions and establish a link between Z-RNA sensing via the Zα2 domain and promotion of influenza-induced PANoptosis and perinatal lethality.

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Hemagglutinin of Influenza A Virus Antagonizes Type I Interferon (IFN) Responses by Inducing Degradation of Type I IFN Receptor 1

Journal of Virology ◽

10.1128/jvi.02749-15 ◽

2015 ◽

Vol 90 (5) ◽

pp. 2403-2417 ◽

Cited By ~ 41

Author(s):

Chuan Xia ◽

Madhuvanthi Vijayan ◽

Curtis J. Pritzl ◽

Serge Y. Fuchs ◽

Adrian B. McDermott ◽

...

Keyword(s):

Signaling Pathway ◽

Influenza A Virus ◽

Influenza A ◽

Type I Interferon ◽

Innate Immune ◽

Host Cells ◽

Type I ◽

Type I Ifn ◽

Type I Ifns ◽

Ha Protein

ABSTRACTInfluenza A virus (IAV) employs diverse strategies to circumvent type I interferon (IFN) responses, particularly by inhibiting the synthesis of type I IFNs. However, it is poorly understood if and how IAV regulates the type I IFN receptor (IFNAR)-mediated signaling mode. In this study, we demonstrate that IAV induces the degradation of IFNAR subunit 1 (IFNAR1) to attenuate the type I IFN-induced antiviral signaling pathway. Following infection, the level of IFNAR1 protein, but not mRNA, decreased. Indeed, IFNAR1 was phosphorylated and ubiquitinated by IAV infection, which resulted in IFNAR1 elimination. The transiently overexpressed IFNAR1 displayed antiviral activity by inhibiting virus replication. Importantly, the hemagglutinin (HA) protein of IAV was proved to trigger the ubiquitination of IFNAR1, diminishing the levels of IFNAR1. Further, influenza A viral HA1 subunit, but not HA2 subunit, downregulated IFNAR1. However, viral HA-mediated degradation of IFNAR1 was not caused by the endoplasmic reticulum (ER) stress response. IAV HA robustly reduced cellular sensitivity to type I IFNs, suppressing the activation of STAT1/STAT2 and induction of IFN-stimulated antiviral proteins. Taken together, our findings suggest that IAV HA causes IFNAR1 degradation, which in turn helps the virus escape the powerful innate immune system. Thus, the research elucidated an influenza viral mechanism for eluding the IFNAR signaling pathway, which could provide new insights into the interplay between influenza virus and host innate immunity.IMPORTANCEInfluenza A virus (IAV) infection causes significant morbidity and mortality worldwide and remains a major health concern. When triggered by influenza viral infection, host cells produce type I interferon (IFN) to block viral replication. Although IAV was shown to have diverse strategies to evade this powerful, IFN-mediated antiviral response, it is not well-defined if IAV manipulates the IFN receptor-mediated signaling pathway. Here, we uncovered that influenza viral hemagglutinin (HA) protein causes the degradation of type I IFN receptor subunit 1 (IFNAR1). HA promoted phosphorylation and polyubiquitination of IFNAR1, which facilitated the degradation of this receptor. The HA-mediated elimination of IFNAR1 notably decreased the cells' sensitivities to type I IFNs, as demonstrated by the diminished expression of IFN-induced antiviral genes. This discovery could help us understand how IAV regulates the host innate immune response to create an environment optimized for viral survival in host cells.

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(In)validating experimentally derived knowledge about influenza A defective interfering particles

Journal of The Royal Society Interface ◽

10.1098/rsif.2016.0412 ◽

2016 ◽

Vol 13 (124) ◽

pp. 20160412 ◽

Cited By ~ 9

Author(s):

Laura E. Liao ◽

Shingo Iwami ◽

Catherine A. A. Beauchemin

Keyword(s):

Mathematical Model ◽

Experimental Method ◽

Influenza A Virus ◽

Influenza A ◽

Infectious Virus ◽

Full Length ◽

Prior Work ◽

Defective Interfering Particles ◽

A Cell ◽

Eclipse Phase

A defective interfering particle (DIP) in the context of influenza A virus is a virion with a significantly shortened RNA segment substituting one of eight full-length parent RNA segments, such that it is preferentially amplified. Hence, a cell co-infected with DIPs will produce mainly DIPs, suppressing infectious virus yields and affecting infection kinetics. Unfortunately, the quantification of DIPs contained in a sample is difficult because they are indistinguishable from standard virus (STV). Using a mathematical model, we investigated the standard experimental method for counting DIPs based on the reduction in STV yield (Bellett & Cooper, 1959, Journal of General Microbiology 21 , 498–509 ( doi:10.1099/00221287-21-3-498 )). We found the method is valid for counting DIPs provided that: (i) an STV-infected cell's co-infection window is approximately half its eclipse phase (it blocks infection by other virions before it begins producing progeny virions), (ii) a cell co-infected by STV and DIP produces less than 1 STV per 1000 DIPs and (iii) a high MOI of STV stock (more than 4 PFU per cell) is added to perform the assay. Prior work makes no mention of these criteria such that the method has been applied incorrectly in several publications discussed herein. We determined influenza A virus meets these criteria, making the method suitable for counting influenza A DIPs.

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