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Event: 2192

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Influenza A Virus production increased

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. More help
IAV production increased
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Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help
Level of Biological Organization
Molecular

Cell term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Cell term
respiratory epithelial cell

Organ term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Organ term
respiratory tract

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor).  Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description.  To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons.  If a desired term does not exist, a new term request may be made via Term Requests.  Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Process Object Action
protein import into nucleus viral RNA-directed RNA polymerase complex occurrence
transcription, RNA-templated segmented viral genome occurrence
viral mRNA export from host cell nucleus occurrence
virus maturation occurrence

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE.Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
IAV infection proliferation KeyEvent Jessica Resnick (send email) Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help

Life Stages

An indication of the the relevant life stage(s) for this KE. More help

Sex Applicability

An indication of the the relevant sex for this KE. More help

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

The major stages of Influenza A virus (IAV) replication, which could each be multiple KE's on their own but are summarized here, include trafficking to the host cell nucleus, replication of vRNAs, viral mRNA transcription, assembly and trafficking of vRNPs, ER targeting and maturation, and HA proteolytic activation at the Golgi or Plasma membrane (1). 

The current model of IAV replication is vRNP entry into the nucleus via the importin-alpha-importin-beta nuclear import pathway within one hour (1-9). However, this step has been shown to contribute to host restriction so it is worth noting that a majority of this work has been performed in immortalized cell lines from various species (1).

Replication of vRNAs occurs in two steps- transcription of the reverse complement (cRNA) followed by transcription of new genome copies using the cRNAs as templates- in the nucleus facilitated by the heterotrimeric viral RNA-dependent RNA polymerase (1). The transcription process is unprimed and reliant on 13 conserved nucleotides at the 5’ end and 12 nucleotides at the 3’ end of each segment that are partially complementary and form a double-stranded “promoter” by base-pairing (10). This “promoter” is recognized by the viral RNA polymerase and the template is transcribed.

Viral mRNA transcription, in contrast, is primed by a process known as “cap- snatching” from host mRNAs (11,12) and polyadenylated through stuttering (13,14). Two segments- M and NS- are spliced (15). NS transcripts have a balanced ratio of spliced to un spliced throughout infection but the ratio of spliced M (M2) increases through infection, although the efficiency differs between strains and also functions in host restriction (16-19).

Following nuclear export, translation of the viral mRNAs is divided between cytosolic ribosomes (internal proteins PB1, PB2, PA, NP, NS1, NS2, and M1) and ER- associated ribosomes (membrane proteins- HA, NA, and M2) (1). The NP and polymerase subunits (PB1, PB2, PA) traffic back to the nucleus to assist in viral mRNA transcription and vRNA replication (1) while NS1 is also trafficked to the nucleus to inhibit interferon signaling (see KE 2180). vRNPs are trafficked to the plasma membrane to meet the other proteins for viral assembly by Rab11, which associates with Pb2 to ensure that all new virions incorporated vRNPs carrying a polymerase (20).

During translocation within the ER, the N-terminus of HA and M2 is directly translocated into the ER lumen, while the NA C-terminus is positioned in the ER lumen (1). Both HA and NA receive multiple N-linked glycans which have been shown to affect activity and antigenicity but mainly function in folding efficiency (21-24). The HA protein is formed through trimerization of independently folded monomers while the NA tetramer results from association of co-translationally formed dimers (21,25,26,27). An abundance of HA and NA is produced during infection to promote oligomerization but consequences of this approach include triggering of the ER stress response, which must be blocked, and syncytia formation, which promotes cell to cell transmission (1,28,29,30)

The HA surface protein is synthesized as an inactive form denoted HA0 (1, 31, 32, 33). HA0 is then proteolytically activated through cleavage into HA1 and HA2 active subunits by trypsin-like proteases (1,31,32,33,34). IAV is fairly promiscuous in its use of these proteases, and the cleavage occurs in a multi- or mono- basic site on the HA0 protein (34,35). Highly pathogenic avian IAVs tend to have a multibasic cleavage site that is targeted by furin (36). Human and low pathogenic avian viruses tend to contain a monobasic cleavage site and utilize any available protease such as TMRPSS2, TMPRSS4, and HAT for proteolytic activation (37,38).

 

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

Technique

Conclusions/ uses

Example Reference

Immunofluorescence microscopy

Visualization of infected cells, can target specific proteins or processes
  1. Martin K, Helenius A. Transport of incoming influenza virus nucleocapsids into the nucleus. J Virol (1991) 65:232–44.
  2. O’Neill RE, Jaskunas R, Blobel G, Palese P, Moroianu J. Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import. J Biol Chem (1995) 270:22701–4. doi:10.1074/jbc.270.39.22701

In situ hybridization

Track localization of RNA viral genomes in cells

Dou D, Hernandez-Neuta I, Wang H, Ostbye H, Qian X, Thiele S, et al. Analysis of IAV replication and co-infection dynamics by a versatile RNA viral genome labeling method. Cell Rep (2017) 20:251–63. doi:10.1016/j.celrep.2017.06.021

In vitro polyadenylation assay

Identify sequences important for polyadenylation

Poon LL, Pritlove DC, Fodor E, Brownlee GG. Direct evidence that the poly(A) tail of influenza A virus mRNA is synthesized by reiterative copying of a U track in the virion RNA template. J Virol (1999) 73:3473–6.

Plasmid expression measured by Northern Blot

Analyze differences in splicing efficiencies

Backstrom Winquist E, Abdurahman S, Tranell A, Lindstrom S, Tingsborg S, Schwartz S. Inefficient splicing of segment 7 and 8 mRNAs is an inherent property of influenza virus A/Brevig Mission/1918/1 (H1N1) that causes elevated expression of NS1 protein. Virology (2012) 422:46–58. doi:10.1016/j.virol.2011.10.004

Single molecule FRET

Molecule- molecule interactions

Robb NC, Te Velthuis AJ, Wieneke R, Tampé R, Cordes T, Fodor E, Kapanidis AN. Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA. Nucleic Acids Res. 2016 Dec 1;44(21):10304-10315. doi: 10.1093/nar/gkw884. Epub 2016 Sep 30. PMID: 27694620; PMCID: PMC5137447.

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  More help

References

List of the literature that was cited for this KE description. More help
  1. Dou, D., Revol, R., Ostbye, H., Wang, H., and Daniels, R., Influenza A Virus Cell Entry, Replication, Virion Assembly and Movement. Front. Immunol.  20 July 2018. https://doi.org/10.3389/fimmu.2018.01581
  2. Martin K, Helenius A. Transport of incoming influenza virus nucleocapsids into the nucleus. J Virol (1991) 65:232–44.
  3. Kemler I, Whittaker G, Helenius A. Nuclear import of microinjected influenza virus ribonucleoproteins. Virology (1994) 202:1028–33. doi:10.1006/viro.1994.1432
  4. O’Neill RE, Jaskunas R, Blobel G, Palese P, Moroianu J. Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import. J Biol Chem (1995) 270:22701–4. doi:10.1074/jbc.270.39.22701
  5. Wang P, Palese P, O’Neill RE. The NPI-1/NPI-3 (karyopherin alpha) binding site on the influenza a virus nucleoprotein NP is a nonconventional nuclear localization signal. J Virol (1997) 71:1850–6.
  6. Cros JF, Garcia-Sastre A, Palese P. An unconventional NLS is critical for the nuclear import of the influenza A virus nucleoprotein and ribonucleoprotein. Traffic (2005) 6:205–13. doi:10.1111/j.1600-0854.2005.00263.x
  7. Wu WW, Weaver LL, Pante N. Ultrastructural analysis of the nuclear localization sequences on influenza A ribonucleoprotein complexes. J Mol Biol (2007) 374:910–6. doi:10.1016/j.jmb.2007.10.022
  8. Chou YY, Heaton NS, Gao Q, Palese P, Singer RH, Lionnet T. Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis. PLoS Pathog (2013) 9:e1003358. doi:10.1371/journal.ppat.1003358
  9. Dou D, Hernandez-Neuta I, Wang H, Ostbye H, Qian X, Thiele S, et al. Analysis of IAV replication and co-infection dynamics by a versatile RNA viral genome labeling method. Cell Rep (2017) 20:251–63. doi:10.1016/j.celrep.2017.06.021
  10. Robb NC, Te Velthuis AJ, Wieneke R, Tampe R, Cordes T, Fodor E, et al. Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA. Nucleic Acids Res (2016) 44:10304–15. doi:10.1093/nar/gkw884
  11. Reich S, Guilligay D, Pflug A, Malet H, Berger I, Crepin T, et al. Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature (2014) 516:361–6. doi:10.1038/nature14009
  12. Plotch SJ, Bouloy M, Ulmanen I, Krug RM. A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell (1981) 23:847–58. doi:10.1016/0092-8674(81)90449-9
  13. Robertson JS, Schubert M, Lazzarini RA. Polyadenylation sites for influenza virus mRNA. J Virol (1981) 38:157–63.
  14. Poon LL, Pritlove DC, Fodor E, Brownlee GG. Direct evidence that the poly(A) tail of influenza A virus mRNA is synthesized by reiterative copying of a U track in the virion RNA template. J Virol (1999) 73:3473–6.
  15. Dubois J, Terrier O, Rosa-Calatrava M. Influenza viruses and mRNA splicing: doing more with less. MBio (2014) 5:e70–14. doi:10.1128/mBio.00070-14
  16. Inglis SC, Brown CM. Differences in the control of virus mRNA splicing during permissive or abortive infection with influenza A (fowl plague) virus. J Gen Virol (1984) 65(Pt 1):153–64. doi:10.1099/0022-1317-65-1-153
  17. Valcarcel J, Portela A, Ortin J. Regulated M1 mRNA splicing in influenza virus-infected cells. J Gen Virol (1991) 72(Pt 6):1301–8. doi:10.1099/0022-1317-72-6-1301
  18. Robb NC, Fodor E. The accumulation of influenza A virus segment 7 spliced mRNAs is regulated by the NS1 protein. J Gen Virol (2012) 93:113–8. doi:10.1099/vir.0.035485-0
  19. Backstrom Winquist E, Abdurahman S, Tranell A, Lindstrom S, Tingsborg S, Schwartz S. Inefficient splicing of segment 7 and 8 mRNAs is an inherent property of influenza virus A/Brevig Mission/1918/1 (H1N1) that causes elevated expression of NS1 protein. Virology (2012) 422:46–58. doi:10.1016/j.virol.2011.10.004
  20. Amorim MJ, Bruce EA, Read EK, Foeglein A, Mahen R, Stuart AD, et al. A Rab11- and microtubule-dependent mechanism for cytoplasmic transport of influenza A virus viral RNA. J Virol (2011) 85:4143–56. doi:10.1128/JVI.02606-10
  21. Wang N, Glidden EJ, Murphy SR, Pearse BR, Hebert DN. The cotranslational maturation program for the type II membrane glycoprotein influenza neuraminidase. J Biol Chem (2008) 283:33826–37. doi:10.1074/jbc.M806897200
  22.  Daniels R, Svedine S, Hebert DN. N-linked carbohydrates act as lumenal maturation and quality control protein tags. Cell Biochem Biophys (2004) 41:113–38. doi:10.1385/CBB:41:1:113
  23. Hebert DN, Zhang JX, Chen W, Foellmer B, Helenius A. The number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin. J Cell Biol (1997) 139:613–23. doi:10.1083/jcb.139.3.613
  24. Powell H,  Pekosz A. Neuraminidase antigenic drift of H3N2 clade 3c.2a viruses alters virus replication, enzymatic activity, and inhibitory antibody binding. Plos Pathogens (2020). https://doi.org/10.1371/journal.ppat.1008411
  25. Saito T, Taylor G, Webster RG. Steps in maturation of influenza A virus neuraminidase. J Virol (1995) 69:5011–7.
  26. da Silva DV, Nordholm J, Madjo U, Pfeiffer A, Daniels R. Assembly of subtype 1 influenza neuraminidase is driven by both the transmembrane and head domains. J Biol Chem (2013) 288:644–53. doi:10.1074/jbc.M112.424150
  27. da Silva DV, Nordholm J, Dou D, Wang H, Rossman JS, Daniels R. The influenza virus neuraminidase protein transmembrane and head domains have coevolved. J Virol (2015) 89:1094–104. doi:10.1128/JVI.02005-14
  28. Hassan IH, Zhang MS, Powers LS, Shao JQ, Baltrusaitis J, Rutkowski DT, et al. Influenza A viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (IRE1) stress pathway. J Biol Chem (2012) 287:4679–89. doi:10.1074/jbc.M111.284695
  29. Roberson EC, Tully JE, Guala AS, Reiss JN, Godburn KE, Pociask DA, et al. Influenza induces endoplasmic reticulum stress, caspase-12-dependent apoptosis, and c-Jun N-terminal kinase-mediated transforming growth factor-beta release in lung epithelial cells. Am J Respir Cell Mol Biol (2012) 46:573–81. doi:10.1165/rcmb.2010-0460OC
  30. Cifuentes-Muñoz N, Dutch RE, Cattaneo R. Direct cell-to-cell transmission of respiratory viruses: The fast lanes. PLoS Pathog. 2018 Jun 28;14(6):e1007015. doi: 10.1371/journal.ppat.1007015. PMID: 29953542; PMCID: PMC6023113.
  31. Klenk HD, Rott R, Orlich M, Blodorn J.Activation of influenza A viruses by trypsin treatment. Virology (1975) 68:426–39. 10.1016/0042-6822(75)90284-6
  32. Huang RT, Rott R, Klenk HD. Influenza viruses cause hemolysis and fusion of cells. Virology (1981) 110:243–7. 10.1016/0042-6822(81)90030-1
  33. Maeda T, Kawasaki K, Ohnishi S. Interaction of influenza virus hemagglutinin with target membrane lipids is a key step in virus-induced hemolysis and fusion at pH 5.2. Proc Natl Acad Sci U S A (1981) 78:4133–7. 10.1073/pnas.78.7.4133
  34. Kido,  H., Okumura,  Y., Yamada,  H., Quang Le,  T. & Yano, M. Proteases essential for human influenza virus entry into cells and their inhibitors as potential therapeutic agents. Curr Pharm Des 13, 405–414 (2007))
  35. Bottcher-Friebertshauser E, Garten W, Matrosovich M, Klenk HD. The hemagglutinin: a determinant of pathogenicity. Curr Top Microbiol Immunol (2014) 385:3–34. 10.1007/82_2014_384 
  36. Stieneke-Grober A, Vey M, Angliker H, Shaw E, Thomas G, Roberts C, et al. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO J (1992) 11:2407–14.
  37. Bottcher E, Matrosovich T, Beyerle M, Klenk HD, Garten W, Matrosovich M. Proteolytic activation of influenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium. J Virol (2006) 80:9896–8. 10.1128/JVI.01118-06 
  38. Chaipan C, Kobasa D, Bertram S, Glowacka I, Steffen I, Tsegaye TS, et al. Proteolytic activation of the 1918 influenza virus hemagglutinin. J Virol (2009) 83:3200–11. 10.1128/JVI.02205-08
  39. Lingwood D, Simons K. Lipid rafts as a membrane-organizing principle. Science (2010) 327:46–50. 10.1126/science.1174621
  40. Chen BJ, Leser GP, Morita E, Lamb RA. Influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles. J Virol (2007) 81:7111–23. 10.1128/JVI.00361-07
  41. Lai JC, Chan WW, Kien F, Nicholls JM, Peiris JS, Garcia JM. Formation of virus-like particles from human cell lines exclusively expressing influenza neuraminidase. J Gen Virol (2010) 91:2322–30. 10.1099/vir.0.019935-0
  42. Yondola MA, Fernandes F, Belicha-Villanueva A, Uccelini M, Gao Q, Carter C, et al. Budding capability of the influenza virus neuraminidase can be modulated by tetherin. J Virol (2011) 85:2480–91. 10.1128/JVI.02188-10
  43. Chlanda P, Schraidt O, Kummer S, Riches J, Oberwinkler H, Prinz S, et al. Structural analysis of the roles of influenza A virus membrane-associated proteins in assembly and morphology. J Virol (2015) 89:8957–66. 10.1128/JVI.00592-15
  44. Elleman CJ, Barclay WS. The M1 matrix protein controls the filamentous phenotype of influenza A virus. Virology (2004) 321:144–53. 10.1016/j.virol.2003.12.009
  45. Rossman JS, Lamb RA. Viral membrane scission. Annu Rev Cell Dev Biol (2013) 29:551–69. 10.1146/annurev-cellbio-101011-155838
  46. Webster RG, Laver WG. Preparation and properties of antibody directed specifically against the neuraminidase of influenza virus. J Immunol(1967) 99:49–55.
  47. Palese P, Compans RW. Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA): mechanism of action. J Gen Virol (1976) 33:159–63. 10.1099/0022-1317-33-1-159