Cellular and Molecular Basis of Infection

As molecular and biochemical sciences have evolved, a new theme has emerged in the literature focusing on understanding influenza infection from this perspective. This starts with chemical profiles of the virus and antigenic characterization of the hemagglutinin and neuraminidase surface proteins typically used to categorize influenza strains into an H#N# subtype, but develops into immunological, biochemical, and genetic study of influenza pathogenesis and function. This type of research still thrives today, with an incredible bank of molecular interactions identified – including interactions with the host cell immune pathways triggered by infection. Understanding the molecular basis for infection and host immune response is essential to creating novel drug therapies and preventing severe disease in influenza patients.

Figure 1. An overview of influenza infection, viral production, and viral release from Krammer et al. 2018.1 Following viral entry (mediated by HA), the incoming virus is incorporated into an endosome. RNPs are transported to the nucleus on release, where they are both replicated and transcribed for viral synthesis. Viral assembly occurs in the cytosol, and viral progeny leave the cell through a budding process mediated by NA. Abbreviations: nuclear export protein (NEP), neuraminidase (NA), hemagglutinin (HA), matrix protein (M1), ion channel (M2), nucleoprotein (NP), polymerase components (PA-x, PB1/PB1-F2, PB2), nonstructural protein 1 (NS1), poly-A tail (AAAA), 5′ cap (CAP), viral ribonucleoprotein complex (vRNP).

Like all viruses, influenza requires the machinery of a host cell to replicate, necessitating infection (in this case, of the respiratory epithelia). The virus typically makes contact with these tissues on inhalation of aerosolized viruses from another infected individual (or animal, though human to human spread is more common with H1N1 once the virus has crossed over to a human host).1 The initial viral-host interaction is mediated by the hemagglutinin (HA) protein (Fig. 2), which binds sialic acids of glycoprotein oligosaccharides on the host cell surface.1 The HA protein, initially referred to as HA0, must be cleaved into HA1 (Fig. 2, blue) and HA2 (Fig. 2, pink) subunits by cellular proteases to be functional. Availability of these proteases in part determines which cell types the virus can successfully infect. Interestingly, H1 HA proteins are cleaved by trypsin-like serine proteases at a monobasic cleavage site more typically associated with human influenza viruses and low pathogenicity in avian influenza viruses,7,8 as opposed to the highly pathogenic avian H5 and H7 subtypes which are cleaved at multi basic sites (MBCS) by ubiquitous furin-like proteases.7

Human influenza virus HAs preferentially bind α-2,6 linked sialic acids expressed on epithelial cells in the upper respiratory tract.1,2,3, 4 α-2,3 linked sialic acids are more common in the lower respiratory tract, typically preventing initial infection in these regions.3 This explains why, in most individuals, influenza presents with relatively mild upper respiratory tract irritation. It is thought that the preference for α-2,6 linked sialic acids over the α-2,3 linked alternative may be because aspartate residues near the binding site participate in additional stabilizing interactions not present with the α-2,3 variant.5,6

Figure 2. 1918 H1N1 hemagglutinin protein in its trimeric form, with H1A subunit in blue and H2A subunit in pink. Side, top, and bottom view. Images generated in UCSF Chimera. PBD 2WRG.16

Upon binding, the virus is internalized by a membranous endosome, which fuses with the viral envelope, releasing vRNPs into the cytosol. This fusion occurs in response to a conformational change in the HA protein, caused by the relatively acidic environment of the endosome.1 

The viral ribonucleoproteins (vRNPs) containing the viral RNAs, nucleoproteins (NPs), and the polymerase components PB1, PB2, and PA are released from the viral envelope and endosome, and trafficked to the nucleus for replication and transcription. To gain entry to the nucleus, NPs bind recruit the host protein importin-α, which is recognized by the importin-β receptor, guiding the RNP to nuclear pores for transport.19 Because influenza viruses cary negative sense RNA, the genomic RNA must be transcribed to positive-sense mRNA by polymerases before translation. Positive sense mRNA transcripts are capped and polyadenylated and transported to the cytosol for transcription.1 Replication is thought to be a significant step with regards to microevolution of the virus because the viral RNA polymerase lacks proofreading activity, leading to frequent mutation.11

In the nucleus, new vRNPs are synthesized and exported for viral assembly. The new virus then fuses with the host cell membrane for release (budding), where it can go on to infect other cells nearby.1 This process is mediated by the neuraminidase (NA) protein (EC 3.2.1.18), a glycosylase which cleaves sialic acids linking the budding virus to the membrane, releasing it.7,9 NA exists as a homotetramer tethered to the viral membrane. Its function relies on a catalytic Tyr residue, and all four monomers contain an active site. The enzyme’s calcium cofactor is thought to stabilize the quaternary structure.19 This enzyme is currently the primary target of antiviral drugs for influenza A viruses.1

Figure 3. H1N1 neuraminidase (NA) protein tetramer, with calcium cofactor in blue. Images generated in UCSF Chimera. PBD 2HTY.18

Interestingly, cholesterol plays an important role in mediating viral infection as well. The virus exploits a characteristic of the host cell membrane called lipid raft domains in both its entry and exit. These often cholesterol rich regions of the membrane are important to many cell signaling processes, including the host adaptive and innate immune response.10 The membrane makeup of certain enveloped viruses, including the influenza virus, is more consistent with these rafts than with the traditional phospholipid membrane, suggesting that these viruses likely exploit these rafts in the process of budding, or release from the cell.10 Thus, the virus relies on the cells cholesterol metabolism for effective infection. The IFITM3 gene mutation actually interferes with this process in the host, inhibiting viral proliferation.11 

This cellular invasion is countered by both innate and adaptive immune response in the host. The frequent genetic drift of the influenza A virus and somewhat ineffective annual vaccines mean that the initial adaptive immune response is relatively weak for influenza infections, as immunological memory is often weak or inaccurate.1 Host innate viral-sensing systems trigger interferon release through a variety of pathways, including the RIG-I-like receptor, toll-like receptor, Jak-STAT, and NOD-like receptor (inflammasome), MAPK, and apoptotic pathways,13 mechanisms which viruses must evade for successful infection and replication. The downstream products of these pathways (interferons and other signaling molecules) are what create most of the symptoms associated with influenza as the body tries to fight the infection.13 Infection may result in cytonecrosis of the respiratory epithelium, which is counteracted by macrophage mediated phagocytosis.11 The immune response is discussed at length on subsequent pages (see: Host Immunity & Preserving the Healthy State).

If the immune response is insufficient in restricting viral proliferation, infection can progress to more complicated cases, commonly involving pneumonia or acute respiratory distress syndrome (ARDS).1 Pneumonia may be primary (viral) or secondary (bacterial).11 Both of these conditions, as well as other complications of influenza, can lead to severe illness and sometimes death.1,11

Summary of Major Scientific Innovations:

  • 1889 – Early pathological studies identify infection of the pharynx and trachea11
  • 1918 – The respiratory epithelium is identified as the primary site of influenza infection11
  • 1941-42 – George K. Hirst suggests existence of the hemagglutinin protein14
    Named for the observation that red blood cells agglutinate in the presence of the virus 
  • 1943 – Early chemical characterization of influenza as a lipoprotein complex15
  • 1950s – Alfred Gottschalk identifies and characterizes the hemagglutinin and neuraminidase proteins13 
  • 1983 – Structure of NA protein published
  • 1988 – Structure of HA protein bound human receptor published16 
  • 1990s-present – Extensive mapping of infection mechanism and host immune pathways 1, 17

Works Cited

  1. Krammer, F.; Smith, G. J. D.; Fouchier, R. A. M.; Peiris, M.; Kedzierska, K.; Doherty, P. C.; Palese, P.; Shaw, M. L.; Treanor, J.; Webster, R. G.; et al. Influenza. Nat. Rev. Dis. Primer 2018, 4 (1), 1–21. https://doi.org/10.1038/s41572-018-0002-y.
  2. Fukuyama, S. and Kawaoka, Y. The pathogenesis of influenza virus infections: the contributions of virus and host factors. Curr Opin Immunol. 2012 23(4)481-486. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3163725/
  3. Denney, L. and Ho, L. The role of respiratory epithelium in host defence against influenza virus infection. Biomed J. 2018 41(4):218-233. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197993/
  4. Weis, W.; Brown, J.H; Cusack, S.; Paulson, J.C.; Skehel, J.J.; and Wiley, D.C. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature 1988, 333, 426-431. https://doi-org.muhlenberg.idm.oclc.org/10.1038/333426a0  
  5. Xu, R.; McBride, R; Nycholat, C.M.; Paulson, J.C.; and Wilson, I.A. Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic. J. Virol. 2011 doi: 10.1128/JVI.06322-11
  6. Xu, R.; and Wilson, I.A. Influenza hemagglutinin from the 2009 pandemic in complex with ligand 3SLN. PBD 2012. http://doi.org/10.2210/pdb3UBQ/pdb 
  7. Schrauwen, E.J.A.; de Graaf, M.; Herfst, S.; Rimmelzwaan, G.F.; Osterhaus, A.; and Fouchier, R.A.M. Determinants of virulence of influenza A virus. Eur J Clin Microbiol Infect Dis. 2014, 33(4): 479-490. https://dx.doi.org/10.1007%2Fs10096-013-1984-8 
  8. Bottcher-Friebertashauser, E.; Freur, C.; Sielaff, F.; Schmidt, S.; Eickmann, M.; Uhlendorff, J.; Steinmetzer, T.; Klenk, H.; and Garten, W. Cleavage of Influenza Virus Hemagglutinin by Airway Proteases TMPRSS2 and HAT Differs in Subcellular Localization And Susceptibility to Protease Inhibitors. J Virol. 2010, 84(11): 5605-5614. https://dx.doi.org/10.1128%2FJVI.00140-10 
  9. KEGG Enzyme. 3.2.1.18. https://www.kegg.jp/dbget-bin/www_bget?ec:3.2.1.18
  10. Mañes, S; del Real, G.; and Martinez, A. Pathogens: raft hijackers. Nature Rev. Immunology 2003, 3, 557-568. https://doi.org/10.1038/nri1129 
  11. Taubenberger, J.K. and Morens, D.M. The Pathology of Influenza Virus Infections. Annu Rev Pathol 2008 3:499-522. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2504709/
  12. Malosh, R.E.; Martin, E.T.; Ortiz, J.R.; and Monto, A.S. The risk of lower respiratory tract infection following influenza virus infection: A systematic and narrative review. Vaccine 2018 36(1):141-147.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736984/
  13. KEGG. Influenza A pathway.  https://www.kegg.jp/kegg-bin/show_pathway?hsa05164+H00398 
  14. Henry, R. and Frederick, M.A. Etymologia: Hemagglutinin and Neuraminidase. Emerg Infec Dis. 2018, 24(10), 1849. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6154157/
  15. Taylor, A.R., Sharp, D.G., McLean, I.W., Beard, D., Beard, J.W., Dingle, J.H., and Feller, E. Jr. Purification and Character of the Swine Influenza Virus. Science. 1943, 98(2557):587-89
  16. Liu, J.; Stevens, D.J.; Haire, L.F.; Walker, P.A.; Coombs, P.J.; Russell, R.J.; Gamblin, S.J.; and Skehel, J.J. Structure of H1 1918 hemagglutinin with human receptor. PBD 2009 http://doi.org/10.2210/pdb2WRG/pdb 
  17. KEGG. Influenza A pathway.  https://www.kegg.jp/kegg-bin/show_pathway?hsa05164+H00398
  18. Russell, R.J.; Haire, L.F.; Stevens, D.J.; Collins, P.J.; Lin, Y.P.; Blackburn, G.M.; Hay, A.J.; Gamblin, S.J.; and Skehel, J.J. N1 Neuraminidase (2HTY). PBD 2006. http://doi.org/10.2210/pdb2HTY/pdb
  19. Dou, D.; Revol, R.; Ostbye, H.; Wang, H.; and Daniels, R. Influenza A Virus Cell Entry, Replication, Virion Assembly and Movement. Front Immunol. 2018, 9: 1581. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062596/

For more information and resources, see Annotated Bibliography.

4 Replies to “Cellular and Molecular Basis of Infection”

  1. Great work! I’m so proud of you! This was a great project that was very easy to follow and had great detail! Can you explain why certain strains of influenza were initially able to infect only one species (e.g. bird, pig) and then became able to infect humans? Why were those strains specific to those species? What changes allow the virus to do this?

    1. Hi Katy!

      Thank you! One of the most common restriction factors preventing efficient transmission of influenza viruses between species is rooted in the hemagglutinin (HA)-host cell interaction. HA binding to host cell surface sialic acids is necessary for the virus to gain entry to the cell, but the details of this interaction vary across species despite the abundance of sialic acids present in all vertebrate systems. In birds, for example, this interaction most typically involves α-2,3 linked sialic acids (SAs), while α-2,6 linked SAs mediate this process in humans (this is driven by relative abundance in the host). Interestingly, pigs share a more even distribution of α-2,3 and α-2,6 linkages in their respiratory tract, allowing them to serve as “mixing vessels” for influenza viruses. Thus, mutations in the HA protein of an avian influenza A virus which promote binding to the α-2,6 moiety rather than the α-2,3 may be necessary to confer pathogenicity in humans. In nature, this could occur either by mutation or recombination during coinfection, or by a combination of both. More recent evidence suggests that optimal pH for viral-endosomal membrane fusion, which releases vRNPs into the cytoplasm, may also serve as such a restriction factor.

  2. This is a great, succinct explanation of the molecular basis of infection for viruses! I enjoyed reading your summary. Maybe you can clear up a few minor questions I had.

    1) Do the different H0 activating proteases that act on H1 HA vs H5 and H7 HAs distinguish the cell types they can infect (due to differential expression of these types of proteases), or just their pathogenicity?

    2) I’m a bit unclear on the steps between synthesis of vRNPs and viral release from the host. How do the vRNPs get recruited to the membrane? Is the viral membrane just a budded vesicle off the host membrane, or is it modified in a special way? Does the virus make its own SNAREs for release, or does it hijack the host’s SNAREs? Perhaps it buds without SNAREs because it doesn’t require vesicle fusion, just exocytosis?

    1. Thank you! And thank you for your comment, these are really interesting questions!. I hope you find these explanations helpful:

      1) While the activating proteases can certainly impact viral replication and pathogenicity, they do also determine what cells are capable of supporting viral growth. This is not, however, a significant point of diversity among influenza A viruses as most IAVs have hemagglutinin proteins capable of being cleaved by the trypsin-like proteases which are available in the respiratory and intestinal epithelia, and only the highly pathogenic H5 and H7 are capable of cleavage by the more ubiquitous proteases.

      2) This process is complex, and not entirely understood, but very interesting! The NS2 protein (non-structural protein 2; encoded in the influenza genome) is thought to mediate the release of vRNPs from the nucleus following replication, and there is evidence that suggests the M1 protein may play a role in recruiting vRNPs to the membrane prior to budding. The membrane of the virus is derived from the host membrane, but typically from specific lipid raft regions which concentrate HA and NA in the membrane to encourage budding. So, you could say that it is modified in some ways as it contains virus-specific components. While I am not particularly familiar with SNAREs, it appears that at least viral entry membrane fusion is mediated by a coiled-coil motif in HA similar the SNARE machinery of many eukaryotes. Budding is thought to be encouraged by an accumulation of viral proteins in that concentrated region of the membrane which force it to pinch away from the rest. So, in that sense, I think what you suggest about fusing without SNAREs is mostly correct.

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