Amino Acid Gymnastics and the Evolution of Influenza Virus Resistance to Oseltamivir

In previous posts, I discussed, respectively, the use of selection to generate an antibody of potential value in treating influenza A virus infections (1) and the relevance of protein dynamics to the evolution of protein function (2).   A recent paper in Science (3) offers evidence suggesting that internal protein dynamics play a crucial role in shaping the evolution and spread of resistance to the influenza neuraminidase inhibitor, oseltamivir (Tamiflu®).

Influenza A viruses express two types of glycoprotein “spikes” on the virion surface: hemagglutinin (HA  or H) and neuraminidase (NA or N).  On the way in, the HA molecules serve to attach the virus to host cells by binding to sialic acid-containing cell surface receptors, thereby initiating entry into the cell where the process of viral replication can begin.  On the way out, the NA molecules cleave host cell sialic acid receptors so that the HA molecules do not prevent the virion from detaching and seeking out new cells to infect.

Bloom et al., investigate the molecular basis for the increase in frequency, since 2007, of oseltamivir-resistant influenza A viruses containing N1 neuraminidase.  Previous clinical testing (4) had revealed that mutation (H274Y) of the histidine (H) at NA amino acid 274 to tyrosine (Y) was associated with resistance to oseltamivir.  Since this mutation was also associated with reduced viral fitness as assessed in vitro in tissue culture and in vivo in mice and ferrets, in multiple published studies (5-7), it was thought that it would not be likely to spread to a clinically-significant degree.  However, during the 2007-2008 influenza season oseltamivir-resistant H1N1 influenza viruses began to be isolated around the world.  Therefore, Bloom et al. (from David Baltimore’s lab at Caltech) hypothesized that additional mutations in the N1 molecules permitted the H274Y mutation to occur, thereby conferring resistance against oseltamivir without compromising viral fitness.

The authors of the Science paper present evidence indicating that the H274Y mutation does not undermine viral fitness by reducing the enzymatic activity per se but by reducing the number of NA molecules that reach the cell (and therefore virion) surface.  They went on to use computational methods to predict a mutation that would restore wild-type levels of NA expression and proceeded to show that this mutation R194G both increased NA expression and fitness to roughly wild-type levels. 

Analysis of a phylogenetic tree for NAs in seasonal H1N1 viruses from 2006 and later led to identification of two candidate mutations (V234M and R222Q) that could have permitted the occurrence of the H274Y mutation without substantial loss of viral fitness among naturally circulating viruses.  Additional studies verified that both of these mutations restored both total NA enzymatic activity and replication ability.

Both the H274Y mutation and the V234M and R222Q mutations appear to mediate their effects by altering interactions among amino acids in the NA polypeptide chains.  In the former case, H274Y prevents a flipping motion of the E276 side chain that is necessary for oseltamivir to bind to NA (8-9, reviewed in 10).  The V234M and R222Q mutations are hypothesized to counter negative effects on folding or stability of NA by H274Y (1).  Thus, in this instance, evolutionary biology, virology, and pharmacology grade into biophysics.

References

1. Greenspan, N. Application of selection to a clinically-important infectious disease. http://evomed.org/?p=145.

2. Greenspan, N. Protein dynamics and evolution. http://evomed.org/?p=151.

3. Bloom JD, Gong LI, Baltimore D. Permissive secondary mutations enable the evolution of influenza oseltamivir resistance. Science. 2010 Jun 4;328(5983):1272-5. PubMed PMID: 20522774.

4. Gubareva LV, Kaiser L, Matrosovich MN, Soo-Hoo Y, Hayden FG. Selection of influenza virus mutants in experimentally infected volunteers treated with oseltamivir. J Infect Dis. 2001 Feb 15;183(4):523-31. Epub 2001 Jan 11. PubMed PMID: 11170976.

5. Ives JA, Carr JA, Mendel DB, Tai CY, Lambkin R, Kelly L, Oxford JS, Hayden FG, Roberts NA. The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo. Antiviral Res. 2002 Aug;55(2):307-17. PubMed PMID: 12103431.

6. Abed Y, Goyette N, Boivin G. A reverse genetics study of resistance to neuraminidase inhibitors in an influenza A/H1N1 virus. Antivir Ther. 2004 Aug;9(4):577-81. PubMed PMID: 15456089.

7. Herlocher ML, Truscon R, Elias S, Yen HL, Roberts NA, Ohmit SE, Monto AS. Influenza viruses resistant to the antiviral drug oseltamivir: transmission studies in ferrets. J Infect Dis. 2004 Nov 1;190(9):1627-30. Epub 2004 Sep 28. PubMed PMID: 15478068.

8. Russell RJ, Haire LF, Stevens DJ, Collins PJ, Lin YP, Blackburn GM, Hay AJ, Gamblin SJ, Skehel JJ. The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature. 2006 Sep 7;443(7107):45-9. Epub 2006 Aug 16. PubMed PMID: 16915235.

9. Collins PJ, Haire LF, Lin YP, Liu J, Russell RJ, Walker PA, Skehel JJ, Martin SR, Hay AJ, Gamblin SJ. Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants. Nature. 2008 Jun 26;453(7199):1258-61. Epub 2008 May 14. PubMed PMID: 18480754.

10. Moscona A. Oseltamivir resistance–disabling our influenza defenses. N Engl J Med. 2005 Dec 22;353(25):2633-6. PubMed PMID: 16371626. https://cpff.ca/wp-content/languages/new/plavix.html
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