Influenza A (H1N1) Virus Resistance to Cyanovirin-N Arises Naturally During Adaptation to Mice and by Passage in Cell Culture in the Presence of the Inhibitor

Authors

Donald F. Smee, Utah State UniversityFollow
Miles K. Wandersee, Utah State University
Melissa B. Checketts, Utah State University
Barry R. O'Keefe, NCI-Frederick
Carrie Saucedo, NCI-Frederick
Michael R. Boyd, University of South Alabama
Vasiliy P. Mishin, University of Virginia
Larisa V. Gubareva, University of Virginia

Document Type

Article

Journal/Book Title/Conference

Antiviral Chemistry and Chemotherapy

Volume

18

Issue

6

Publisher

International Medical Press

Publication Date

12-2007

First Page

317

Last Page

327

Abstract

Influenza A/New Caledonia/20/99 (H1N1) virus was studied for development of resistance to cyanovirin-N (CVN). CVN neutralizes virus infectivity by binding to specific high-mannose oligosaccharides on the viral haemagglutinin 1 (HA1) subunit. During virus adaptation to mice in the absence of CVN treatment the virus became resistant to CVN (CVN-MR virus), as did virus passaged in cell culture in the presence of CVN (CVN-R virus). The CVN-R virus possessed a single amino acid change at position 94a (Asn94aAsp) of HA1 that eliminated this glycosylation site. The CVN-MR virus at mouse passage 7 was a mixture of clones, consisting of a single mutation (Asp225Gly) and double mutations (Asn63Ser+Asp225Gly or Asn94a+Asp225Gly), eliminating glycosylation sites. CVN did not bind well to the CVN-R and CVN-MR viruses. Propagating these viruses in cells treated with 1 mM deoxymannojirimycin (dMJ, mannosidase inhibitor) increased sensitivity to CVN, suggesting that glycans attached at other sites on HA1 that typically are not high-mannosidic became so due to dMJ treatment. Further evaluation showed that the Asp225Gly mutant virus was sensitive to the inhibitor and did not kill mice or induce weight loss. The CVN-R virus was also avirulent to mice. The double-mutant CVNMR viruses were resistant to CVN and caused deaths and severe weight loss in mice. CVN-R virus subjected to mouse adaptation acquired the 225 mutation and a lethal phenotype. Thus, the 225 mutation in the HA receptor-binding site in combination with a loss of glycan at Asn (63 or 94a) are important for mouse adaptation in this virus. The mutations reported here causing resistance to CVN are consistent with its known mode of action.

Comments

Originally published by International Medical Press. Publisher's PDF available through remote link.

Recommended Citation

Smee, D.F., M.K. Wandersee, M.B. Checketts, B.R. O'Keefe, C. Saucedo, M.R. Boyd, V.P. Mishin, and L.V. Gubareva 2008. Influenza A (H1N1) virus resistance to cyanovirin-N arises naturally during adaptation to mice and by cell culturing in the presence of the inhibitor Antiviral Chem. Chemother. 18:317-327.