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.
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 CVN-MR 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.
Smee
Donald F
DF
Institute for Antiviral Research, Utah State University, Logan, UT, USA. dsmee@cc.usu.edu
Wandersee
Miles K
MK
Checketts
Melissa B
MB
O'Keefe
Barry R
BR
Saucedo
Carrie
C
Boyd
Michael R
MR
Mishin
Vasiliy P
VP
Gubareva
Larisa V
LV
D000222:Adaptation, Physiological / Q000187:drug effects*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000494:pharmacology*; D002352:Carrier Proteins / Q000494:pharmacology*; D002460:Cell Line; D024882:Drug Resistance, Viral / Q000187:drug effects* / Q000235:genetics; D005838:Genotype; D006031:Glycosylation / Q000187:drug effects; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D009981:Influenza B virus / Q000187:drug effects / Q000502:physiology; D008361:Mannosidases / Q000037:antagonists & inhibitors; D051379:Mice; D009154:Mutation / Q000187:drug effects; D010641:Phenotype; D012692:Serial Passage
D000998:Antiviral Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; C107733:cyanovirin N; D008361:Mannosidases