Rameix-Welti Marie-Anne, Munier Sandie, Le Gal Sebastien, Cuvelier Frederique, Agou Fabrice, Enouf Vincent, Naffakh Nadia, van der Werf Sylvie
Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, Département de Virologie, Paris, France.
Antivir Ther. 2011;16(4):597-603. doi: 10.3851/IMP1804.
During the 2007-2008 season, A(H1N1) viruses naturally resistant to oseltamivir due to an H275Y substitution in the neuraminidase emerged and spread in the human population. The neuraminidase of 2007-2008 A(H1N1) viruses has an increased affinity for sialic acids as compared with the N1 of previously circulating viruses.
Using site-directed mutagenesis analysis and an enzymatic assay on cells transiently expressing the viral neuraminidase, the amino acid changes that could account for the particular enzymatic properties of the neuraminidase of 2007-2008 A(H1N1) viruses were explored. The affinity for the substrate (K(m)) and the inhibition constants for inhibitors (K(i)) were determined for wild-type and mutated neuraminidases. Reverse genetics was used to produce 6:2 reassortant viruses expressing haemagglutinin and neuraminidase derived from A(H1N1) viruses of the 2007-2008 season or from a previously circulating H1N1 virus, in an A/WSN/33 background.
The D344N substitution characteristic of the N1 of 2007-2008 A(H1N1) viruses was identified as a major determinant of its increased affinity for sialic acids. According to the viral plaque phenotype of the 6:2 reassortant viruses, the H275Y mutation was deleterious when the surface glycoproteins were derived from the H1N1 virus isolated in 2004, but not when they were derived from A(H1N1) viruses of the 2007-2008 season.
The D344N substitution, by modifying the enzymatic property of the N1, may have favoured the emergence and spread of viruses naturally resistant to oseltamivir.
在2007 - 2008年流感季节，由于神经氨酸酶中H275Y替换而对奥司他韦天然耐药的A(H1N1)病毒在人群中出现并传播。与先前流行病毒的N1相比，2007 - 2008年A(H1N1)病毒的神经氨酸酶对唾液酸的亲和力增加。
利用定点诱变分析和对瞬时表达病毒神经氨酸酶的细胞进行酶活性测定，探索了可能导致2007 - 2008年A(H1N1)病毒神经氨酸酶特殊酶活性的氨基酸变化。测定了野生型和突变型神经氨酸酶对底物的亲和力（K(m)）和对抑制剂的抑制常数（K(i)）。采用反向遗传学方法，在A/WSN/33背景下，产生表达来自2007 - 2008年季节A(H1N1)病毒或先前流行的H1N1病毒的血凝素和神经氨酸酶的6:2重配病毒。
2007 - 2008年A(H1N1)病毒N1的D344N替换被确定为其对唾液酸亲和力增加的主要决定因素。根据6:2重配病毒的病毒蚀斑表型，当表面糖蛋白来自2004年分离的H1N1病毒时，H275Y突变是有害的，但当它们来自2007 - 2008年季节的A(H1N1)病毒时则不是。
D344N替换通过改变N1的酶活性，可能促进了对奥司他韦天然耐药病毒的出现和传播。
