Downloaded from www.microbiologyresearch.org by IP: 54.160.90.203 On: Thu, 30 Jun 2016 18:44:47 Journal of General Virology (2002), 83, 2683–2692. Printed in Great Britain ................................................................................................................................................................................................................................................................................... A release-competent influenza A virus mutant lacking the coding capacity for the neuraminidase active site Larisa V. Gubareva, 1,2 Marina S. Nedyalkova, 1,2 Dmitri V. Novikov, 1 K. Gopal Murti, 3 Erich Hoffmann 3 † and Frederick G. Hayden 1 1 Department of Internal Medicine, University of Virginia, 1300 Jefferson Park Avenue, Jordan Hall Room 2231, PO Box 800473, Charlottesville, VA 22908, USA 2 DI Ivanovsky Institute of Virology, 16 Gamaleya Str., Moscow 123098, Russia 3 Department of Virology and Molecular Biology, St Jude Children’s Research Hospital, 332 North Lauderdale Str., Memphis, TN 38108, USA Both influenza A virus surface glycoproteins, the haemagglutinin (HA) and neuraminidase (NA), interact with neuraminic acid-containing receptors. The influenza virus A/Charlottesville/31/95 (H1N1) has shown a substantially reduced sensitivity to NA inhibitor compared with the A/WSN/33 (H1N1) isolate by plaque-reduction assays in Madin–Darby canine kidney (MDCK) cells. However, there was no difference in drug sensitivity in an NA inhibition assay. The replacement of the HA gene of A/WSN/33 with the HA gene of A/Charlottesville/31/95 led to a drastic reduction in sensitivity of A/WSN/33 to NA inhibitor in MDCK cells. Passage of A/Charlottesville/31/95 in cell culture in the presence of an NA inhibitor resulted in the emergence of mutant viruses (delNA) whose genomes lacked the coding capacity for the NA active site. The delNA mutants were plaque- to-plaque purified and further characterized. The delNA-31 mutant produced appreciable yields ( 10 6 p.f.u./ml) in MDCK cell culture supernatants in the absence of viral or bacterial NA activity. Sequence analysis of the delNA mutant genome revealed no compensatory substitutions in the HA or other genes compared with the wild-type. Our data indicate that sialylation of the oligo- saccharide chains in the vicinity of the HA receptor-binding site of A/Charlottesville/31/95 virus reduces the HA binding efficiency and thus serves as a compensatory mechanism for the loss of NA activity. Hyperglycosylation of HA is common in influenza A viruses circulating in humans and has the potential to reduce virus sensitivity to NA inhibitors. Introduction Both influenza A virus surface glycoproteins, the haemag- glutinin (HA) and neuraminidase (NA), interact with neura- minic acid-containing receptors. The HA binds to neuraminic Author for correspondence : Larisa Gubareva (at University of Virginia). Fax 1 434 924 9065. e-mail LVG9Bvirginia.edu †Present address : MedImmune, Inc., 297 North Bernando Avenue, Mountain View, CA 94043, USA. The GenBank accession numbers of the sequences reported in this paper are A/Charlottesville/31/95 : PB1 AF398865, PB2 AF398866, PA AF398862, HA AF398878, NP AF398867, defective NA AF398870, M AF398876, NS AF398877 ; A/Charlottesville/28/95 : PA AF398864, HA AF398874, defective NA AF398873. acid-containing receptors and the NA destroys HA receptors by cleaving the terminal neuraminic acid residues from adjacent oligosaccharide chains. Both viral glycoproteins are sialylated by cellular enzymes, and the activity of viral NA is required to prevent self-aggregation of progeny virions and to promote release from the cellular membrane (Colman, 1994). Each subunit of the NA homotetramer consists of a cytoplasmic tail, a transmembrane domain, a stalk and a head region. The crystal structure of the NA head, which contains the enzyme active site, has been extensively studied and used in rational drug design (Colman, 1994). The recently developed NA inhibitors zanamivir and oseltamivir have demonstrated therapeutic benefit in clinical trails, and the inhibitor RWJ-270201 (BCX- 1812) is undergoing clinical evaluation (Gubareva et al., 2000). Influenza viruses have demonstrated a broad range of sensitivities to NA inhibitors in cell culture, despite the efficient 0001-8453  2002 SGM CGID