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Journal of Virology, July 2000, p. 6316-6323, Vol. 74, No. 14
Institut für Virologie,
Philipps-Universität, 35011 Marburg,1 and
Institut für Mikrobiologie und Molekularbiologie, Justus
Liebig-Universität Giessen, 35392 Giessen,2 Germany
Received 15 December 1999/Accepted 25 April 2000
The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1)
carries two N-linked oligosaccharides attached to Asn123 and Asn149 in
close vicinity to the receptor-binding pocket. In previous studies in
which HA mutants lacking either one (mutants G1 and G2) or both (mutant
G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity
(M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol. 71:8377-8384, 1997). We have now investigated the effect of
these mutations on virus growth using recombinant viruses generated by
an RNA polymerase I-based reverse genetics system. Two reassortants of
influenza virus strain A/WSN/33 were used as helper viruses to obtain
two series of HA mutant viruses differing only in the neuraminidase
(NA). Studies using N1 NA viruses revealed that loss of the
oligosaccharide from Asn149 (mutant G2) or loss of both
oligosaccharides (mutant G1,2) has a pronounced effect on virus growth
in MDCK cells. Growth of virus lacking both oligosaccharides from
infected cells was retarded, and virus yields in the medium were
decreased about 20-fold. Likewise, there was a reduction in plaque size
that was distinct with G1,2 and less pronounced with G2. These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1
recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that
N-glycans flanking the receptor-binding site of the HA molecule are
potent regulators of influenza virus growth, with the glycan at Asn149
being dominant and that at Asn123 being less effective. In addition, we
show here that HA and NA activities need to be highly balanced in order
to allow productive influenza virus infection.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Interdependence of Hemagglutinin Glycosylation and Neuraminidase
as Regulators of Influenza Virus Growth: a Study by Reverse
Genetics

*
Corresponding author. Mailing address: Institut
für Virologie, Philipps-Universität Marburg, Postfach 2360, 35011 Marburg, Germany. Phone: 49/6421/28-66253. Fax: 49/6421/28-68962.
E-mail: Klenk{at}mailer.uni-marburg.de.
Present address: Robert-Koch-Institut, 13353 Berlin, Germany.
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