R. Wagner et al., Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics, J VIROLOGY, 74(14), 2000, pp. 6316-6323
The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries
two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicin
ity 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 the
se glycans regulate receptor binding affinity (M, Ohuchi, R. Ohuchi, A. Fel
dmann, and H. D. Klenk, J. Virol, 71:8377-8384, 1997). We have now investig
ated the effect of these mutations on virus growth using recombinant viruse
s generated by an RNA polymerase I-based reverse genetics system. Two reass
ortants of influenza virus strain A/WSN/33 were used as helper viruses to o
btain 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 i
n plaque size that was distinct with G1,2 and less pronounced,vith G2, Thes
e effects could be attributed to a highly impaired release of mutant progen
y 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 rece
ptor. These results demonstrate that N-glycans flanking the receptor bindin
g 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 effe
ctive. In addition, we show here that HA and NA activities need to be highl
y balanced in order to allow productive influenza virus infection.