N-glycans attached to hemagglutinin in the head region and the stem domain control growth of influenza viruses by different mechanisms

Abstract

Background: The hemagglutinin (HA) of fowl plague virus A/FPV/Ro/34 (H7N1) contains seven N-glycans, two of which are flanking the receptor binding site and three being located in the stem domain. In the present study, we have elucidated the functional role of these five glycans in the course of virus replication in different hosts. Methods: Using a RNA polymerase I-based reverse genetics system, we generated recombinant viruses in which the HA mutants were stably incorporated in the background of the influenza reassortants WSN-HK and HK-WSN. Results: Growth of viruses lacking the glycans from the HA head region was impaired in MDCK cells as well as in embryonated chicken eggs. This restriction was due to limited release of progeny viruses from host cells as a result of the enhanced receptor affinity of the mutated HA. Accordingly, the high activity N2-subtype neuraminidase (NA) was able to partly overcome this effect, while the low activity N1-subtype NA was not. Thus, HA and NA activities need to be highly balanced to promote influenza viruses growth. When glycans were eliminated from the HA stem, the resulting viruses showed temperature sensitive growth in cell culture and in chicken eggs. The degree of growth restriction was dependent on the position from which the glycan had been removed. The viruses had a decreased pH stability, indicating that the stem glycans might maintain the HA in a fusion competent conformation. Viruses lacking the stem glycan from Asn 28 could not be rescued, indicating that this glycan is essential for the formation of replication competent viruses. Conclusions: Our results demonstrate that N-glycans linked to distinct HA domains regulate influenza virus growth at different stages of the replication cycle. Moreover, the deletion of glycan attachment sites from HA represents a powerful experimental strategy for the generation of attenuated influenza viruses.