POSTREASSORTMENT CHANGES IN INFLUENZA-A VIRUS HEMAGGLUTININ RESTORINGHA-NA FUNCTIONAL MATCH

An important function of influenza virus neuraminidase (NA) is the rem oval of sialic acid residues from virion components in order to preven t the aggregation of virus particles. In previous communications we ha ve reported that reassortant Viruses containing the NA gene of A/USSR/ 90/77 (H1N1) virus end HA genes of H3, H4, H10, or H13 subtypes had a tendency to virion aggregation at 4 degrees C and that the virion clus ters irreversibly dissociated after the treatment with bacterial neura minidase. It was concluded that in such reassortants the removal of si alic acid residues is inefficient. Nonaggregating variants of the reas sortants were selected in the course of serial passages in embryonated chicken eggs. In the present paper a reassortant virus, R2, having th e HA gene of A/Duck/Ukraine/1/63 (H3N8) virus and the other genes of A /USSR/90/77 (H1N1) virus, as well as its non-aggregating passage varia nts and both parent viruses, have been studied in order to reveal the presence of unremoved sialic acid residues in the virions. An assay of sialic acid content by high-performance liquid chromatography with fl uorescent detection has revealed the presence of sialic acid in the pu rified virus preparations of A/USSR/90/77 (H1N1) virus and the R2 reas sortant and its nonaggregating variants, whereas only trace amounts of sialic acid have been detected in the A/Duck/Ukraine/1/63 (H3N8) pare nt virus. The data obtained with the use of the labeled ''indicator'' virus suggest that the unremoved sialic acid residues are present at t he virion surface. The nonaggregating variants have been shown to poss ess a lower affinity toward high-molecular-weight sialic acid-containi ng substrates compared to the initial reassortant R2. Sequencing of HA genes has revealed amino acid changes in the nonaggregating variants compared to the initial reassortant. One substitution, N248D in HA1, i s the same in two independently selected nonaggregating variants. The presented data suggest that the complete removal of sialic acid residu es by viral NA from the virion components is not obligatory for the ab sence of virus particle aggregation: the latter may be achieved tin th e reassortants and, presumably, in the wild-type virus) through a bala nce between the degree of HA affinity toward the sialic acid-containin g receptors and the extent of the removal of sialic acid residues by N A. (C) 1998 Academic Press.