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In this study, we investigated the role of the conserved neuraminidase (NA) cytoplasmic tail residues in influenza virus replication. Mutants of influenza A virus (A/WSN/33 [H1N1]) with deletions of the NA cytoplasmic tail region were generated by reverse genetics. The resulting viruses, designated NOTAIL, contain only the initiating methionine of the conserved six amino-terminal residues. The mutant viruses grew much less readily and produced smaller plaques than did the wild-type virus. Despite similar levels of NA cell surface expression by the NOTAIL mutants and wild-type virus, incorporation of mutant NA molecules into virions was decreased by 86%. This reduction resulted in less NA activity per virion, leading to the formation of large aggregates of progeny mutant virions on the surface of infected cells. A NOTAIL virus containing an additional mutation (Ser-12 to Pro) in the transmembrane domain incorporated three times more NA molecules into virions than did the NOTAIL parent but approximately half of the amount incorporated by the wild-type virus. However, aggregation of the progeny virions still occurred at the cell surface. All NOTAIL viruses were attenuated in mice. We conclude that the cytoplasmic tail of NA is not absolutely essential for virus replication but exerts important effects on the incorporation of NA into virions and thus on the aggregation and virulence of progeny virus. In addition, the relative abundance of long filamentous particles formed by the NOTAIL mutants, compared with the largely spherical wild-type particles, indicates a role for the NA cytoplasmic tail in virion morphogenesis.

This work had two purposes: (i) to determine in vivo whether liver phenylalanine hydroxylase (PAH) is regulated by its substrates phenylalanine and tetrahydrobiopterin (BH4) as studies with purified enzyme suggest and (ii) to investigate in vivo the relationship between PAH activity and BH4 turnover. We found there are two BH4 pools in hepatocytes, one that is metabolically available (free BH4) and one that is not (bound BH4). Bound BH4 appears bound to PAH; the PAH-BH4 complex has much less catalytic activity and is less readily phenylalanine activated than uncomplexed enzyme. Interconversion of activated and unactivated PAH and bound and free BH4 is driven by phenylalanine; and free BH4 concentration is determined by the state of activation and activity of PAH. In hepatocytes, BH4 and PAH (subunit) concentrations are equal, all intracellular BH4 appears to be available to PAH, and free BH4 turns over rapidly (t1/2 approximately 1 hr). There is no evidence for feedback inhibition of BH4 synthesis; the BH4 synthetic rate appears high when free BH4 concentration is high and low when free BH4 is low. The data provide support in vivo that phenylalanine and BH4 are positive and negative regulators of the activity and activation state of PAH in the proposed manner; they also imply that regulation of BH4 turnover and PAH activity are linked processes, which are both controlled by phenylalanine concentration.