Degradation of Mutant Influenza Virus Hemagglutinins Is Influenced by Cytoplasmic Sequences Independent of Internalization Signals

Abstract

A mutant influenza virus hemagglutinin, HA؉8, having a carboxyl-terminal extension of 8 amino acids that included 4 aromatic residues, was internalized within 2 min of arriving at the cell surface and was degraded quickly by a process that was inhibited by ammonium chloride. Through second-site mutagenesis, the internalization sequence of HA؉8 was found to closely resemble the internalization signals of the transferrin receptor or large mannose 6-phosphate receptor. Comparison of the intracellular traffic of HA؉8 and a series of other HA mutants that differed in their rates of internalization revealed a relation between the amount of the protein on the plasma membrane at steady state and the internalization rate that would be predicted if most of each protein recycled to the cell surface. However, there was no simple correlation between the internalization rate and the rate of degradation, indicating that transport to the compartment where degradation occurred was not simply a function of the concentration of the proteins in early endosomes. The internal populations of both HA؉8, which was degraded with a t 1/2 of 1.9 h, and HA-Y543, which was degraded with a t 1/2 of 2.9 h, were found by cell fractionation and density-shift experiments to reside in early endosomes with little accumulation in lysosomes. A fluid-phase marker reached lysosomes 3-4-fold faster than these proteins were degraded. Degradation of these mutant HAs involved a rate-determining step in early endosomes that was sensitive to some feature of the protein that depended upon sequence differences in the cytoplasmic domain unrelated to the internalization signal.

Dynamic or Stable Interactions of Influenza Hemagglutinin Mutants with Coated Pits

“…This is in line with former observations (25) on HA-Y543, which demonstrated temperature-dependent dynamic interactions with coated pits. As for HAϩ4, whose internalization is extremely slow (28), this mutant did not show detectable reduction in its lateral mobility, and diffused in the membrane in an unrestricted manner, just like HA wt.…”

“…The mutants are depicted in Table I. The derivation of the mutants HAϩ8 (containing a carboxyl-terminal extension of eight amino acids with four aromatic residues, generating a strong internalization signal), HAϩ4 (which is HAϩ8 minus the last four residues), and HAϩ8-S548,S554 (HAϩ8 with the tyrosines at positions 548 and 554 replaced by serines) is detailed elsewhere (28). HA-Y543, which is a point mutant of HA wt (cysteine 543 replaced by tyrosine), was described previously (9).…”

“…They were grown postinfection for 36 -38 h (for HA wt and HAϩ4) or 44 -46 h (for HAϩ8 and HAϩ8-S548,S554). The longer incubations after infection used for the latter two mutants were selected to achieve surface density closer to that of HA wt, as the percentage of these mutants at the cell surface is significantly lower (28). The cells were washed twice with cold HBSS containing 20 mM HEPES and 2% BSA (HBSS/HEPES/BSA, pH 7.2), and labeled with anti-HA TMR-FabЈ (100 g/ml, 30 min, 4°C).…”

“…The absence of an internalization signal in wild-type HA (HA wt), which is excluded from coated pits (9,25), enables a direct comparison between specific internalization signals introduced one at a time in each mutant. We have focused on HA mutants that were internalized at different rates (28) and varied in their tendency to form higher complexes (larger than the trimeric HA wt). Our results indicate that, depending on the specific internalization signal, the HA mutants can shift all the way from no interactions to stable entrapment in coated pits, and that a shift from transient to stable entrapment correlates with significantly higher internalization rates.…”

Structural Requirements for Major Histocompatibility Complex Class II Invariant Chain Endocytosis and Lysosomal Targeting

“…This suggests that the rate-limiting step for lysosomal targeting is not endocytosis. Further support for this idea comes from studies on mutant hemagglutinins by Zwart et al (54), who demonstrated that targeting to the latter stages of endocytic pathway does not simply correlate with the concentration of mutant hemagglutinins in the early endosome (54). With their hemagglutinin mutants, they demonstrated that there was no correlation between internalization rate and degradation rate and, furthermore, that the signal for these two processes were distinct.…”