ENDOCYTOSIS OF CHIMERIC INFLUENZA-VIRUS HEMAGGLUTININ PROTEINS THAT LACK A CYTOPLASMIC RECOGNITION FEATURE FOR COATED PITS

The influenza virus A/Japan/305/57 hemagglutinin (HA) can be converted from a protein that is essentially excluded from coated pits into one that is internalized at approximately the rate of uptake of bulk memb rane by replacing the HA transmembrane and cytoplasmic sequences with those of either of two other glycoproteins (Roth et al., 1986. J. Cell Biol. 102:1271-1283). To identify more precisely the foreign amino ac id sequences responsible for this change in HA traffic, DNA sequences encoding the transmembrane (TM) or cytoplasmic (CD) domains of either the G glycoprotein of vesicular stomatitis virus (VSV) or the gC glyco protein of herpes simplex virus were exchanged for those encoding the analogous regions of wild type HA (HA wt), HA-HA-G and HA-HA-gC, chime ras that contain only a foreign CD, resembled HA wt in having a long r esidence on the cell surface and were internalized very slowly, HA-HA- gC was indistinguishable from HA in our assays, whereas twice as much HA-HA-G was internalized as was HA wt, However, HA-G-HA, containing on ly a foreign TM, was internalized as efficiently as was HA-G-G, a chim eric protein with transmembrane and cytoplasmic sequences of VSV G pro tein. Conditions that blocked internalization through coated pits also inhibited endocytosis of the chimeric proteins. Although the external domains of the chimeras were less well folded than that of the wild t ype HA, denaturation of the wild type HA external domain by treatment with low pH did not increase the interaction of HA with coated pits. H owever, mutation of four amino acids in the TM of HA allowed the prote in to be internalized, indicating that the property that allows HA to escape endocytosis resides in its TM. These results indicate that poss ession of a cytoplasmic recognition feature is not required for the in ternalization of all cell surface proteins and suggest that multiple m echanisms for internalization exist that operate at distinctly differe nt rates.