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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.

The cellular localization and virion association of the human cytomegalovirus (HCMV) UL97 protein were studied. UL97 protein demonstrated early nuclear localization followed by late perinuclear accumulation. It was found to be a structural virion constituent detected in all three enveloped forms of extracellular viral particles and shown to be phosphorylated by the virion-associated protein kinase. UL97 protein immunoprecipitated from virions and from infected cells demonstrated protein kinase activity manifested by autophosphorylation. This activity was reduced in the presence of a ganciclovir-resistance mutation at residue 460, implicated in nucleotide binding. A mutant virus, from which the proposed UL97 kinase catalytic domain had been deleted, could not be propagated in the absence of a helper wild-type virus. The characterization of UL97 protein as a virion-associated protein kinase which appears essential for viral replication, provides further insight into HCMV replication and could identify a potential novel target for antiviral therapy.

Abstract. 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 membrane 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. Toddeutify more precisely the foreign amino acid 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 glycoprotein 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, chimeras that contain only a foreign CD, resembled HA wt in having a long residence 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 only a foreign TM, was internalized as efficiently as was HA-G-G, a chimeric protein with transmembrane and cytoplasmic sequences of VSV G protein. 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 type HA, denaturation of the wild type HA external domain by treatment with low pH did not increase the interaction of HA with coated pits. However, mutation of four amino acids in the TM of HA allowed the protein to be internalized, indicating that the property that allows HA to escape endocytosis resides in its TM. These results indicate that possession of a cytoplasmic recognition feature is not required for the internalization of all cell surface proteins and suggest that multiple mechanisms for internalization exist that operate at distinctly different rates.Part of the process of communicating with their external environment, cells maintain an active traffic of membranes moving between the cell surface and internal organelles. Solutes are absorbed through fluid-phase endocytosis, nutrients and hormones are concentrated and internalized by receptor-mediated endocytosis, and resident proteins of the plasma membrane are removed for degradation in lysosomes. Cellular pathogens, like many viruses, use these endocytic processes to enter the cell. It is clear that there are at least several distinct pathways for this traffic (Watts and Marsh, 1992), but the exact number of pathways and the relations between them are not currently known. However, in cells that are not undergoing major changes in shape, such as those that occur during locomotion or phagocytosis, a major pathway

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