Characterization of the human cytomegalovirus UL97 gene product as a virion-associated protein kinase.

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.

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 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). To identify 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.

Degradation of mutant influenza virus hemagglutinins is influenced by cytoplasmic sequences independent of internalization signals.

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 t1/2 of 1.9 h, and HA-Y543, which was degraded with a t1/2 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.

Odorant signal termination by olfactory UDP glucuronosyl transferase.

The onset of olfactory transduction has been extensively studied, but considerably less is known about the molecular basis of olfactory signal termination. It has been suggested that the highly active cytochrome P450 monooxygenases of olfactory neuroepithelium are termination enzymes, a notion supported by the identification and molecular cloning of olfactory-specific cytochrome P450s (refs. 13-16). But as reactions catalysed by cytochrome P450 (refs 17, 18) often do not significantly alter volatility, lipophilicity or odour properties, cytochrome P450 may not be solely responsible for olfactory signal termination. In liver and other tissues, drug hydroxylation by cytochrome P450 is frequently followed by phase II biotransformation, for example by UDP glucuronosyl transferase (UGT), resulting in a major change of solubility and chemical properties. We report here the molecular cloning and expression of an olfactory-specific UGT. The olfactory enzyme, but not the one in liver microsomes, shows preference for odorants over standard UGT substrates. Furthermore, glucuronic acid conjugation abolishes the ability of odorants to stimulate olfactory adenylyl cyclase. This, together with the known broad spectrum of drug-detoxification enzymes, supports a role for olfactory UGT in terminating diverse odorant signals.

The effects of foreign transmembrane domains on the biosynthesis of the influenza virus hemagglutinin.

Eleven chimeric proteins were created in which the transmembrane, the cytoplasmic, or both topological domains of the influenza virus hemagglutinin (HA) were replaced with those from five other glycoproteins. All of the chimeric HAs reached the cell surface but appeared to differ in the degree to which they were stably folded. Comparisons of the rates of folding, passage into the Golgi, and arrival at the plasma membrane of wild-type HA and the chimeric proteins suggest that formation of a stable HA trimer is not an absolute requirement for export from the endoplasmic reticulum. In addition, there appear to be at least two steps at which the rate of transport can be altered during exocytosis, one occurring before and the other after the trimming of oligosaccharides by Golgi mannosidases. Certain of the chimeras differed from HA in their ability to pass through each of these steps. Replacement of the HA transmembrane domain with the analogous sequences from other proteins affected folding and transport of the chimeric HAs in ways that suggest that the HA transmembrane sequences form a specific structure in the membrane that differs from that formed by analogous sequences from the other proteins.

A single amino acid change in the cytoplasmic domain allows the influenza virus hemagglutinin to be endocytosed through coated pits.

Through site-specific mutagenesis, three of the ten amino acids of the cytoplasmic domain of the influenza virus hemagglutinin (HA) were individually changed to tyrosines. None of these changes had significant effect on the rate of export, the rate of folding, or the antigenicity of the mutant HAs. However, one of these mutations, substituting tyrosine for cysteine at amino acid 543, changed HA from a protein that was endocytosed at a very low rate to a protein that readily entered coated pits, was internalized, and apparently recycled to the cell surface. Replacement of cysteine 543 with phenylalanine or serine did not increase the rate of internalization of HA. Phosphorylation of the mutant HA bearing a tyrosine at position 543 was not detected. These results indicate a specific and local role for the tyrosine introduced into the cytoplasmic domain of HA that is necessary for interaction of the protein with coated pits.

5-Hydroxytryptamine in platelets of torsion dystonia patients.

In an effort to explore possible changes in 5-hydroxytryptamine (5-HT) metabolism in idiopathic torsion dystonia (ITD), platelets from patients were studied. Platelet 5-HT concentrations did not differ from those in matched controls. 5-HT uptake by the platelets was also studied, and the results demonstrated significantly higher Km values, but Vmax values were normal among patients. Inhibition by imipramine of 5-HT uptake by platelets taken from ITD patients was also normal. The therapeutic implications of the low affinity of 5-HT to its platelet receptors are discussed.