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The interaction of the catalytic subunit of herpes simplex virus DNA polymerase with the processivity subunit, UL42, is essential for viral replication and is thus a potential target for antiviral drug discovery. We have previously reported that a peptide analogous to the C-terminal 36 residues of the catalytic subunit, which are necessary and sufficient for its interaction with UL42, forms a monomeric structure with partial alpha-helical character. This peptide and one analogous to the C-terminal 18 residues specifically inhibit UL42-dependent long chain DNA synthesis. Using multidimensional (1)H nuclear magnetic resonance spectroscopy, we have found that the 36-residue peptide contains partially ordered N- and C-terminal alpha-helices separated by a less ordered region. A series of "alanine scan" peptides derived from the C-terminal 18 residues of the catalytic subunit were tested for their ability to inhibit long-chain DNA synthesis and by circular dichroism for secondary structure. The results identify structural aspects and specific side chains that appear to be crucial for interacting with UL42. These findings may aid in the rational design of new drugs for the treatment of herpesvirus infections.

T cell receptor signaling results both in T cell proliferation and apoptosis. A key enzyme at the intersection of these downstream pathways is phosphatidylinositol 3'-kinase (PI 3-kinase). In a previous report, we showed that the p85alpha subunit of the PI 3-kinase preferentially associated with the CD3-zeta membrane-proximal immunoreceptor tyrosine-based activation motif of the zeta chain (zetaA-ITAM) (Exley, M., Varticovski, L., Peter, M., Sancho, J., and Terhorst, C. (1994) J. Biol. Chem. 269, 15140-15146). Here, we demonstrate that tyrosine phosphorylation of CD3-epsilon can recruit the PI 3-kinase enzyme in a T cell activation-dependent manner. In vivo studies with Jurkat cells stably transfected with a CD8-CD3-epsilon chimera (termed CD8-epsilon) shows that ligation of endogenous CD3-epsilon or CD8-epsilon by specific antibodies induces tyrosine phosphorylation of CD3-epsilon or CD8-epsilon, respectively. Increased tyrosine phosphorylation correlates with increased binding of p85alpha PI 3-kinase and recruitment of PI 3-kinase enzymatic activity to CD3-epsilon or CD8-epsilon proteins. Mutagenesis studies in COS-7 cells, transiently transfected with CD8-epsilon, p85alpha, and Fyn cDNAs in various combinations, show that both Tyr170 and Tyr181 within the CD3-epsilon-ITAM are required for efficient binding of p85alpha PI 3-kinase. Thus, replacement of Tyr170 by Phe (Y170F), or Tyr181 by Phe (Y181F) significantly reduces binding of p85alpha PI 3-kinase, whereas it does not affect binding of Fyn. Further in vitro experiments suggest that a direct binding of the tandem SH2 domains of p85alpha PI 3-kinase to the two phosphorylated tyrosines in a single CD3-epsilon-ITAM may occur. The data also support a model in which a single CD3 subunit can recruit distinct effector molecules by means of TCR-mediated differential ITAM phosphorylation.

The rates of protein and lipid synthesis of Mycoplasma capricolum were essentially synchronous during growth and depended on the sterol supplement in the media increasing in the order cholesterol (0.5 microgram/ml) < lanosterol (10 microgram/ml) < lanosterol (10 microgram/ml) + cholesterol (0.5 microgram/ml) < cholesterol (10 microgram/ml). The effect of lanosterol plus low cholesterol on macromolecular synthesis was synergistic. Whereas protein and lipid synthesis were brought virtually to a halt by cholesterol starvation, DNA synthesis continued for about 8 h. Increasing the palmitate and elaidate concentrations 4-fold in the lanosterol-supplemented media raised the growth rate even in the absence of the small amount of cholesterol (0.5 microgram/ml) needed otherwise for the synergistic effect on growth. Studies of the kinetics of fatty acid uptake by resting cells showed that the apparent Km (17 microM) of oleate uptake in lanosterol-grown cells was specifically lowered to 3 microM, a value equal to that seen in cholesterol-grown cells, by the inclusion of a synergistic amount of cholesterol in the growth media. By contrast, the apparent Km for palmitate uptake was the same (2 microM) for all three cell types. The results are consistent with the membrane cholesterol serving in a dual role, one as a bulk component and another more specific function involving the regulation of unsaturated fatty acid uptake and thereby phospholipid biosynthesis.

Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph was grown on media supplemented with [3H]palmitate or [3H]oleate. The isolated bacterial membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the more than 50 membrane polypeptides revealed by Coomassie blue staining, approximately 25 were labeled with [3H]palmitate and only about 6 were labeled with [3H]oleate. Exhaustive delipidation of the membranes with chloroform:methanol did not alter the labeling pattern. Treatment of delipidated membranes by mild alkaline hydrolysis released up to 71% of the [3H]palmitate and 93% of the [3H]oleate. The data suggest that numerous membrane proteins of M. capricolum are covalently modified by acylation with saturated and unsaturated fatty acids. Cerulenin, a specific inhibitor of fatty acid synthesis had no effect on the labeling of mycoplasma membrane proteins by either [3H]palmitate or [3H]oleate. A small amount of membrane-associated cholesterol previously shown to stimulate sequentially the synthesis of unsaturated phospholipid, RNA, and protein (Dahl, J. S., and Dahl, C. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 692-696) specifically enhances the acylation of certain proteolipids by oleate but not by palmitate.

Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph, contains a large number of membrane proteins covalently modified by both saturated and unsaturated fatty acids (Dahl, C.E., Dahl, J.S., and Bloch, K. (1983) J. Biol. Chem. 258, 11814-11818). Pulse-chase experiments show that the radioactivity in the fatty acid moieties of the acyl proteins increases rather than decreases during a 4.5-h incubation period suggesting that a large intracellular pool of metabolites such as phospholipid serves as the donor for protein acylation. We find that cells incubated for 4 h in a growth medium containing [3H]palmitate-labeled phosphatidylglycerol or 2-[3H]palmitoyl dipalmitoylphosphatidylcholine show the same labeling pattern as cells incubated for 4 h in a complete growth medium with [3H]palmitate. Exogenously added phospholipids are not hydrolyzed to free fatty acid during the labeling period. Acylation of proteins is inhibited in cells treated with chloramphenicol showing that there is no pool of proacyl protein in the cell. Labeling of membrane proteins also occurs with [3H]glycerol. Glycerol is incorporated primarily into the same proteins as oleate suggesting that acylation by unsaturated fatty acid may involve a protein bound diglyceride moiety. Palmitate, on the other hand, appears to bind to other sites along the polypeptide chain in addition to the diglyceride moiety.