The membrane domain of a bacteriophage assembly protein. Transmembrane-directed proteolysis of a membrane-spanning fusion protein.

A tripartite fusion construct encoding the amino-terminal half of EcoRI endonuclease followed by amino acids 217-299 of the filamentous bacteriophage gene I protein (pI) attached to the enzymatic portion of alkaline phosphatase results in the production of two proteins. The larger protein, pIf, is the complete tripartite fusion protein while the smaller protein, pIf*, results from internal initiation of translation at pI methionine 241. Both pIf and pIf* span the Escherichia coli inner membrane via a 20-amino-acid hydrophobic stretch of pI with their amino termini in the cytoplasm and their carboxyl-terminal alkaline phosphatase domains in the periplasm. The alkaline phosphatase moiety of approximately 70% of pIf is released into the periplasm by in vivo proteolysis, but only about 10% of pIf* is cleaved. Neither DegP, OmpT, nor protease III are responsible for the cleavage in vivo, and leader peptidase is unable to cleave the fusion protein in vitro. Deletion and substitution analyses demonstrate that the degree of periplasmic cleavage depends on the sequence of the cytoplasmic domain of the fusion proteins. Possible mechanisms for this transmembrane-directed cleavage event are compared to proposed models for signal transduction.

The membrane domain of a bacteriophage assembly protein. Membrane insertion and growth inhibition.

The gene I protein (pI) of the f1 filamentous bacteriophage is a non-capsid protein that is required for the assembly of the bacteriophage. It spans the Escherichia coli inner membrane once with its amino terminus in the cytoplasm and its carboxyl-terminal portion in the periplasm. The presence of moderate amounts of this protein in the membrane results in rapid inhibition of cell growth, probably from a loss of membrane potential. Previous observations defined a 55-amino-acid sequence within pI required for its membrane insertion which includes a 20-residue hydrophobic stretch preceded by a 13-residue positively charged amphiphilic helix. To define the minimal sequence required for membrane translocation and for growth inhibition, a deletion analysis was performed on a tripartite fusion construct containing the 55-residue pI sequence flanked upstream by the amino-terminal portion of EcoRI endonuclease and downstream by the enzymatic portion of alkaline phosphatase. Only the 20-residue hydrophobic stretch immediately preceded by 1 arginine residue is required for membrane insertion of the fusion proteins. This region also sufficed to inhibit cell growth provided it contained protein domains exposed in both the cytoplasm and periplasm. It was not possible to separate the domains required for membrane insertion and cell growth inhibition. No requirement for the positively charged amphiphilic helix was detected either for membrane insertion or growth inhibition, suggesting that it plays a role in phage assembly and not membrane insertion.

Membrane localization and topology of a viral assembly protein.

The gene I protein (pI) of the filamentous bacteriophage f1 is required for the assembly of this virus. Antibodies specific to either the amino or carboxyl terminus of this protein were used to determine the location and topology of the gene I protein in f1-infected bacteria. pI is anchored in the inner membrane of Escherichia coli cells via a 20-amino-acid hydrophobic stretch, with its carboxyl-terminal 75 residues located in the periplasm and its amino-terminal 253 amino acids residing in the cytoplasm. By using the carboxyl-terminal pI antibody, a smaller protein, pI*, is also detected in f1-infected cells at a ratio of one to two molecules per molecule of pI. Analysis of proteins produced from a gene I amber mutant plasmid or bacteriophage suggests that pI* is most likely the result of an in-frame internal translational initiation event at methionine 241 of the 348-amino-acid pI. pI* is shown to be an integral inner membrane protein inserted in the same orientation as pI. The relation of the cellular locations of pI and pI* to some of the proposed functions of pI is discussed.

Novel polyaminolipids enhance the cellular uptake of oligonucleotides.

Two new polyaminolipids have been synthesized for the purpose of improving cellular uptake of oligonucleotides. The amphipathic compounds are conjugates of spermidine or spermine linked through a carbamate bond to cholesterol. The polyaminolipids are relatively nontoxic to mammalian cells. In tissue culture assays, using fluorescent-tagged or radiolabeled triple helix-forming oligonucleotides, spermine-cholesterol and spermidine-cholesterol significantly enhance cellular uptake of the oligomers in the presence of serum. Spermine-cholesterol is comparable with DOTMA/DOPE (a 1:1 (w/w) formulation of the cationic lipid N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) and the neutral lipid dioleylphosphatidylethanolamine (DOPE)) in increasing cellular uptake of oligonucleotides, while spermidine-cholesterol is more efficient. The internalized oligonucleotides are routed to the nucleus as early as 20 min after treatment, suggesting that the polyaminolipids increase the permeability of cellular membranes to oligonucleotides. At later times, much of the incoming oligonucleotides are sequestered within punctate cytoplasmic granules, presumably compartments of endosomal origin. Coadministration with polyaminolipids markedly improves the cellular stability of the oligonucleotides; more than 80% of the material can be recovered intact up to 24 h after addition to cells. In the absence of the polyaminolipids, nearly all of the material is degraded within 6 h. These data suggest that the new polyaminolipids may be useful for the delivery of nucleic acid-based therapeutics into cells.

Intramolecular G-quartet motifs confer nuclease resistance to a potent anti-HIV oligonucleotide.

We have identified a potentially therapeutic anti-human immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of deoxyguanosines and thymidines (T30177, also known as AR177: 5'-g.tggtgggtgggtggg.t-3', where asterisk indicates phosphorothioate linkage). In acute assay systems using human T-cells, T30177 and its total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium production by 50% at 0.15 and 0.3 microM, respectively. Under physiological conditions, the sequence and composition of the 17-mer favors the formation of a compact, intramolecularly folded structure dominated by two stacked guanine quartet motifs that are connected by three loops of TGs. The molecule is stabilized by the coordination of a potassium ion between the two stacked quartets. We now show that these guanine quartet-containing oligonucleotides are highly resistant to serum nucleases, with t1/2 of 5 h and >4 days for T30175 and T30177, respectively. Both oligonucleotides were internalized efficiently by cells, with intracellular concentrations reaching 5-10-fold above the extracellular levels after 24 h of incubation. In contrast, single-base mutated variants or random sequence control oligonucleotides that could not form the compactly folded structure had markedly reduced half-lives (t1/2 from approximately 3 to 7 min), low cellular uptake, and no sequence-specific anti-HIV-1 activity. These data suggest that the tertiary structure of an oligonucleotide is a key determinant of its nuclease resistance, cellular uptake kinetics, and biological efficacy.