Antiviral activity of the zinc ionophores pyrithione and hinokitiol against picornavirus infections

We have discovered two metal ion binding compounds, pyrithione (PT) and hinokitiol (HK), that efficiently inhibit human rhinovirus, coxsackievirus, and mengovirus multiplication. Early stages of virus infection are unaffected by these compounds. However, the cleavage of the cellular eukaryotic translation initiation factor eIF4GI by the rhinoviral 2A protease was abolished in the presence of PT and HK. We further show that these compounds inhibit picornavirus replication by interfering with proper processing of the viral polyprotein. In addition, we provide evidence that these structurally unrelated compounds lead to a rapid import of extracellular zinc ions into cells. Imported Zn²⁺ was found to be localized in punctate structures, as well as in mitochondria. The observed elevated level of zinc ions was reversible when the compounds were removed. As the antiviral activity of these compounds requires the continuous presence of the zinc ionophore PT, HK, or pyrrolidine-dithiocarbamate, the requirement for zinc ions for the antiviral activity is further substantiated. Therefore, an increase in intracellular zinc levels provides the basis for a new antipicornavirus mechanism.     

Inhibition by zinc of rhinovirus protein cleavage: interaction of zinc with capsid polypeptides.

Zinic ions rapidly inhibit virus production in HeLa cells infected with human rhinovirus type 1A and lead to the accumulation of human rhinovirus type 1A precursor polypeptides. The degree to which cleavage of these precursors is inhibited is directly dependent on the quantity of cell-associated zinc. Proteolysis resumes after the removal of zinc-containing medium, and the accumulated viral precursors are cleaved predominantly to stable virus polypeptides. The precursors stabilized at the lowest zinc levels are those that contain capsid protein sequences. Furthermore, added zinc is bound to human rhinovirus type 1A capsids and prevents them from forming crystals. Zinc-resistant mutants display antigenic alterations in coat proteins. These results suggest that zinc complexes with rhinovirus coat proteins and alters them so that they cannot function as substrates for proteases or as reactants in the assembly of the virus particles.     

Failure To Cleave Murine Leukemia Virus Envelope Protein Does Not Preclude Its Incorporation in Virions and Productive Virus-Receptor Interaction

It is thought that complete cleavage of retroviral envelope protein into mature surface protein (SU) and transmembrane protein (TM) is critical for its assembly into virions and the formation of infectious virus particles. Here we report the identification of highly infectious, cleavage-deficient envelope mutant proteins. Substitution of aspartate for lysine 104, arginines 124 and 126, or arginines 223 and 225 strongly suppressed cleavage of the envelope precursor and yet allowed efficient incorporation of precursor molecules as the predominant species in virions that were almost as infectious as the wild-type virus. These results indicate that cleavage of the envelope precursor into mature SU and TM is not necessary for assembly into virions. Moreover, they call into question how many mature envelope protein subunits are required to complete virus entry, suggesting that a very few molecules suffice. The failure of host cell proteases to cleave these mutant proteins, whose substitutions are distal to the actual site of cleavage, suggests that the envelope precursor is misfolded, sequestering the cleavage site. In agreement with this, all cleavage mutant proteins exhibited significant losses of receptor binding, suggesting that these residues play roles in proper envelope protein folding. We also identified a charged residue, arginine 102, whose substitution suppressed envelope cleavage and allowed precursor incorporation but resulted in virions that were virtually noninfectious and that exhibited the greatest reduction in receptor binding. Placement of these cleavage mutations into envelope proteins of targeted retroviral vectors for human gene therapy may prevent loss of the modified surface proteins from virions, improving their infectivity and storage hardiness.     

Role of divalent metal ions in the hammerhead RNA cleavage reaction.

A hammerhead self-cleaving domain composed of two oligoribonucleotides was used to study the role of divalent metal ions in the cleavage reaction. Cleavage rates were measured as a function of MgCl2, MnCl2, and CaCl2 concentration in the absence or presence of spermine. In the presence of spermine, the rate vs metal ion concentration curves are broader, and lower concentrations of divalent ions are necessary for catalytic activity. This suggests that spermine can promote proper folding of the hammerhead and one or more divalent ions are required for the reaction. Six additional divalent ions were tested for their ability to support hammerhead cleavage. In the absence of spermine, rapid cleavage was observed with Co2+ while very slow cleavage occurred with Sr2+ and Ba2+. No detectable specific cleavage was observed with Cd2+, Zn2+, or Pb2+. However, in the presence of 0.5 mM spermine, rapid cleavage was observed with Zn2+ and Cd2+, and the rate with Sr2+ was increased, indicating that while these three ions could not promote proper folding of the hammerhead they were able to stimulate cleavage. These results suggest certain divalent ions either participate directly in the cleavage mechanism or are specifically involved in stabilizing the tertiary structure of the hammerhead. Additionally, an altered divalent metal ion specificity was observed when a unique phosphorothioate linkage was inserted at the cleavage site. The substitution of a sulfur for a nonbridging oxygen atom substantially reduced the affinity of an important Mg2+ ion necessary for efficient cleavage. In contrast, the reaction proceeds normally with Mn2+, presumably due to its ability to coordinate with both oxygen and sulfur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correct proteolytic cleavage is required for the cell adhesive function of uvomorulin

All Ca2(+)-dependent cell adhesion molecules are synthesized as precursor polypeptides followed by a series of posttranslational modifications including proteolytic cleavage. The mature proteins are formed intracellularly and transported to the cell surface. For uvomorulin the precursor segment is composed of 129-amino acid residues which are cleaved off to generate the 120-kD mature protein. To elucidate the role of proteolytic processing, we constructed cDNAs encoding mutant uvomorulin that could no longer be processed by endogenous proteolytic enzymes and expressed the mutant polypeptides in L cells. Instead of the recognition sites for endogenous proteases, these mutants contained either a recognition site of serum coagulation factor Xa or a new trypsin cleavage site. The intracellular proteolytic processing of mutant polypeptides was inhibited in both cases. The unprocessed polypeptides were efficiently expressed on the cell surface and had other features in common with mature uvomorulin, such as complex formation with catenins and Ca2(+)-dependent resistance to proteolytic degradation. However, cells expressing unprocessed polypeptides showed no uvomorulin-mediated adhesive function. Treatment of the mutant proteins with the respective proteases results in cleavage of the precursor region and the activation of uvomorulin function. However, other proteases although removing the precursor segment were ineffective in activating the adhesive function. These results indicate that correct processing is required for uvomorulin function and emphasize the importance of the amino-terminal region of mature uvomorulin polypeptide in the molecular mechanism of adhesion.     

Effect of Zn2+ during reactivation and refolding of urea-denatured creatine kinase

The courses of refolding and reactivation of urea-denatured creatine kinase (CK) (ATP:creatine N-phosphotrans-ferase, EC 2.7.3.2) have been studied in the absence and presence of zinc ions. The presence of Zn²⁺ at low concentrations blocks the reactivation and refolding of urea-denatured CK and keeps it in a partially folded state. The partially folded state proved to be a monomeric state which resembles the molten globule state in the CK folding pathway. During refolding in the presence of Zn²⁺ , creatine kinase forms aggregates with the aggregation dependent on zinc concentration and temperature. In the presence of EDTA, the partially folded creatine kinase can be reactivated and refolded following a biphasic course, suggesting the existence of a monomeric intermediate during the refolding of CK. The results also suggest that low concentrations of zinc ions might be toxic to some proteins such as creatine kinase by disrupting their proper folding.     

The processing of a cathepsin L precursor in vitro

Subcultured rat fibroblasts secreted a cathepsin L precursor when maintained for 24 h in serum-free medium containing 20 mM ammonium ions. The precursor was identified by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using polyclonal antibodies to cathepsin L. The molecular mass of the precursor was found to be approximately 39 kDa, which confirms the result originally reported by Y. Nishimura et al. (1988, Arch. Biochem. Biophys. 263, 107-116). Treatment of the precursor containing medium with cathepsin D at pH values ranging from 3.5 to 5.5 caused a limited cleavage of the precursor molecule. The resultant polypeptides are an unstable intermediate form with Mr 35,000 and a stable single chain form of cathepsin L showing a Mr about 32,500. The cathepsin D-mediated conversion was strongly accelerated by Hg2+ ions. A further proteolytic cleavage of the 32.5-kDa polypeptide has not been observed. The enzymatic activity toward Z-Phe-Arg-NHMec at pH 5.5 increased during the conversion, indicating that active cathepsin L was formed from an inactive precursor molecule.     

Luminometric method for screening retroviral protease inhibitors

We have developed a sensitive luminometric assay for determining the activity of retroviral proteases that uses proteolytic cleavage of polypeptide substrate immobilized on Ni-NTA HisSorb Strips microplates. The protease substrate derived from the Gag precursor protein of Mason-Pfizer monkey virus (M-PMV) was conjugated with horseradish peroxidase (HRP), which catalyzes oxidation of luminol in the assay. The cleavage of the substrate was monitored as a decrease in luminescent signal caused by the release of the cleavage product conjugated to HRP. Testing of a set of M-PMV protease inhibitors confirmed that this method is sufficiently sensitive and specific for high-throughput screening of retroviral protease inhibitors.     

Proteolytic mechanisms in amyloid-beta metabolism: therapeutic implications for Alzheimer's disease

The accumulation of the amyloid-beta peptide, the main constituent of the "amyloid plaque", is widely considered to be the key pathological event in Alzheimer's disease. Amyloid-beta is produced from the amyloid precursor protein through the action of the proteases beta-secretase and gamma-secretase. Alternative cleavage of amyloid precursor protein by the enzyme alpha-secretase precludes amyloid-beta production. In addition, several proteases are involved in the degradation of amyloid-beta. This review focuses on the proteolytic mechanisms of amyloid-beta metabolism. An increasingly detailed understanding of proteolysis in both amyloid-beta deposition and clearance has identified some of these proteases as potential therapeutic targets for Alzheimer's disease. A more complex knowledge of these proteases takes us one step closer to developing "disease-modifying" therapies, but these advances also emphasize that significant challenges must be overcome before clinically effective drugs to treat Alzheimer's disease become a reality.     

Folding pathway mediated by an intramolecular chaperone: propeptide release modulates activation precision of pro-subtilisin

Propeptides of several proteases directly catalyze the protein folding reaction. Uncatalyzed folding traps these proteases into inactive molten-globule-like conformers that switch into active enzymes only when their cognate propeptides are added in trans. Although tight binding and proteolytic susceptibility forces propeptides to function as single turnover catalysts, the significance of their inhibitory function and the mechanism of activation remain unclear. Using pro-subtilisin as a model, we establish that precursor activation is a highly coordinated process that involves synchronized folding, autoprocessing, propeptide release, and protease activation. Our results demonstrate that activation is controlled by release of the first free active protease molecule. This triggers an exponential cascade that selectively targets the inhibitory propeptide in the autoprocessed complex as its substrate. However, a mutant precursor that enhances propeptide release can drastically reduce the folding efficiency by altering the synergy between individual stages. Our results represent the first demonstration that propeptide release, not precursor folding, is the rate-determining step and provides the basis for the proposed model for precise spatial and temporal activation that allows proteases to function as regulators of biological function.     

Regulation of bone morphogenetic protein-4 activity by sequence elements within the prodomain

Bone morphogenetic protein-4 (BMP-4) is synthesized as a large precursor protein, which undergoes proprotein convertase-mediated proteolytic maturation along the secretory pathway to release the active ligand. Pro-BMP-4 is initially cleaved at a consensus furin motif adjacent to the mature ligand domain (the S1 site), and this allows for subsequent cleavage at an upstream motif (the S2 site). This sequential cleavage liberates a small, evolutionarily conserved, prodomain fragment (the linker peptide) of unknown fate and function. Here we show that the linker domain is essential for proper folding, exit from the endoplasmic reticulum, and thus cleavage of the BMP-4 precursor when overexpressed in Xenopus oocytes and embryos but not in cultured mammalian cells. Mature BMP-4 synthesized from a precursor in which the S1 site is non-cleavable, such that the linker domain remains covalently attached to the ligand, has little or no activity in vivo. Finally, analysis of folding, cleavage, and bioactivity of chimeric precursors containing the BMP-7 prodomain and BMP-4 mature domain, or vice versa, with or without the BMP-4 linker domain revealed that the linker domain is only functional in the context of the BMP-4 prodomain, and that differential cleavage around this domain can regulate the activity of a heterologous ligand.     

Factors affecting composition and thermostability of mengovirus virions.

The composition of mengovirus virions produced by infected cells varies with the incubation temperature. Virons produced at 37.0 or 39.5 degrees contain four major polypeptides (alpha, beta, gamma, and delta) and one minor polypeptide (beta'). Virons produced at 31.5 degrees C contain two additional polypeptides (D1 and E). The virions of two temperature-sensitive (ts) and thermolabile mutants of mengovirus (ts25 and ts88) contain an increased amount of polypeptide beta', with a corresponding decrease in polypeptide beta when compared with the wild-type mengovirus.     

Protein quality control in the bacterial periplasm

The proper functioning of extracytoplasmic proteins requires their export to, and productive folding in, the correct cellular compartment. All proteins in Escherichia coli are initially synthesized in the cytoplasm, then follow a pathway that depends upon their ultimate cellular destination. Many proteins destined for the periplasm are synthesized as precursors carrying an N-terminal signal sequence that directs them to the general secretion machinery at the inner membrane. After translocation and signal sequence cleavage, the newly exported mature proteins are folded and assembled in the periplasm. Maintaining quality control over these processes depends on chaperones, folding catalysts, and proteases. This article summarizes the general principles which control protein folding in the bacterial periplasm by focusing on the periplasmic maltose-binding protein.     

A new approach for alteration of protease functions: pro-sequence engineering

Several proteases, including the bacterial serine protease subtilisins, require the assistance of the N-terminal pro-sequence of precursors to produce active, mature enzymes. Upon completion of folding, the pro-sequence is autocatalytically degraded because it is not necessary for the activity or stability of folded, mature cognates of the original enzymes. Therefore, the pro-sequence functions as an intramolecular chaperone that guides correct folding of the protease domain. Interestingly, Shinde et al. proposed a new theory of "protein memory" in which an identical polypeptide can fold into an altered conformation with different secondary structure, stability and specificities through a mutated pro-sequence [Shinde et al. (1997) Nature 389:520–522]. We also showed that the autoprocessing efficiency was improved by modifications in the pro-sequence of mutant subtilisins with altered substrate specificity. Further, the pro-sequence from a subtilisin homologue was found to chaperone the intramolecular folding of denatured subtilisin. These results indicate that engineering of the pro-sequence, i.e., site-directed and/or random mutagenesis, chimeras and gene shuffling between members of the family, would be a useful method for improving the functions of autoprocessing proteases. Conventional protein engineering techniques have thus far employed mutagenesis in the protease domain to modify the enzymatic properties. This new approach, which we term "pro-sequence engineering", is not only an important tool for studying the mechanism of protein folding, but also a promising technology for creating unique proteases with various beneficial properties.     

The role of proteolytic cleavage of viral glycoproteins in the pathogenesis of influenza virus infections

Infectivity, tropism, spread, and pathogenicity of influenza viruses are based on the interplay between the fusogenic glycoproteins and appropriate host endoproteases. The hemagglutinin (HA) of influenza A and B viruses and the HEF (hemagglutinating, esterase, fusion) glycoprotein of influenza C virus receive their full biological activity by proteolytic cleavage of a precursor molecule at a definite cleavage site. The amino acid motifs at the cleavage site and the availability of suitable proteases are critical for the clinical manifestation of the infection. Prototype cleavage proteases, including bacterial enzymes, are described.     

Regulation of hepatitis C virus polyprotein processing by signal peptidase involves structural determinants at the p7 sequence junctions

The hepatitis C virus genome encodes a polyprotein precursor that is co- and post-translationally processed by cellular and viral proteases to yield 10 mature protein products (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Although most cleavages in hepatitis C virus polyprotein precursor proceed to completion during or immediately after translation, the cleavages mediated by a host cell signal peptidase are partial at the E2/p7 and p7/NS2 sites, leading to the production of an E2p7NS2 precursor. The sequences located immediately N-terminally of E2/p7 and p7/NS2 cleavage sites can function as signal peptides. When fused to a reporter protein, the signal peptides of p7 and NS2 were efficiently cleaved. However, when full-length p7 was fused to the reporter protein, partial cleavage was observed, indicating that a sequence located N-terminally of the signal peptide reduces the efficiency of p7/NS2 cleavage. Sequence analyses and mutagenesis studies have also identified structural determinants responsible for the partial cleavage at both the E2/p7 and p7/NS2 sites. Finally, the short distance between the cleavage site of E2/p7 or p7/NS2 and the predicted transmembrane alpha-helix within the P' region might impose additional structural constraints to the cleavage sites. The insertion of a linker polypeptide sequence between P-3' and P-4' of the cleavage site released these constraints and led to improved cleavage efficiency. Such constraints in the processing of a polyprotein precursor are likely essential for hepatitis C virus to post-translationally regulate the kinetics and/or the level of expression of p7 as well as NS2 and E2 mature proteins.     

Random circular permutation of DsbA reveals segments that are essential for protein folding and stability

One of the key questions in protein folding is whether polypeptide chains require unique nucleation sites to fold to the native state. In order to identify possible essential polypeptide segments for folding, we have performed a complete circular permutation analysis of a protein in which the natural termini are in close proximity. As a model system, we used the disulfide oxidoreductase DsbA from Escherichia coli, a monomeric protein of 189 amino acid residues. To introduce new termini at all possible positions in its polypeptide chain, we generated a library of randomly circularly permuted dsbA genes and screened for active circularly permuted variants in vivo. A total of 51 different active variants were identified. The new termini were distributed over about 70 % of the polypeptide chain, with the majority of them occurring within regular secondary structures. New termini were not found in approximately 30 % of the DsbA sequence which essentially correspond to four alpha-helices of DsbA. Introduction of new termini into these "forbidden segments" by directed mutagenesis yielded proteins with altered overall folds and strongly reduced catalytic activities. In contrast, all active variants analysed so far show structural and catalytic properties comparable with those of DsbA wild-type. We suggest that random circular permutation allows identification of contiguous structural elements in a protein that are essential for folding and stability.     

Expression, purification, and characterization of blasticidin S deaminase (BSD) from Aspergillus terreus: the role of catalytic zinc in enzyme structure

We established an efficient overproduction-purification system for blasticidin S deaminase (BSD) using the cDNA cloned from Aspergillus terreus. The estimated molecular mass of the purified enzyme indicated BSD was a tetramer. This tetrameric form was very resistant to denaturation by SDS and showed heat-modifiable behavior on SDS-PAGE; i.e., BSD migrated much slower (as a single band of 36 kDa) in its active conformation than its completely denatured polypeptide (13 kDa) if heat treatment in 2% SDS was not performed before electrophoresis. As predicted from the presence of the catalytic zinc-coordinating sequence motif conserved in the cytosine nucleoside/nucleotide deaminase family, BSD also contained one zinc per deaminase subunit. However, the predicted catalytic function appeared not to be the only role of this zinc in the enzyme. First, titration of the zinc-chelating -SH groups with p-hydroxymercuriphenylsulfonate led to dissociation of the BSD tetramer into unstable monomers or dimers. Second, depletion of zinc on reconstitution of chemically denatured BSD (with either guanidine-HCl or acidic pH) resulted in improper folding of the polypeptide. These results suggest that zinc also plays a structural role in maintenance of the protein structure. When we introduced mutations at Glu-56 (the proposed active site) and Cys-91 (a proposed catalytic zinc-binding Cys) in BSD, none of the resulting mutants (E56D, E56Q, C91A, C91S, and C91H) showed any detectable activity, as judged with the spectrophotometric assay. Replacements of Cys-91 resulted in gross perturbation of the enzyme structure although the catalytically essential Glu-56 was not necessarily required for proper folding of the enzyme. These results further support our proposal that the catalytic zinc coordinated by the conserved sequence motif is also structural in BSD.