Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus.

Neutralizing polyclonal antibody to respiratory syncytial virus (RSV) has been shown to be an effective prophylactic agent when administered intravenously in high-risk infants. This study describes the generation of a humanized monoclonal antibody, MEDI-493, that recognizes a conserved neutralizing epitope on the F glycoprotein of RSV. The affinity of MEDI-493 was found to be equal to or slightly better than an isotype-matched chimeric derivative of the parent antibody. In plaque reduction, microneutralization, and fusion-inhibition assays, MEDI-493 was significantly more potent than the polyclonal preparation. Broad neutralization of a panel of 57 clinical isolates of the RSV A and B subtypes was demonstrated. Pretreatment of cotton rats with MEDI-493 resulted in 99% reduction of lung RSV titers at a dose of 2.5 mg/kg, corresponding to a serum concentration of 25-30 microg/mL. Further, MEDI-493 did not induce increased RSV infection or pathology in either a primary or a secondary challenge.

Mutations in the B subunit of Escherichia coli DNA gyrase that affect ATP-dependent reactions.

We have previously reported specific labeling of Escherichia coli DNA gyrase by the ATP affinity analog pyridoxal 5'-diphospho-5'adenosine (PLP-AMP), which resulted in inhibition of ATP-dependent reactions. The analog was found to be covalently bound at Lys103 and Lys110 on the gyrase B subunit (Tamura, J. K., and Gellert, M. (1990) J. Biol. Chem. 265, 21342-21349). In this study, the importance of these two lysine residues is examined by site-directed mutagenesis. Substitutions of Lys 103 result in the loss of ATP-dependent functions. These mutants are unable to supercoil DNA, to hydrolyze ATP, or to bind a nonhydrolysable ATP analog, 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP). The ATP-independent functions of gyrase, such as relaxation of negatively supercoiled DNA and oxolinic acid-induced cleavage of double-stranded DNA, are unaffected by these mutations, suggesting that the mutant B subunits are assembling correctly with the A subunits. Gyrase with substitutions of Lys110 retains all activities. However, the affinity of ATP is decreased. The DNA supercoiling activity of gyrase A2B2, tetramers reconstituted with varying ratios of inactive mutant and wild-type gyrase B subunits is consistent with a mechanism of DNA supercoiling that requires the interdependent activity of both B subunits in ATP binding and hydrolysis.

Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas.

Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy.

Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes.

Sequence motifs within the nonstructural protein NS3 of members of the Flaviviridae family suggest that this protein possesses nucleoside triphosphatase (NTPase) and RNA helicase activity. The RNA-stimulated NTPase activity of this protein from prototypic members of the Pestivirus and Flavivirus genera has recently been established and enzymologically characterized. Here, we experimentally demonstrate that the NS3 protein from a member of the third genus of Flaviviridae, human hepatitis C virus (HCV), also possesses a polynucleotide-stimulated NTPase activity. Characterization of the purified HCV NTPase activity showed that it exhibited reaction condition optima with respect to pH, MgCl2, and salt identical to those of the representative pestivirus and flavivirus enzymes. However, each NTPase also possessed several unique properties when compared with one another. Notably, the profile of polynucleotide stimulation of the NTPase activity was distinct for the three enzymes. The HCV NTPase was the only one whose activity was significantly enhanced by a deoxyribopolynucleotide. Additional distinguishing features among the three enzymes relating to the kinetic properties of their NTPase activities are discussed. These studies provide a foundation for investigation of the putative RNA helicase activity of these proteins and for further study of the role of the NS3 proteins of members of the Flaviviridae in the replication cycle of these viruses.

RNA-stimulated NTPase activity associated with the p80 protein of the pestivirus bovine viral diarrhea virus.

The genomic RNA of pestiviruses contains a single large open frame coding for virion structural proteins and viral nonstructural polypeptides. Based on the presence of specific amino acid sequence motifs, pestivirus nonstructural protein p80 was predicted to be both a serine-type proteinase and a nucleoside triphosphatase (NTPase)/RNA helicase. We previously demonstrated p80 possesses the former activity (Wisherchen and Collett, Virology 184, 341-350, 1991). Here, we provide experimental evidence that this protein is also an RNA-stimulated NTPase. Employing immunoaffinity chromatography, we partially purified a p80 protein analog (p87) from recombinant baculovirus-infected insect cells. We show this preparation contained a specific NTPase activity. This activity was not found in material similarly purified from lysates of baculovirus-infected insect cells not expressing the p87 protein. That the NTPase activity was associated with the p87 polypeptide was demonstrated in two ways. First, the NTPase activity was shown to be completely inhibited by monoclonal antibodies specific to the p80 polypeptide, but was unaffected by monoclonal antibodies to unrelated antigens. Second, radiolabeled ATP could be specially cross-linked to the p87 polypeptide. NTP hydrolysis by the p87 protein was stimulated by the presence of particular single-strand RNA molecules. Initial enzymologic characterization of the pestivirus p80 NTPase is presented, and the presumptive role of this activity in pestivirus replication is discussed.

RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria.

The nonstructural protein NS3 of the prototypic flavivirus, yellow fever virus, was investigated for possession of an NTPase activity. The entire NS3 protein coding sequence and an amino-terminal truncated version thereof were engineered into Escherichia coli expression plasmids. Bacteria harboring these plasmids produced the expected polypeptides, which upon cell disruption were found in an insoluble aggregated material considerably enriched for the NS3-related polypeptides. Solubilization and renaturation of these materials, followed by examination of their ability to hydrolyze ATP, revealed an ATPase activity present in both the full-length and amino-terminal truncated NS3 preparations but not in a similarly prepared fraction from E. coli cells engineered to express an unrelated polypeptide. The amino-terminal truncated NS3 polypeptide was further enriched to greater than 95% purity by ion-exchange and affinity chromatography. Throughout the purification scheme, the ATPase activity cochromatographed with the recombinant NS3 polypeptide. The enzymatic activity of the purified material was shown to be a general NTPase and was dramatically stimulated by the presence of particular single-stranded polyribonucleotides. These results are discussed in view of similar activities identified for proteins of other positive-strand RNA viruses.

Slow interaction of 5'-adenylyl-beta,gamma-imidodiphosphate with Escherichia coli DNA gyrase. Evidence for cooperativity in nucleotide binding.

We have examined the kinetics of interaction between Escherichia coli DNA gyrase and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP) in the presence and absence of ATP. In the absence of ATP, [alpha-32P]ADPNP binds extremely slowly to gyrase, with an apparent second-order rate constant (k1) of 120 M-1 min-1. Similarly, the limited negative supercoiling of closed-circular DNA caused by ADPNP binding is slow, requiring at least 2 h to reach completion in the presence of 100 microM ADPNP. A very slow but detectable rate of dissociation of ADPNP from gyrase was measured, with a rate constant of 3.5 x 10(-4) min-1. The calculated dissociation constant for ADPNP is thus 2.9 microM. ADPNP is a potent competitive inhibitor of ATP-dependent DNA supercoiling. Inhibition is established much more rapidly than can be accounted for by the slow rate of ADPNP binding in the absence of ATP. We have found that ATP can accelerate the rate of [32P]ADPNP binding by more than 15-fold (k1 = 1,850 M-1 min-1). The ATP-promoted rate enhancement requires the presence of DNA; in the absence of DNA, ATP has no effect on the rate of binding. Relaxed closed-circular, nicked-circular, and linear pBR322 DNA are all equally effective cofactors for ATP-stimulated binding of ADPNP. After a short lag, the presence of ATP also greatly speeds up ADPNP dissociation from gyrase bound initially to closed-circular DNA, with the restoration of DNA supercoiling activity. This effect is not observed in the presence of nicked-circular or linear DNA, suggesting that ADPNP dissociates more rapidly from gyrase bound to supercoiled DNA. The results of ADPNP binding provide evidence for cooperative interactions between the nucleotide binding sites. To account for these data, a model is proposed for the interaction of nucleotides at the two ATP binding sites on DNA gyrase.

Characterization of the ATP binding site on Escherichia coli DNA gyrase. Affinity labeling of Lys-103 and Lys-110 of the B subunit by pyridoxal 5'-diphospho-5'-adenosine.

We have labeled the adenosine triphosphate binding site of Escherichia coli DNA gyrase with the ATP affinity analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP strongly inhibits the ATP-ase and DNA supercoiling activities of DNA gyrase, with 50% inhibition occurring at 7.5 microM inhibitor. ATP and ADP compete with PLP-AMP for binding and protect the enzyme against inhibition. The labeling appears to proceed by a Schiff base complex between the 4-formyl group of the pyridoxyl moiety of PLP-AMP and a protein primary amino group, since the inhibition and reagent labeling are reversible unless the complex is treated with NaBH4. Complete inactivation is estimated to occur upon the covalent incorporation of 2 mol of inhibitor/mol of gyrase. The Km for ATP was found to be unchanged for partially inhibited enzyme samples, suggesting an all-or-none type of inhibition. A 3H-labeled peptide spanning residues 93-131 of the B protein was isolated from a V-8 protease digest. Radioactive peaks corresponding to Lys-103 and Lys-110 were found during the Edman degradation, suggesting that these amino acids form part of the ATP binding site. A comparison of the amino acid sequence in this region with the sequences of other type II topoisomerases indicates the possible location of a common ATP binding domain.

The adenine nucleotide binding site on yeast hexokinase PII. Affinity labeling of Lys-111 by pyridoxal 5'-diphospho-5'-adenosine.

The adenine nucleotide analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), is shown to be a potent and specific inhibitor of yeast hexokinase PII. Evidence that the analog binds specifically at the ATP binding site includes the demonstration that glucose binding enhances PLP-AMP binding and that PLP-AMP and ATP bind competitively with an apparent Ki(PLP-AMP) = 23 microM. In addition, from the relationship between the degree of inhibition and extent of modification, it is estimated that the incorporation of 1 mol of PLP-AMP/mol of subunit is required for complete inhibition. Borohydride reduction of the Schiff's base complex formed between hexokinase and [3H]PLP-AMP gives a stable product. The reduced derivative was digested with trypsin and a single radioactive peptide was isolated by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis identified Lys-111 as the modified residue. Taking into account the known structures of the binary complexes (Shoham, M., and Steitz, T. A. (1980) J. Mol. Biol. 140, 1-14), the results suggest that Lys-111, located in the smaller of the two lobes of hexokinase, moves into the active site upon formation of the ternary complex.

Affinity labeling of nucleotide-binding sites on kinases and dehydrogenases by pyridoxal 5'-diphospho-5'-adenosine.

A new adenine nucleotide analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), has been synthesized. The effectiveness of PLP-AMP as an affinity probe has been tested using a number of nucleotide-binding enzymes. In comparison to reaction with pyridoxal 5'-phosphate, PLP-AMP binds more tightly and exhibits greater specificity of labeling for most enzymes tested. PLP-AMP is a very potent inhibitor of yeast alcohol dehydrogenase and rabbit muscle pyruvate kinase, with complete inhibition obtained upon incorporation of 1 mol of reagent/mol of catalytic subunit. The reagent is also a potent inhibitor of yeast hexokinase and phosphoglycerate kinase. When modified in the absence of substrates, these enzymes require 2 mol of reagent/mol of active site for complete inhibition. However, when modified in the presence of sugar substrates, this stoichiometry decreases to 1.1 for the hexokinase-glucose complex and 1.4 for the phosphoglycerate kinase . 3-phosphoglycerate complex. The most potent inhibition by PLP-AMP was observed with rabbit muscle adenylate kinase. Half-maximal inhibition was obtained at a concentration of approximately 1 microM. In contrast to these examples, PLP-AMP, as well as pyridoxal 5'-phosphate, fails to act as a potent or specific inhibitor of beef heart mitochondrial F1-ATP-ase. The high specificity of labeling and the ability of nucleotide substrates to decrease the rate of inactivation of the kinases and dehydrogenase are consistent with the modification of active site residues. The complete reversibility of both modification and inactivation in the absence of reduction by NaBH4 and the absorption spectra of modified enzymes prior to and following reduction indicate reaction with lysyl residues. We conclude that PLP-AMP holds considerable promise as an affinity label for exploring the structure and mechanism of nucleotide-binding enzymes.

Changes in chemical properties of mitochondrial adenosinetriphosphatase upon removal of tightly bound nucleotides.

The removal of tightly bound nucleotides from mitochondrial F1-ATPase was found to affect the inhibition by ADP and chemical reactivity toward 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-C1) and sulfhydryl reagents. Preincubation of nucleotide-depleted F1 with 40 microM ADP in the presence of ethylenediaminetetraacetic acid (EDTA) resulted in a 51% inhibition of the steady-state level of ATPase activity whereas only a 25% inhibition was observed for native F1. Both partially inhibited states of the enzyme could be reversed by the subsequent addition of ATP. Measurement of [14C]ADP binding to nucleotide-depleted F1 in the presence of EDTA reveals three equivalent ADP binding sites with a Kd of 0.45 microM, and a fourth site of lower affinity. The sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) were found to inhibit the ATPase activity of nucleotide-depleted F1 but not native F1 or nucleotide-depleted F1 in the presence of ADP or ATP. Polyacrylamide gel electrophoresis of nucleotide-depleted F1 labeled with [14C]NEM gave a 2-fold increase in incorporation into the (alpha + beta) subunits and a 7-fold increase in label in the gamma subunit after 90 min compared to when ADP was present during the reaction. ADP binding to the noncatalytic sites enhanced the rate of inhibition of nucleotide-depleted F1 by NBD-C1 about 2-fold while retarding the subsequent intramolecular transfer from an essential phenol group to an amino group about 2.8-fold. The results suggest a conformational change in F1 caused by changes in nucleotide--protein interaction at the noncatalytic sites.