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D E Hruby

Publications and source records attributed to D E Hruby.

At least 55 records · Page 3Linked to original sources
PubMed1993

Further characterization of an adenosine-containing modification of vaccinia virus proteins.

Three vaccinia virus (VV) core proteins which become labeled when virus is grown in the presence of radiolabeled adenosine or orthophosphate were identified as the major viral core proteins 4A, 4B, and 25K on the basis of comigration with [35S]methionine-labeled viral proteins and immunoprecipitation with monospecific polyclonal antisera. Boronate affinity chromatography and HPLC analysis suggested that a cis-diol-containing adenosine compound is present on this set of viral proteins. The replication of VV in tissue culture cells was prevented by the ADP-ribosylation inhibitors nicotinamide (NIC), 3-aminobenzamide (3-AB), and meta-iodobenzylguanidine (MIBG). None of these compounds significantly affected viral DNA synthesis at lower drug concentrations, although at higher concentrations of the three drugs a reduction in viral DNA synthesis was evident. Total VV protein synthesis also decreased at higher inhibitor levels, and the proteolytic processing of the major virion core proteins was greatly diminished as well. The three inhibitors also affected labeling of viral core proteins and cellular histone proteins by [8-14C]adenosine. In addition, mature, infectious virus particles were not formed in the presence of either 60 mM NIC or 3-AB, or 0.6 mM MIBG. These results provide evidence that the major VV core proteins are subject to modification by an adenosine compound, and suggest the possibility that this modification might represent ADP-ribosylation.

Adenosine↗
PubMed1993

An NH2-terminal peptide from the vaccinia virus L1R protein directs the myristylation and virion envelope localization of a heterologous fusion protein.

The vaccinia virus L1R gene product is a late protein destined for insertion into the envelope of intracellular virus particles. Because this protein is co-translationally modified by the addition of myristic acid to the penultimate NH2-terminal glycine residue, it was of interest to identify the modification signal within the L1R protein and to assess the relevance of myristylation to protein localization. To this end, a family of chimeric reporter genes containing 0-13 codons from the NH2 terminus of the L1R open reading frame fused in-frame to the bacterial chloramphenicol acetyltransferase gene was constructed. The encoded proteins were tested as myristylation substrates in cell-free extracts and infected cells. The results obtained in vitro and in vivo were similar and suggested that although the NH2-terminal 5 amino acids of the L1R protein were the minimum signal required to observe modification by myristate, 12 amino acids were required to obtain wild type levels of myristylation with a modulating role played by the intervening amino acid residues. Furthermore, subcellular fractionation of infected cells expressing the fusion proteins indicated that the NH2 terminus of the L1R protein was capable of targeting the fusion proteins to membrane-containing fractions only if myristylated. In particular, the myristylated fusion protein containing the first 12 amino acids of the L1R protein abutted to the chloramphenicol acetyltransferase protein was found associated with the envelope of intracellular vaccinia virus particles.

Acyltransferases↗
PubMed1993

Expression and single-step purification of enzymatically active vaccinia virus thymidine kinase containing an engineered oligohistidine domain by immobilized metal affinity chromatography.

A method has been developed for the controlled expression in Escherichia coli and rapid purification of an enzymatically active vaccinia virus (VV) thymidine kinase protein containing an engineered oligohistidine domain. The nucleotide sequence that encodes the VV thymidine kinase open reading frame was inserted into a plasmid expression vector (pET-16b, Novagen Inc., Madison, WI) under the control of a strongly repressed bacteriophage T7 promoter and high efficiency translational signals. The construct (pET-16b:TK) directs the synthesis of a fusion protein (His-TK) with an N-terminal histidine decapeptide fused to the VV thymidine kinase polypeptide. Upon induction of E. coli strain BL21(DE3)pLysS with isopropyl beta-D-thiogalactoside, accumulation of large quantities of a 22-kDa protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein reacted with polyclonal antiserum raised against a TrpE-VVTK fusion protein. The predominantly soluble fusion protein (approximately 13% of the total soluble bacterial protein) was purified to homogeneity from crude bacterial extracts in a single-step by immobilized metal chelate affinity chromatography (Ni(2+)-nitrilotriacetic acid-agarose) under nondenaturing conditions and was shown to have thymidine kinase activity. The yield of the purification scheme was about 15 mg recombinant protein/liter of bacterial culture. The availability of purified VV TK protein should greatly facilitate biochemical studies on its enzymatic activity, as well as analyses of its structural and functional domains.

Amino Acid Sequence↗
PubMed1993

Isolation and analysis of vaccinia virus previrions.

Vaccinia virus (VV) virion morphogenesis is a complex sequence of events that occurs late in viral infection that is essential for the production of mature progeny. Electron microscopy studies have identified multiple morphogenic forms of virus particles, apparently assembled in a sequence from immature to mature particles that correlates with distinct physical changes. This assembly process is, however, rather poorly understood at the molecular level. To better characterize the multiple forms of VV previrions, sucrose log gradient fractionation of VV-infected cells was used to separate radiolabeled immature and mature forms of the virus. Depending on time postinfection that the infected cells were harvested, four distinct peaks of acid-precipitable counts could be detected that displayed different rates of sedimentation. Using pulse-chase analysis procedures, the labeled peaks were shown to have precursor-product relationships as slower sedimenting entities chased to faster sedimenting ones with time. These peaks were referred to as A, B, C, and V particles, with A being the initial precursor form found near the top of the gradient and V being the fastest sedimenting product. As the previrions mature, they migrated faster in the gradient and became infectious and resistant to treatment with DNase I. The core protein composition of the A particles was predominantly uncleaved precursors, with only small amounts of the mature core proteins 4a, 4b, 25K, and 23K evident. However, as the sedimentation rate of the particles increased, proteolytic maturation proceeded such that C particles were composed almost exclusively of mature core proteins. Together these results indicate that several distinct and separable forms of VV previrions exist, that VV core protein precursors are associated with the previrions prior to cleavage, and that maturation of the core proteins is coordinately linked to the conversion from noninfectious previrions to infectious viral particles.

Cell Line↗
PubMed1993

Viral acylproteins: greasing the wheels of assembly.

Viruses take advantage of the host's protein modification and targeting pathways to modify their own proteins and to ensure that they assume active configurations and locate appropriately for assembly. In many viruses, one recurrent theme in such processes is exploitation of cellular protein acylation pathways for the addition of myristic and palmitic acid to capsid or envelope proteins.

Acylation↗
PubMed1993

trans processing of vaccinia virus core proteins.

The three major vaccinia virus (VV) virion proteins (4a, 4b, and 25K) are proteolytically matured from larger precursors (P4a, P4b, and P25K) during virus assembly. Within the precursors, Ala-Gly-X motifs have been noted at the putative processing sites, with cleavage apparently taking place between the Gly and X residues. To identify the sequence and/or structural parameters which are required to define an efficient cleavage site, a trans-processing assay system has been developed by tagging the carboxy terminus of the P25K polypeptide (precursor of 25K) with an octapeptide FLAG epitope, which can be specifically recognized by a monoclonal antibody. By using transient expression assays with cells coinfected with VV, the proteolytic processing of the chimeric gene product (P25K:FLAG) was monitored by immunoblotting procedures. The relationship between the P25K:FLAG precursor and the 25K:FLAG cleavage product was established by pulse-chase experiments. The in vivo cleavage of P25K:FLAG was inhibited by the drug rifampin, implying that the reaction was utilizing the same pathway as authentic VV core proteins. Moreover, the 25K:FLAG protein was found in association with mature virions in accord with the notion that cleavage occurs concomitantly with virion assembly. Site-directed mutagenesis of the Ala-Gly-Ala motif at residues 31 to 33 of the P25K:FLAG precursor to Ile-Asp-Ile blocked production of the 25K:FLAG product. The efficiency of 25K:FLAG production (33.71%) is, however, approximately only half of the production of 25K (63.98%) within VV-infected cells transfected with pL4R:FLAG. One explanation for the lower efficiency of 25K:FLAG production was suggested by the observation in the immunofluorescent-staining experiment that 25K:FLAG-related proteins were not specifically localized to the virus assembly factories (virosomes) within VV-infected cells, although virosome localization was prominent for P25K-related polypeptides. Since VV core protein proteolytic processing is believed to take place during virion maturation, only the P25K:FLAG which was assembled into immature virions could undergo proteolytic maturation. Furthermore during these experiments, a potential cleavage intermediate (25K') of P25K was identified. Amino acid residues 17 to 19 (Ala-Gly-Ser) of the P25K precursor were implicated as the intermediate cleavage site, since no 25K':FLAG product was produced from a mutant precursor in which the sequence was altered to Ile-Asp-Ile. Taken together, these results provide biochemical and genetic evidence to support the hypothesis that the Ala-Gly-X cleavage motif plays a critical role in VV virion protein proteolytic maturation.

Amino Acid Sequence↗
PubMed1993

Vaccinia virus: a novel approach for molecular engineering of peptide vaccines.

One of the more promising new approaches for producing prophylactic vaccines involves the genetic engineering of vaccinia virus (VV) strains to express a single protein, a portion of a protein, or a single epitope from a heterologous pathogen. As an example, the application of this approach to the prevention of streptococcal-related diseases is considered. Experiments are described in which several generations of VV-based recombinant vaccines were constructed that expressed various derivatives of the M protein gene from Streptococcus pyogenes (serotype 6). These recombinants were used to demonstrate the high-level expression, stability, and antigen authenticity of the bacterial proteins expressed in infected tissue culture cells. When tested in a mouse model system, immunization with the VV recombinants expressing specific subdomains of the M protein conferred protection to challenge from both homologous and heterologous serotypes of pathogenic streptococci.

Animals↗
PubMed1992

Vaccinia and cowpox viruses encode a novel secreted interleukin-1-binding protein.

Supernatants from vaccinia virus (VV)-infected CV-1 cells were examined and found to contain a 33 kd protein capable of binding murine interleukin-1 beta (mIL-1 beta). A VV open reading frame (ORF) that exhibits 30% amino acid identity to the type II IL-1 receptor was expressed in CV-1-EBNA cells and shown specifically to bind mIL-1 beta. A similar ORF from cowpox virus was expressed and also specifically bound mIL-1 beta. A recombinant VV was constructed in which this ORF was disrupted (vB15RKO). Supernatants from vB15RKO-infected cells did not contain an IL-1-binding protein. Supernatants from VV-infected CV-1 cells were capable of inhibiting IL-1-induced murine lymphocyte proliferation in vitro while supernatants from vB15RKO infected cells did not. Intracranial inoculation of mice with vB15RKO suggests that this ORF is involved in VV virulence. The possible role of a virus-encoded IL-1-binding protein in the pathology of a poxvirus infection and its relationship to other poxvirus-encoded immune modulators is discussed.

Animals↗
PubMed1992

A single amino acid substitution abolishes feedback inhibition of vaccinia virus thymidine kinase.

Exploitation of differences in the substrate specificity of the type I and type II thymidine kinases (EC 2.7.1.21, TK) expressed by the Herpesviridae and Poxviridae (and human cells), respectively, has lead to the development of effective antiherpetic drugs such as acyclovir and gancyclovir. Analysis of type I TK protein sequences reveals a consensus sequence which corresponds to domain IV of type II TK proteins such as that encoded by vaccinia virus (VV). The type I descriptor (Xpho - + + Xpho) differs at the second position from the type II consensus sequence (Xpho Xphi + + Xpho) by having an aspartic acid residue (D) substituted for a glutamine (Q). To test the hypothesis that this substitution may be responsible for the observed differences in substrate specificity of these enzymes and as a approach to identify the nucleoside binding site of the type II VV TK, site-directed mutagenesis was employed to alter glutamine 114 (Q114) within domain IV of VV TK to a histidine (Q114H) or an aspartic acid (Q114D). All of the mutant enzymes retained full enzymatic activity as compared to wild-type VV TK when thymidine or bromodeoxyuridine were used as substrates. However, unlike the wild-type herpes simplex (type 1) TK enzyme, neither wild-type nor domain IV VV TK mutants were able to phosphorylate acyclovir or cytidine substrates. Surprisingly, the domain IV VVTK mutants displayed a dramatic loss of feedback inhibition by dTTP. Mutations of the Q114 position also lead to a difference in ATP binding as demonstrated by an altered elution pattern of Q114H and Q114D from an ATP-agarose affinity column with dTTP. Taken together, these results suggest that domain IV of VV TK is not involved directly in substrate discrimination but instead participates in feedback inhibition by dTTP.

Acyclovir↗
PubMed1992

Site-directed mutagenesis of a conserved domain in vaccinia virus thymidine kinase. Evidence for a potential role in magnesium binding.

Alignment of prokaryotic and vertebrate type II thymidine kinases (TK) (EC 2.7.1.21), such as that encoded by vaccinia virus (VVTK), reveals three conserved regions: designated domains I, III, and VII. Domains I and III of VVTK contain residues which closely resemble segments A (ATP) and B (Mg2+), respectively, of a Mg.ATP binding descriptor proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J. (1982) EMBO J. 1, 945-951). In support of this hypothesis, domain I of the VVTK enzyme has previously been identified as the ATP binding site (Black and Hruby, 1990b). With regard to Mg2+ binding, several features of the VVTK domain III suggest that it may be responsible for this activity: 1) sequence similarity to a magnesium binding motif proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J. (1982) EMBO J. 1, 945-951); 2) alignment of the predicted secondary structure of type II TK enzymes with other magnesium-binding enzymes such as adenylate kinase, EF-TU, and p21 reveals a conserved aspartic acid residue preceded by several hydrophobic residues with domain III; and 3) the conserved VVTK domain III aspartic acid residue (D82) aligns with D93 residue of adenylate kinase which is has been shown by NMR to participate in Mg2+ binding (Yan, H., and Tsai, M.-D., Biochemistry, in press). To directly examine the potential contribution of the conserved domain III D82 residue of VVTK in magnesium binding, site-directed mutagenesis was performed on positions D82 and G84 to generate four mutants, N82, L82, I82, and V84. Each mutant was analyzed for enzyme activity, divalent cation requirements, tetramer formation, and ATP binding ability. The results obtained were consistent with D82 playing a direct role in Mg2+ binding and suggest that while the aspartic acid does not appear to participate directly with ATP binding it may instead act to facilitate ATP hydrolysis by binding Mg2+ which aids to correctly position ATP for nucleophilic attack.

Amino Acid Sequence↗
PubMed1992

Evidence for multiple species of vaccinia virus-encoded palmitylated proteins.

When cells were infected with vaccinia virus in the presence of [3H]palmitic acid, radiolabel was incorporated into six viral proteins with apparent molecular weights of 92, 41, 37, 26, 17, and 14 kDa, all of which are expressed at late times during the infection cycle. The [3H]palmitate-labeled fatty acid moieties from the modified proteins were isolated, converted to p-nitrophenacyl derivatives, and subjected to reverse phase HPLC analysis which confirmed the identity of the fatty acid group as palmitic acid. Furthermore, the radiolabeled palmitate-protein bonds were sensitive to treatment with neutral hydroxylamine, suggesting that association of the fatty acid moieties with these proteins occurs via a thioester linkage. Previous studies by other investigators have identified the 37-kDa protein as the major antigen present in the outer membrane of extracellular enveloped virions, and demonstrated that the protein is modified by palmitic acid but is not glycosylated (G. Hiller and K. Weber J. Virol. (1985) 55, 651-659). Growth of vaccinia virus in the presence of tunicamycin indicated that the 41- and 26-kDa palmitylated proteins were also subject to modification by glycosylation, whereas like the 37-kDa protein, the 92-, 17-, and 14-kDa species did not appear to be glycosylated. Subcellular fractionation studies provided evidence that all of the viral palmitylated proteins were membrane-associated. Extraction of purified vaccinia virus with NP-40 and DTT demonstrated that the palmitylated proteins were associated with one of the viral membranes rather than the core of the virion. Viewing these results together with the previous reports of myristylated VV proteins (Franke et al. J. Virol. (1989) 63, 4285-4291), suggests that acylation of VV proteins represents a major modification pathway utilized by VV proteins during the assembly of progeny virions.

Chromatography, High Pressure Liquid↗
PubMed1991

Assembly and analysis of a functional vaccinia virus "amplicon" containing the C-repeat region from the M protein of Streptococcus pyogenes.

Previous studies have shown that when inoculated intranasally into mice, vaccinia virus (VV) recombinants expressing the carboxyl half of the Streptococcus pyogenes M protein [which contains the C-repeat region (CRR)] could elicit a protective immune response against subsequent challenge by both homologous and heterologous serotypes of pathogenic group A streptococci. In the present study, an insertion plasmid was constructed that contained three tandem in-frame repeats of a 310-base-pair DNA sequence encoding the CRR from streptococcal M6 protein under control of a constitutive viral promoter. The plasmid was used to introduce the bacterial sequences into the VV genome by homologous recombination. Surprisingly, the recombinant VV:CRR3X virus that was isolated appeared to represent not an individual recombinant virus but a complex mixture of variants that contained from 1 to greater than 20 tandem copies of the CRR region at the insertion site. This genomic complexity was mirrored at the transcriptional level in that a nested set of coterminal transcripts was detected in VV:CRR3X-infected cells, which increased in size from 1400 to 6600 bases by increments of approximately 300 bases. All transcripts containing two or more CRR inserts appeared functional, as Western (immuno) blot analyses of VV:CRR3X-infected cell extracts revealed a family of CRR-related proteins with apparent molecular masses that increased from 30 kDa upward in increments of 10 kDa. All data are consistent with the hypothesis that variation in the VV:CRR3X recombinants is from random crossover events that occur within the CRR region during viral DNA replication. These results suggest that the genomic diversity generated by the "recombinogenic" properties of vaccinia recombinants containing tandem foreign inserts could be used to facilitate induction of a broadly protective immune response against antigenically diverse pathogenic agents.

Amino Acid Sequence↗
PubMed1991

The multistep proteolytic maturation pathway utilized by vaccinia virus P4a protein: a degenerate conserved cleavage motif within core proteins.

The most abundant vaccinia virus (VV) core protein found within the virion is protein 4a, which represents approximately 14% of the particle's dry weight. The 4a protein is synthesized as a 102.5-kDa precursor, which is proteolytically processed to a 62-kDa product concomitant with virion assembly. To identify the pathway by which P4a is converted into 4a, immunological reagents which are specific for subregions of the P4a precursor were developed and used in concert with peptide mapping and protein sequencing procedures. The results obtained suggest that the 891 amino acid P4a precursor is cleaved at two locations, between residues 614 and 615 and 697 and 698. Both the large amino-terminal 4a protein (residues 1-614) and the carboxy-terminal-derived 23-kDa protein (residues 698-891) become major virion constituents. The location and fate of the small internal peptide (residues 615-697) is not known. Interestingly, an analysis of the predicted amino acid sequences at the sites of cleavage within the P4a precursor indicated the presence of an Ala-Gly decreases Thr motif flanking the 697-698 site and an Ala-Gly decreases Ser motif flanking the 614-615 site. Since both of these signals are quite similar to the Ala-Gly decreases Ala signal previously identified as the cleavage point within the VV P4b and P25K core protein precursors (VanSlyke et al., 1991.J. Gen. Virol. 72, 411-416), this suggests that processing of all three core protein precursors may be coordinately linked and/or catalyzed by the same proteinase during viral assembly.

Amino Acid Sequence↗
PubMed1991

Proteolytic maturation of vaccinia virus core proteins: identification of a conserved motif at the N termini of the 4b and 25K virion proteins.

Three structural proteins (4a, 4b and 25K) located within the virion core of vaccinia virus are cleavage products of precursor polypeptides (P4a, P4b and P25K) synthesized late in viral infection. Pulse-chase labelling experiments revealed that cleavage of the core proteins lags considerably behind precursor synthesis and that processing requires continuous protein synthesis. The N-terminal sequences of 4b and 25K, but not 4a, were determined by microsequencing core proteins isolated from purified virions. Comparison of these data with the predicted amino acid sequence of P4b and P25K revealed a conserved Ala-Gly-Ala motif flanking the apparent N termini of both proteins, as well as several additional sequence similarities between the P4b and P25K precursors both upstream and downstream of the putative cleavage site. The Ala-Gly-Ala tripeptide signal was also found in the same region of the amino acid sequences of the homologous proteins of fowlpox virus.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of the Escherichia coli thymidine kinase gene provides evidence for conservation of functional domains and quaternary structure.

Using lambda bacteriophage clones from the Kohara Escherichia coli library spanning minutes 25.5 to 28.5 on the E. coli chromosome (strain W3110), two overlapping DNA fragments were identified which were able to confer thymidine kinase (TK) enzyme activity to a TK- strain of E. coli (KY895). This genetic complementation assay was used in concert with subcloning procedures to identify the minimal region (a 900 bp EcoRI-SalI fragment) which contained the E. coli thymidine kinase gene (tdk). The nucleotide sequence of the EcoRI-SalI fragment and a small portion of the adjoining downstream fragment was determined. Computer analysis of the derived sequence indicated the presence of a rightward-reading open reading frame of 615 bp which was capable of encoding a 205-amino-acid polypeptide with a predicted Mr of 23458 daltons. The in vivo transcriptional activity of this locus was confirmed by Northern blot hybridization analysis of RNA isolated from E. coli JM101 or KY895 which detected a 650-nucleotide RNA transcribed from this region. This places the tdk gene at approximately minute 27.35 on the E. coli W3110 chromosome, about 15 kb downstream from the narG locus and approximately 25 kb upstream of the trp operon. Although the predicted Mr of the E. coli TK protein was 23.5 kDa, gel-filtration analyses suggested that, like eukaryotic thymidine kinases, the active form of this enzyme is a multimeric complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Identification of the ATP-binding domain of vaccinia virus thymidine kinase.

Although small in size (20 kDa), the vaccinia virus (VV) thymidine kinase protein (EC 2.7.1.21 TK) is a relatively complex enzyme which must contain domains involved in binding both substrates (ATP and thymidine) and a feedback inhibitor (dTTP), as well as sequences directing the association of individual protein monomers into a functional tetrameric enzyme. Alignment of predicted amino acid sequences of the thymidine kinase genes from a variety of sources was used to identify highly conserved regions as a first step toward locating potential regions housing essential domains. A conserved domain (domain I) near the amino terminus of VV TK protein had characteristics consistent with a nucleotide-binding site. Analysis of the nucleotide substrate specificity of VV TK indicated that ATP acts as the major phosphate donor for thymidine phosphorylation while GTP, CTP, and UTP were inefficient substrates. Site-directed mutagenesis was performed on domain I to generate 11 mutant enzymes. Comparison of the wild-type and mutant proteins with regard to enzyme activity revealed that two of the mutant enzymes, T18 and S19, exhibited enhanced enzyme activity (3.73-fold and 1.35-fold, respectively) relative to the control. The other mutations introduced led to greatly reduced levels of enzyme activity which correlated with a reduced or altered ability of the mutant enzymes to bind ATP as determined by ATP-agarose affinity chromatography. Wild-type VV TK bound to an ATP affinity column could also be eluted with dTTP. Glycerol gradient separation of wild-type TK in the presence or absence of dTTP indicated that dissociation of the tetrameric complex was not the means by which enzymatic inhibition was achieved. Taken together, these results suggest that (i) domain I (amino acids 11-22) of the VV TK corresponds to the ATP-binding site, and (ii) that dTTP is able to interfere with ATP binding, either directly or indirectly, and thereby inhibit enzymatic activity without dissociating the native enzyme.

Adenosine Triphosphate↗