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Biomedical subjects

D E Hruby

Publications and source records attributed to D E Hruby.

At least 37 records · Page 2Linked to original sources
PubMed1997

Secretion of Porphyromonas gingivalis fimbrillin polypeptides by recombinant Streptococcus gordonii.

The fimbriae of Porphyromonas gingivalis plays an important role in the pathogenesis of periodontal disease. A structural subunit of the P. gingivalis fimbriae, fimbrillin, has been shown to promote adherence of the bacteria to host surfaces and also induce an immune response. Biologically active domains of fimbrillin responsible for adherence or eliciting immune responses have been determined. In a previous study, we engineered the human oral commensal organism Streptococcus gordonii to express such biologically active domains on the surface of the bacteria as a vaccine delivery system. In this study we report an alternative approach of secreting fimbrillin polypeptide domains into the medium by modification of the surface-expression system described earlier. Such recombinant S. gordonii, in addition to being a source for antigen presentation to trigger a protective immune response, may have the added advantage of directly blocking the fimbriae-mediated adherence of P. gingivalis to the oral cavity following implantation. This approach can also be utilized for secreting other biologically important therapeutic molecules on mucosal surfaces for modulating local microenvironments.

Bacterial Proteins↗
PubMed1997

Palmitylation of the vaccinia virus 37-kDa major envelope antigen. Identification of a conserved acceptor motif and biological relevance.

Computer-assisted alignment of known palmitylproteins was used to identify a potential peptide motif, TMDX1-12AAC(C)A (TMD, transmembrane domain; X, any amino acid; C, cysteine acceptor residues; A, aliphatic residue) responsible for directing internal palmitylation of the vaccinia virus 37-kDa major envelope antigen, p37. Site-directed mutagenesis was used to confirm this motif as the site of modification and to produce a nonpalmitylated version of the p37 protein. Comparative phenotypic analysis of the wild-type and mutant p37 alleles confirmed that the p37 protein is involved in viral envelopment and egress, and suggested that attachment of the palmitate moiety was essential for correct intracellular targeting and protein function.

Acylation↗
PubMed1997

Chlamydia psittaci IncA is phosphorylated by the host cell and is exposed on the cytoplasmic face of the developing inclusion.

Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole called an inclusion. Chlamydia psittaci (strain GPIC) produces a 39 kDa protein (IncA) that is localized to the inclusion membrane. While IncA is present as a single 39 kDa species in purified reticulate bodies, two additional higher M(r) forms are found in C. psittaci-infected cells. This finding suggested that IncA may be post-translationally modified in the host cell. Here we present evidence that IncA is a serine/threonine phosphoprotein that is phosphorylated by host cell enzymes. This conclusion is supported by the following experimental findings: (i) treatment of infected cells with inhibitors of host cell phosphatases or kinases altered the electrophoretic migration pattern of IncA; (ii) treatment with calf intestinal alkaline phosphatase eliminated the multiple-banding pattern of IncA, leaving only the protein band with the lowest relative molecular weight; and (iii) radioimmunoprecipitation of lysates of [32P]-orthophosphate-labelled infected HeLa cells with anti-IncA antisera demonstrated that the two highest M(r) IncA bands were phosphorylated. A vaccinia-virus recombinant expressing incA was used to determine if HeLa cells can phosphorylate IncA in the absence of a chlamydial background. IncA in lysates of these cells migrated identically to that seen in C. psittaci-infected cells, indicating the host cell was responsible for the phosphorylation of the protein. Microinjection of fluorescently labelled anti-IncA antibodies into C. psittaci-infected HeLa cells resulted in immunostaining of the outer face of the inclusion membrane. Collectively, these results demonstrate that IncA is phosphorylated by the host cell, and regions of IncA are exposed at the cytoplasmic face of the inclusion.

Animals↗
PubMed1997

Development of a radioactive protein A-based assay for analysis of surface protein expression in gram-positive bacteria.

This paper describes an immunochemical method which uses radioactive protein A for the detection and analysis of streptococcal M6 protein epitopes on the surface of recombinant Streptococcus gordonii. With this assay, recombinant S. gordonii cells expressing a portion of the M6 protein on their surfaces show a 75-fold increase in bound radioactivity over cells of the control S. gordonii parental strain. Furthermore, use of the assay to monitor the amount of M6 protein present on the surface of the S. gordonii recombinant during growth in culture demonstrated that expression is highest at late log phase, with the protein being sloughed off during stationary phase. This simple assay allows analysis of surface protein without any protein purification or sophisticated instrumentation. As such, it should be broadly applicable to following the expression of most surface-accessible bacterial proteins.

Antigens, Bacterial↗
PubMed1997

Identification and analysis of three myristylated vaccinia virus late proteins.

Previous studies have shown that at least three vaccinia virus (VV) late proteins (with apparent molecular asses of 37, 35, and 25 kDa) label with myristic acid. Time course labeling of VV-infected cells with [3H]myristic acid reveals at least three additional putative myristylproteins, with apparent molecular masses of 92, 17, and 14 kDa. The 25-kDa protein has previously been identified as that encoded by the L1R open reading frame, leaving the identities of the remaining proteins to be determined. Sequence analysis led to the preliminary identification of the 37-, 35-, and 17-kDa proteins as G9R, A16L, and E7R, respectively. Using synthetic oligonucleotides and PCR techniques, each of these open reading frames was amplified by using VV DNA as a template and then cloned individually into expression vectors behind T7 promoters. These plasmid constructs were then transcribed in vitro, and the resulting mRNAs were translated in wheat germ extracts and radiolabeled with either [35S]methionine or [3H]myristic acid. Each wild-type polypeptide was labeled with [35S]methionine or [3H]myristic acid in the translation reactions, while mutants containing an alanine in place of glycine at the N terminus were labeled only with [35S]methionine, not with myristic acid. This result provided strong evidence that the open reading frames had been correctly identified and that each protein is myristylated on a glycine residue adjacent to the initiating methionine. Subcellular fractionations of VV-infected cells suggested that A16L and E7R are soluble, in contrast to L1R, which is a membrane-associated protein.

Animals↗
PubMed1996

Expression of functional Porphyromonas gingivalis fimbrillin polypeptide domains on the surface of Streptococcus gordonii.

Genetically engineering bacteria to express surface proteins which can antagonize the colonization of other microorganisms is a promising strategy for altering bacterial environments. The fimbriae of Porphyromonas gingivalis play an important role in the pathogenesis of periodontal diseases. A structural subunit of the P. gingivalis fimbriae, fimbrillin, has been shown to be an important virulence factor, which likely promotes adherence of the bacterium to saliva-coated oral surfaces and induces host responses. Immunization of gnotobiotic rats with synthetic peptides based on the predicted amino acid sequence of fimbrillin has also been shown to elicit a specific immune response and protection against P. gingivalis-associated periodontal destruction. In this study we engineered the human oral commensal organism Streptococcus gordonii to surface express subdomains of the fimbrillin polypeptide fused to the anchor region of streptococcal M6 protein. The resulting recombinant S. gordonii strains expressing P. gingivalis fimbrillin bound saliva-coated hydroxyapatite in a concentration-dependent manner and inhibited binding of P. gingivalis to saliva-coated hydroxyapatite. Moreover, the recombinant S. gordonii strains were capable of eliciting a P. gingivalis fimbrillin-specific immune response in rabbits. These results show that functional and immunologically reactive P. gingivalis fimbrillin polypeptides can be expressed on the surface of S. gordonii. The recombinant fimbrillin-expressing S. gordonii strains may provide an effective vaccine or a vehicle for replacement therapy against P. gingivalis. These experiments demonstrated the feasibility of expressing biologically active agents (antigens or adhesin molecules) by genetically engineered streptococci. Such genetically engineered organisms can be utilized to modulate the microenvironment of the oral cavity.

Animals↗
PubMed1996

The vaccinia virus 4c and A-type inclusion proteins are specific markers for the intracellular mature virus particle.

Gel analysis of vaccinia virus particles purified by buoyant [correction of bouyant] density demonstrates a protein with an estimated molecular mass of 59 kDa, which is apparently restricted to the intracellular mature virion (IMV) form. Western blotting (immunoblotting) and immunoprecipitation procedures identify the protein as the vaccinia virus 4c protein, which facilitates occlusion of poxvirus particles within cowpox cytoplasmic inclusions. Western blotting procedures also identify the truncated A-type inclusion protein of vaccinia virus as a specific marker for IMV particles. Kinetic analyses of virion maturation and 4c production suggest that peak enveloped virion production occurs before peak IMV production in the virus replication cycle and that 4c production is concomitant with maturation of IMV. The implications for a distinct and evolutionarily conserved function of IMV in viral pathogenesis are discussed.

Animals↗
PubMed1995

Analysis of the role of the amino-terminal peptide of vaccinia virus structural protein precursors during proteolytic processing.

Several VV structural proteins are produced by the removal of amino-terminal peptides from their cognate precursors. In the experiments reported here, directed genetic approaches were used to investigate the possible role of these terminal peptides in protein processing. As a model system, the FLAG epitope-tagged P25K precursor was used to prepare constructs in which the 31-amino-acid P25K N-terminal peptide was removed or replaced by heterologous sequences, while the -A-G*-A- cleavage motif was retained. Only a trace amount of the leaderless P25KF(delta 31) polypeptide was found within the mature virions, implying that proteolytic processing is necessary for the incorporation of the 25K product into mature virions. In trans-processing assays, significant levels of the 25K product were generated from wild-type P25KF and P4b:25KF, which consists of the 61-amino-acid P4b terminal peptide, and from P4b:25KF with 15, 30, or 44 residues of the amino terminus deleted. In contrast, only a small amount of 25K was produced from the TK:25KF, which contains the amino-terminal 30 residues of VV thymidine kinase, a protein which is not cleaved under normal circumstances. Furthermore, it has been hypothesized that a hydrophobic residue is required at position P4 relative to the -A-G*-A- motif for the cleavage to take place. An intermediate level of the 25K product was detected from the TK:25KF(Q29V) mutant which has the glutamine residue at P4 replaced with a valine residue, suggesting that the hydrophobic P4 residue and additional substrate determinants in the N-terminal peptide region are required for the proteolytic processing reaction to occur normally. Taken together, these data suggest that the amino-terminal peptides of the VV core proteins are to some extent interchangeable and that the residues proximal to the AGA site are of most importance.

Amino Acid Sequence↗
PubMed1995

Brefeldin A inhibits vaccinia virus envelopment but does not prevent normal processing and localization of the putative envelopment receptor P37.

The fungal metabolite Brefeldin A was found to inhibit the production of the infectious enveloped form of vaccinia virus, although production of the infectious intracellular form was not affected. Electron microscopic analysis and caesium chloride density centrifugation of progeny virions indicates that the drug block is not due to retention and accumulation of enveloped virions within the cell. Biochemical analysis of the candidate envelopment receptor for vaccinia virus, viral protein P37, shows that the drug has no discernible effect on palmitylation of this protein and does not prevent or alter its association with intracellular membranes. This suggests that P37 may not in fact be the receptor on intracellular membranes for vaccinia virus envelopment, and leaves open the question of what function this molecule performs in the envelopment process. 24AI 20563

Antifungal Agents↗
PubMed1995

Physical and molecular genetic analysis of the multistep proteolytic maturation pathway utilized by vaccinia virus P4a protein.

Three potential AG*X cleavage sites have previously been identified in vaccinia virus (VV) P4a precursor, namely AG*N, AG*S and AG*T. Utilization of the COOH-proximal AG*T site in P4a leads to release of a 23 kDa product ('23K'). Here we propose that cleavage at the AG*S site alone is responsible for release of 4a, based on peptide mapping and microsequencing which demonstrated that the NH2 terminus of 4a is co-terminal with P4a, thus indicating that the AG*N site is excluded from proteolytic processing. Proteolysis of P4a at AG*S and AG*T to yield 4a and 23K should theoretically also liberate an intervening 9 kDa peptide ('9K'), although efforts to isolate this peptide have been repeatedly unsuccessful. To investigate the fate of this intervening peptide, mutation of the P4a coding sequence at the AG*S or AG*T site, followed by transient expression in VV-infected cells, lead to the synthesis of unique 4a-9K or 9K-23K chimeric protein products. This implies that neither end of the intervening 9K peptide is intrinsically destabilizing, and that its supposed degradation may be suppressed when it remains associated with 4a or 23K.

Amino Acid Sequence↗
PubMed1994

Proteolytic cleavage of vaccinia virus virion proteins. Mutational analysis of the specificity determinants.

Previous studies have suggested that cleavage of vaccinia virus core protein precursors occurs within the consensus tripeptide motif -A-G decreases X-. As an approach to delineate the sequence and structural features of the precursor polypeptides that are responsible for directing site-specific scission within this element, site-directed mutagenesis procedures were employed in concert with an in vivo trans-processing assay of the P25K: FLAG reporter plasmid. The results obtained suggest that residue occupancy at the P1' site (following the nomenclature of Schechter and Berger (Schechter, I., and Berger, A. (1976) Biochem. Biophys. Res. Commun. 27, 157-162), the positions at the amino- and carboxyl-proximal residues are indicated as P1, P2, etc., and P1', P2', etc., respectively) was extremely permissive, with only a proline substitution blocking cleavage. In contrast, the permissible occupancy of the P1 (serine or alanine) and P2 (cysteine, serine, or asparagine) sites was extremely restricted. Analysis of P1/P2 double mutants supported this conclusion and suggested additional levels of combinatorial stringency. Insertion or deletion of sequences immediately adjacent (amino- or carboxyl-terminal) to the -A-G-X- motif completely abrogated cleavage, suggesting the presence of additional important structural determinants. Mutation of the conserved proline or basic amino acid residues in these regions had no effect on cleavage, whereas it appeared that the presence of a hydrophobic residue in the P4 site was required.

Amino Acid Sequence↗
PubMed1994

Immunolocalization of vaccinia virus structural proteins during virion formation.

Proteolytic processing of vaccinia virus core proteins is an essential step in the formation of mature virions and occurs during the process of virion morphogenesis. In order to investigate how the vaccinia virus (VV) structural proteins become integrated into virus particles during normal maturation, immunological reagents were generated against the three major VV core proteins 4a, 4b, and 25K and their precursor molecules P4a, P4b, and P25K. These sera were used in conjunction with immunofluorescent and immunogold labeling of VV-infected tissue culture cells. The immunofluorescent results indicated that all three core precursors and their cleavage products were localized to virosomes. As the infection progressed, punctate staining with these sera became spread throughout the cytoplasm which suggested that individual virion particles were being recognized. Immunoelectron microscopy showed that the core proteins were localized to the center of both immature and mature virus particles. This result was in contrast to the situation observed using antisera directed against L65, a protein previously implicated in the assembly of the viral membrane. Immunogold staining of L65 showed that it was initially located along the inner side of the immature virion membrane and remained with the membrane even as the viroplasm began to condense toward the center of the virus particle. In order to determine whether the core protein localization observed was the result of precursors, products, or both, a synthetic peptide strategy was used to generate an antiserum that recognized only P4a in immunoprecipitation reactions. Immunogold labeling with this reagent indicated that P4a was found in the viroplasm of immature particles and in low levels in the mature virion. Intracellular localization of core and L65 proteins during virion morphogenesis is discussed.

Amino Acid Sequence↗
PubMed1994

Differential utilization of a conserved motif for the proteolytic maturation of vaccinia virus proteins.

Several of the vaccinia virus core proteins are synthesized as large precursor proteins which are subsequently processed to smaller products during the course of viral maturation. Amino acid alignment of these proteins reveals a conserved Ala-Gly-X motif (AG*X) at their confirmed cleavage sites. To better understand the regulation of cleavage site selection, the sequence of the entire vaccinia virus genome was searched for the occurrence of this AG*X motif in predicted open reading frames. Of the 82 sites found, 19 resembled cleavage sites which have previously been shown to be actively processed, namely AG*A of P25K and P4b, and AG*S and AG*T of P4a. To test the universality of the AG*X motif utilization, immunological methods in concert with N-terminal microsequencing procedures have been used to determine which of the subset of predicted proteins containing AG*A sites are utilized in vivo. Of the seven AG*A-containing substrates, four were cleaved and three were not. Considering all the known AG*X processing events, it appears that only those proteins expressed at late times during infection and associated with the assembling virion are candidate substrates for proteolytic cleavage. Such proteins include P4a, P4b, P25K, and the newly identified P21K and P17K (derived from genes A17L and A12L, respectively). Although proteins such as DNA polymerase, P37K, and a host range protein contain a consensus cleavage site, they are excluded from processing. This proteolytic exclusion presumably occurs because these proteins do not meet both of the above criteria, which suggests that temporal expression or compartmentalization (substrate presentation) in the assembling virion may play a regulatory role in proteolysis.

Amino Acid Sequence↗
PubMed1994

Uses of vaccinia virus in vaccine delivery.

The construction of vaccinia-based vaccines has been hampered by a lack of information on both the mechanisms of vaccinia-induced immunity in humans and the effect of prior exposure to vaccinia on the course of an immune response to a non-vaccinia antigen. Recent studies have investigated the immune responses induced by this virus in humans and the ability of recombinant viruses to successfully induce immunity to diverse pathogens with diverse routes of infection. In addition to the previously described ability of vaccinia to induce immune responses in experimental animals, the virus has been shown to encode modulators of immune function that may, in the future, permit the use of virus to induce qualitatively different immune responses to particular heterologous antigens.

Animals↗
PubMed1994

Characterization of the vaccinia virus L1R myristylprotein as a component of the intracellular virion envelope.

In many cases, virus-encoded acylproteins appear to localize to specific cellular and viral membranes and to be directly involved with the processes of virus morphogenesis and/or egress from the infected cell. It was therefore of interest to determine whether the major vaccinia virus (VV) myristylprotein, L1R, is specifically associated with one or more of the membranes enveloping various infectious forms of VV virions. To this end, single-membraned intracellular virions (INV) and extracellular enveloped virions (EEV), which are surrounded by at least two distinct membranes, were purified from VV-infected cell lysates. The location of the VV L1R protein was determined by using a monospecific anti-L1R serum to detect the L1R protein by immunoblot in INV- and EEV-containing fractions, by examining the proteinase K sensitivity of the L1R protein in intact INV and EEV particles, and by immunoelectron microscopy. The data obtained clearly indicate that although the L1R protein is a constituent of both the INV and EEV particles, it is exclusively found in the inner INV-specific membrane. These results are discussed with regard to the potential role of the VV L1R protein in the primary intracellular envelopment of infectious VV particles.

Animals↗
PubMed1994

Conditional lethal expression of the vaccinia virus L1R myristylated protein reveals a role in virion assembly.

Within vaccinia virus-infected cells, the product of the L1R open reading frame is covalently modified by myristic acid at the penultimate NH2-terminal glycine residue. Previously we have shown that while the L1R protein is a constituent of both intracellular mature virus particles and extracellular enveloped virions which are released from the infected cell, it is associated exclusively with the primary membranes surrounding the virion core. Given this rather specific localization, it was of interest to study the potential role of this essential gene in virus replication and morphogenesis. To this end, we have constructed a recombinant vaccinia virus in which expression of the L1R gene can be transcriptionally repressed. Without the inducer isopropylthiogalactopyranoside (IPTG), synthesis of the L1R protein was blocked, resulting in a total inhibition of plaque formation. Velocity sedimentation of viral particles labeled in the presence of [3H]thymidine, grown in the absence of IPTG, revealed a substantial reduction in viral DNA incorporation into virions. Likewise, proteolysis of the major core proteins p4a, p4b, and p25K, believed to occur during the final stages of virion maturation, was severely impaired. In the absence of L1R expression, only immature virions could be detected by electron microscopy. Transient expression of a plasmid containing the full-length L1R gene driven by its own promoter was able to complement and rescue the defective phenotype. However, a plasmid bearing a mutation in the myristyl acceptor glycine residue was unable to biologically rescue the recombinant, and the protein was not detected in purified virions.trans complementation using a truncated, myristylated form of the L1R protein partially rescued the defective mutant. Collectively, these data suggest that myristic acid mediates essential interactions of the L1R protein with viral membranes and/or other virion components that lead to the productive assembly, maturation, and release of particles.

Animals↗
PubMed1994

A transcriptionally controlled trans-processing assay: putative identification of a vaccinia virus-encoded proteinase which cleaves precursor protein P25K.

Vaccinia virus maturation into infectious particles appears to be dependent on the proteolytic processing of at least five viral proteins, each containing a conserved AG*X cleavage motif and each requiring proper association with the previrion particle. To identify the responsible proteinase, a transcriptionally controlled trans-processing assay was developed to monitor cleavage at the permissive AG*S site of the P25K core protein precursor. This assay led to the putative identification of a VV proteinase encoded by open reading frame G1L. The predicted protein contains an HXXEH sequence which is a direct inversion of the active site consensus sequence present in thermolysin and other metalloendopeptidases. Site-directed mutation of this consensus sequence suggests that the G1L protein may be a novel, virus-encoded metalloendoproteinase, although confirmation of this activity must await the development of a suitable cell-free processing assay.

Amino Acid Sequence↗