Protein IIIa Research Articles

890. Capsid Protein IIIa as a Locale for Incorporation of Heterologous Peptides on Adenovirus Type 5

890. Capsid Protein IIIa as a Locale for Incorporation of Heterologous Peptides on Adenovirus Type 5

Visualization of α-Helices in a 6-Ångstrom Resolution Cryoelectron Microscopy Structure of Adenovirus Allows Refinement of Capsid Protein Assignments

The structure of adenovirus was determined to a resolution of 6 A by cryoelectron microscopy (cryoEM) single-particle image reconstruction. Docking of the hexon and penton base crystal structures into the cryoEM density established that alpha-helices of 10 or more residues are resolved as rods. A difference map was calculated by subtracting a pseudoatomic capsid from the cryoEM reconstruction. The resulting density was analyzed in terms of observed alpha-helices and secondary structure predictions for the additional capsid proteins that currently lack atomic resolution structures (proteins IIIa, VI, VIII, and IX). Protein IIIa, which is predicted to be highly alpha-helical, is assigned to a cluster of helices observed below the penton base on the inner capsid surface. Protein VI is present in approximately 1.5 copies per hexon trimer and is predicted to have two long alpha-helices, one of which appears to lie inside the hexon cavity. Protein VIII is cleaved by the adenovirus protease into two fragments of 7.6 and 12.1 kDa, and the larger fragment is predicted to have one long alpha-helix, in agreement with the observed density for protein VIII on the inner capsid surface. Protein IX is predicted to have one long alpha-helix, which also has a strongly indicated propensity for coiled-coil formation. A region of density near the facet edge is now resolved as a four-helix bundle and is assigned to four copies of the C-terminal alpha-helix from protein IX.

Cryoelectron Microscopy of Protein IX-Modified Adenoviruses Suggests a New Position for the C Terminus of Protein IX

Recombinant human adenovirus is a useful gene delivery vector for clinical gene therapy. Minor capsid protein IX of adenovirus has been of recent interest since multiple studies have shown that modifications can be made to its C terminus to alter viral tropism or add molecular tags and/or reporter proteins. We examined the structure of an engineered adenovirus displaying the enhanced green fluorescent protein (EGFP) fused to the C terminus of protein IX. Cryoelectron microscopy and reconstruction localized the C-terminal EGFP fusion between the H2 hexon and the H4 hexon, positioned between adjacent facets, directly above the density previously assigned as protein IIIa. The original assignment of IIIa was based largely on indirect evidence, and the data presented herein support the reassignment of the IIIa density as protein IX.

The adenovirus capsid: major progress in minor proteins

Human adenoviruses have been the subject of intensive investigation since their discovery in the early 1950s: they have served as model pathogens, as probes for studying cellular processes and, more recently, as efficient gene-delivery vehicles for experimental gene therapy. As a result, a detailed insight into many aspects of adenovirus biology is now available. The capsid proteins and in particular the hexon, penton-base and fibre proteins (the so-called major capsid proteins) have been studied extensively and their structure and function in the virus capsid are now well-defined. On the other hand, the minor proteins in the viral capsid, i.e. proteins IIIa, VI, VIII and IX, have received much less attention. Only the last few years have witnessed a sharp increase in the number of studies on their structure and function. Here, a review of the minor capsid proteins is provided, with a focus on new insights into their position and role in the capsid and the opportunities that they provide for improving human adenovirus-derived gene-delivery vectors.

Structural Stability of Adenovirus Type 5

Thermally induced structural changes in adenovirus type 5 (Ad) in the presence of either 2 or 10% sucrose were investigated using a variety of biophysical techniques. In solutions containing 2% sucrose, a highly cooperative transition in the structure of the virus was observed at 45 degrees C as detected by tryptophan fluorescence, derivative UV absorption spectroscopy, circular dichroism (CD), and dynamic and static light scattering. This transition resulted in (at least partial) disassembly of the virus and a concomitant increase in the accessibility of the viral DNA to the fluorescent dye, TOTO-1. Capsid disassembly was verified by transmission electron microscopy, which showed ruptured icosahedral vertices near 45 degrees C followed by complete capsid disassembly at higher temperatures. SDS-PAGE of thermally treated Ad suggests that the penton base (protein III) and protein IIIa (located in the peripentonal region) are significantly more labile than other capsid proteins and may be the initial instigators in capsid disassembly. Additional discrete structural transitions were observed in viral proteins using the aforementioned spectroscopic techniques. Thermally induced rearrangements of the condensed DNA at higher temperatures were also detected by the appearance of "psi"-like features in the CD spectra as well as a dramatic decrease in accessibility of DNA to TOTO-1. These transitions corresponded to discrete endothermic events that are also detected by differential scanning calorimetry. By increasing the concentration of sucrose to 10%, secondary and tertiary structural features of adenoviral proteins were significantly stabilized, although loss of quaternary structure at 45 degrees C was still observed.

Unscheduled expression of capsid protein IIIa results in defects in adenovirus major late mRNA and protein expression

Adenovirus gene expression is to a large extent regulated at the level of alternative RNA splicing. For example, in the major late region 1 (L1) unit, a common 5′ splice site can be joined to two alternative 3′ splice sites, resulting in the formation of the so-called 52,55K (proximal 3′ splice site) or the IIIa (distal 3′ splice site) mRNAs. Whereas, the 52,55K mRNA is expressed both early and late during infection, the IIIa mRNA is strictly confined to the late phase of the infectious cycle. We have previously shown that IIIa mRNA splicing is subjected to a tight viral control of IIIa 3 splice site usage. In an attempt to determine why adenovirus uses elaborate mechanisms to confine IIIa mRNA production to the late phase of infection, we characterized the phenotype of a recombinant adenovirus expressing the IIIa protein from an inducible tetracycline regulated gene cassette. The results show that expression of the IIIa protein during the early phase of infection results in a significant reduction in late viral protein synthesis and a moderate block to viral DNA replication. Interestingly, unscheduled IIIa protein expression resulted in a perturbation of the accumulation of alternatively spliced L1 mRNAs. Thus, 52,55K mRNA accumulation was inhibited while no effects on endogenous IIIa mRNA expression was detected.

Polypeptide composition of an adenovirus type 5 used in cancer gene therapy

For cancer gene therapy, a recombinant adenovirus serotype 5 named RPR/INGN201 has been constructed by susbtitution of the E1 region with human tumor suppressor gene p53. The protein components of RPR/INGN201 virions were separated by reversed-phase HPLC and were individually identified by electrospray time-of-flight mass spectrometry and N-terminal sequencing, both on intact proteins and on their proteolytic fragments after trypsin digestion. Twenty-five peptide components of the proteome (including fiber) with greater than 0.25–0.5% contribution to the protein content of the virus were identified and characterized. Fiber was confirmed to be partially glycosylated (both the non-glycosylated and the monoglycosylated states were identified), and two proteins were isolated and identified as phosphorylation derivatives, namely protein V (non-phosphorylated and monophosphorylated) and protein IIIa (mono- and diphosphorylated). This new analytical tool proved to be very useful not only for refining our current knowledge of the polypeptide repertoire of purified infectious virions but also for monitoring and very rapidly identifying structural modifications resulting from changes in the manufacturing process. It was also used successfully for the characterization of various adenoviral constructs.

Protein ligands of the human adenovirus type 2 outer capsid identified by biopanning of a phage-displayed peptide library on separate domains of wild-type and mutant penton capsomers.

A filamentous phage-displayed random hexapeptide library was screened on the adenovirus type 2 (Ad2) penton capsomer and its separate domains, penton base, full-length fiber, fiber shaft and fiber knob. Affinity supports were designed to immobilize the penton ligate with a preferred orientation, via immuno-adsorption to pre-coated antibody. Three classes of phagotopes were distinguished in the eluates from the penton and fiber domains. (i) The first class represented peptide sequences identified in certain Ad2 capsid proteins, protein IIIa, protein pVIII, penton base and penton fiber. Data from specific ligand elution of phages bound to fiber and penton base wild-types and mutants suggested that the region overlapping the RLSNLLG motif at residues 254-260 in the penton base and the FNPVYP motif at residues 11-16 in the fiber tail formed mutual interacting sites in the penton capsomer. (ii) The second class consisted of phagotopes homologous to peptide sequences found in host cell membrane proteins involved in receptor or adhesion functions. One of the most abundant species corresponded to a conserved motif present in the beta-strand B of type III modules of human fibronectin. In addition, phages which were screened for their failure to bind to penton base RGD mutants were found to carry consensus motifs to peptide sequences present in the RGD recognition site of human integrin beta subunits. (iii) The third class comprised peptide motifs common to both viral and cellular proteins, suggesting that a mechanism of ligand exchange could occur during virus entry and uncoating, and virus assembly and release.

Protein IIIa of Rhizobium leguminosarum is probably a porin

The cloning, sequencing and expression of the gene encoding the 36-kilodalton (kDa) outer membrane protein of Rhizobium leguminosarum has been recently described in the literature (De Maagd Ra et al, (1992) J Bacteriol 174, 214–221). We present evidence that this protein is a porin from a sub-type covalently bound to the peptidoglycan.

Antibody-mediated uncoating of adenovirus in vitro.

In a virus destabilization assay in vitro it was demonstrated that exposure of adenovirus to proteins will non-specifically protect the virus from being uncoated following transfer to low pH and hypotonic conditions. Such uncoating was also fully inhibited upon pretreatment of virus with 0.05% of the non-ionic detergent polyoxyethylenesorbitan monolaurate (Tween 20). However, in the presence of low concentrations of Tween 20 it was shown that monospecific immunoglobulins, directed against the fiber antigen and polyspecific antibodies produced in response to intact virions, were able to overcome the detergent-protecting effect of uncoating. Immunoglobulins directed towards the remaining outer-capsid components, the hexon, the penton base and the protein IIIa, revealed no such effects. The antifiber-mediated uncoating was paralleled by an aggregation of the virions. The data suggest that the virion-stabilizing effect of salt is enhanced by the hydrophobic action of a non-ionic detergent. Under these conditions the interaction between antifiber antibodies and fibers of the virion will trigger a destabilization of the virion upon transfer to low pH and hypotonic conditions.

Antibody-mediated uncoating of adenovirus in vitro

In a virus destabilization assay in vitro it was demonstrated that exposure of adenovirus to proteins will non-specifically protect the virus from being uncoated following transfer to low pH and hypotonic conditions. Such uncoating was also fully inhibited upon pretreatment of virus with 0.05% of the non-ionic detergent polyoxyethylenesorbitan monolaurate (Tween 20). However, in the presence of low concentrations of Tween 20 it was shown that monospecific immunoglobulins, directed against the fiber antigen and polyspecific antibodies produced in response to intact virions, were able to overcome the detergent-protecting effect of uncoating. Immunoglobulins directed towards the remaining outer-capsid components, the hexon, the penton base and the protein IIIa, revealed no such effects. The antifiber-mediated uncoating was paralleled by an aggregation of the virions. The data suggest that the virion-stabilizing effect of salt is enhanced by the hydrophobic action of a non-ionic detergent. Under these conditions the interaction between antifiber antibodies and fibers of the virion will trigger a destabilization of the virion upon transfer to low pH and hypotonic conditions.

Cloning, nucleotide sequencing, and expression in Escherichia coli of a Rhizobium leguminosarum gene encoding a symbiotically repressed outer membrane protein.

We describe the cloning of a gene from Rhizobium leguminosarum biovar viciae strain 248 encoding protein IIIa, the 36-kDa outer membrane protein forming a part of the outer membrane protein antigen group III. The expression of this antigen group is repressed in the bacteroid form during symbiosis (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J. J. Lugtenberg, J. Bacteriol. 171:1136-1142, 1989). A cosmid clone expressing the strain 248-specific MAb38 epitope of this antigen group in a nonrelated strain was selected by a colony blot assay. Sequencing revealed one large open reading frame encoding a 39-kDa protein. N-terminal amino acid sequencing of the purified 36-kDa outer membrane protein IIIa revealed that the isolated gene, now designated ropA, is the structural gene for this protein and that the mature protein was formed by processing of the 22-residue N-terminal signal sequence. The gene is preceded by a promoter that was active in R. leguminosarum but not in Escherichia coli. This promoter, which showed no homology to known promoter sequences, was located approximately by determination of the transcription start site. The region upstream of the putative promoter was shown to contain two potential binding sites for integration host factor protein. Expression of protein IIIa under control of the inducible lac promoter in E. coli shows that, of its earlier described properties, the peptidoglycan linkage of protein IIIa is specific for R. leguminosarum but that outer membrane localization and calcium-stabilized oligomer formation can to a large extent also occur in E. coli.

A Variant Form of Thrombasthenia

We encountered two siblings with abnormal bruising since infancy. Studies revealed functional platelet defects characterized by a lack of platelet aggregation and adenosine triphosphate release on exposure to adenosine diphosphate and collagen as well as variable responses with ristocetin (at concentrations of less than or equal to 1.25 g/L) and arachidonic acid. In addition, little or no platelet aggregation was observed after exposure to hexadimethrine bromide (Polybrene), the calcium ionophore A23187, and the thromboxane/endoperoxide analogue U46619. The membrane proteins IIIa and Ib were present, as determined with monoclonal antibody testing, and no platelet-associated IgG was found. Platelet analysis with routine electron microscopy and ultrastructural cytochemistry revealed normal morphologic features and no deficiencies in the number of alpha granules dense bodies and other organelles. The platelet abnormality may represent a new variant of thrombasthenia.

Protein phosphorylation in guinea-pig myenteric ganglia and brain: Presence of calmodulin kinase II, protein kinase C and cyclic AMP kinase and characterization of major phosphoproteins

The aim of this study was to demonstrate the presence of calmodulin-stimulated protein kinase II, protein kinase C, and cyclic AMP-stimulated protein kinase in isolated myenteric ganglia and to characterize the major ganglia phosphoproteins using biochemical and immunochemical techniques. Ganglia from the small intestine of guinea-pigs were isolated, disrupted by sonication in Triton X-100, and phosphorylated. The phosphoprotein patterns obtained were compared with those of synaptosomes from guinea-pig and rat cerebral cortex. Myenteric ganglia were as rich in protein kinase C and cyclic AMP-stimulated protein kinase as brain tissue, but the level of calmodulin-stimulated protein kinase II was relatively lower. The α subunit of calmodulin-stimulated protein kinase II was detected by immunoblotting and the β subunit by autophosphorylation. The ratio of β to α subunit was considerably higher in ganglia than in brain and ganglia β subunit had a lower apparent molecular weight than the brain enzyme. A number of neuronal phosphoproteins were found in ganglia including the 87,000 mol. wt phosphoprotein, synapsins 1a and 1b, and proteins IIIa and IIIb. A phosphoprotein of 48,000 mol. wt had many of the characteristics of the B-50 protein but was not the same. In addition, a number of other phosphoproteins not previously identified in neurons were found in ganglia including those with apparent molecular weights of 60,000 and 58,000 that were the major calmodulin kinase substrates. The guinea-pig enteric nervous system has been extensively studied but, unlike other parts of the mammalian nervous system, little is known about the intracellular mechanisms underlying its functions. A technique for isolating myenteric ganglia is now available and we have used this preparation to characterize the major protein kinases and phosphoproteins present in this tissue. The results obtained will allow the phosphorylation of the various proteins to be investigated after physiological or pharmacological manipulation of myenteric ganglia in situ and in vivo.

Purification and characterization of wild-type and ts112 mutant protein IIIa of human adenovirus 2 expressed in Escherichia coli

The expression of the protein IIIa gene from human adenovirus type 2 (Ad2) in Escherichia coli has been described previously ( M. Cuillel, M. Milleville, and J. C. D'Halluin, 1987, Gene 55, 295–301). The same construct has now been used to express a protein IIIa gene from an Ad2 mutant ts112 whose functional mutation occurs in this gene. The mutant virus is defective at nonpermissive temperatures in the latest stage of virus maturation. Both the wild-type and ts112 recombinant proteins are produced in E. coli in an insoluble form, but are readily solubilized in urea. They have the same molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), they sediment as a monomeric species in sucrose gradient centrifugation, and proteolytic digestion reveals a similar pattern for both proteins. Hydrodynamic studies and electron microscopy show that both proteins have an elongated shape, which can be approximated to a cylinder of 20 nm in length and 2.8 nm in diameter. The only well-established difference between the mutant and the wild-type recombinant protein is the higher solubility of the mutant.

Characterization of the adenovirus proteinase: substrate specificity.

Peptides were synthesized based on the cleavage sites in the adenovirus type 2 proteins pVI and pVII. The synthetic peptides were incubated with disrupted, purified adenovirus as a source of proteinase and specific cleavages were monitored by fast protein liquid chromatography and amino acid analysis. Using this approach it was established that all the peptides cleaved were of the form M(L)XGX decreases G or M(L)XGG decreases X. Thus we have shown that the adenoviral proteinase recognizes a specific secondary structure formed by a sequence of at least five amino acids, the main determinants of specificity being two and four residues to the N-terminal side of the bond cleaved. We were able to examine the relevant structural features of the peptide substrates by utilizing the CHEM-X molecular modelling package. Using our consensus sequence we were able to predict the cleavage sites in the viral proteins pVIII, pre-terminal protein (pTP), 11K and IIIa. Octapeptides containing the predicted sites in pVIII and the pTP were synthesized and shown to be cleaved by the proteinase.

Association of higher molecular weight ribonucleoproteins of 7 S particle preparations with multimers of transcription factor IIIA

High molecular weight (HMW) fractions of Xenopus laevis 7 S ribonucleoprotein (RNP) particle preparations were analyzed for RNA and protein content. RNA/protein ratios, amino acid analyses and Western blots reveal that the major HMW fraction from a non-denaturing polyacrylamide gel (band b) contains two molecules of transcription factor protein IIIA (TFIIIA) to one 5 S RNA. Another HMW band appears to contain 4 molecules of TFIIIA to one 5 S RNA. Yet another RNP band (band a) contains 5 S RNA and a protein unrelated to TFIIIA. Thus, native 7 S particle preparations contain 5 S RNA bound to multimeric forms of TFIIIA as well as to an unrelated protein. The presence of additional TFIIIA molecules associated with 7 S particles may have significance in the sequestering of TFIIIA during transcriptional regulation of the 5 S gene. Protein dimer; Nucleic acid-binding protein; TFIIIA; RNP, 7 S

Phosphoproteins localized to presynaptic terminal linked to persistence of long-term potentiation (LTP): quantitative analysis of two-dimensional gels

Previous findings suggest: (1) that altering protein kinase C (PKC) activity alters the persistence of long-term potentiation (LTP) in the intact hippocampal formation; and (2) that PKC activity is directly correlated with persistence of LTP in vivo as measured by the vitro phosphorylation of two major PKC substrates in adult hippocampus, protein F1 and 80k. Using quantitative analysis of two-dimensional gels, we report here two additional phosphoproteins of 72 and 55 kDa which were directly correlated to persistence of LTP induced in the intact dorsal hippocampal formation. The phosphorylation of both proteins in response to addition of different kinase stimulators was distinct from that of protein F1 and 80k. Moreover, neither protein was a substrate for exogenous PKC. The physicochemical properties of these phosphoproteins suggest they are identical to the previously described synaptic vesicle proteins IIIa and IIIb, and as such are immunologically indistinguishable. Because proteins IIIa and IIIb are known to be phosphorylated by a Ca 2+/calmodulin (CaM)-stimulated kinase, and protein F1 is known to be a plasma membrane-associated protein (P-57) which releases bound CaM in response to phosphorylation by PKC, the present findings suggest a potential mechanism in which PKC-mediated changes in plasma membrane protein produce CaM kinase-mediated changes in synaptic vesicle proteins through a phosphorylation cascade. These membrane/vesicle alteration are postulated to underlie the increased synaptic efficacy which marks persistent LTP.

Cholinergic regulation of protein III phosphorylation in bovine adrenal chromaffin cells

Protein IIIa (Mr approximately 74,000) and protein IIIb (Mr approximately 55,000), referred to collectively as protein III, are synaptic vesicle-associated phosphoproteins found in all regions of the rat nervous system and in the rat adrenal medulla. In the present study, the presence of protein III and the regulation of its phosphorylation were examined in chromaffin cells isolated from bovine adrenal medullae. Protein III was present in chromaffin cells isolated and purified from bovine adrenal medullae. The levels of protein III were moderately enriched in purified chromaffin cells compared with whole adrenal medullae. Preincubation of chromaffin cells with 32PO4 led to the endogenous phosphorylation of protein III, and phosphopeptide maps of chromaffin cell protein III were similar to those of protein III from bovine cerebral cortex. Treatment of the chromaffin cells with ACh produced calcium-dependent increases in both the phosphorylation of protein III and the release of 3H-norepinephrine. These effects of ACh were mimicked by nicotine but not by muscarine. Other secretagogues (elevated K+, veratridine, Ba2+) also increased both the phosphorylation of protein III and the release of 3H-norepinephrine. However, detailed characterization of the secretagogue-induced increases in protein III phosphorylation and 3H-norepinephrine secretion suggested that protein III phosphorylation was more directly associated with an increase in intracellular calcium than with secretion per se.

Use of Ar + plasma etching to localize structural proteins in viruses: Studies with adenovirus 2

The experiments described here were undertaken to test the idea that low energy Ar + plasma etching could be employed as the basis of a method to order viral structural polypeptides according to their physical proximity to the virus surface. Since low energy (500 eV) Ar + ions do not penetrate deeply into virus surfaces. one expects that the outermost proteins will be damaged before internal ones when intact virions are irradiated. To test this expectation, we exposed adenovirus 2 to a 500-eV Ar + plasma and then employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis to assess the extent of damage to the major structural polypeptides. Gel analyses showed that the proteins exposed on the virus surface (proteins II, III, and IV) were degraded rapidly during the first 10 s of irradiation while protein VII, the major core polypeptide, was almost completely protected. Proteins located between the capsid and the core, such as proteins IIIa and VI, were degraded at intermediate rates. Quantitative measurements demonstrated that the observed decay rate differences were not due simply to differences in protein target size; distance to the virion surface made an important contribution. The plasma etching technique, therefore, appears to have considerable potential for the structural analysis of viruses and other macromolecular assemblies where the proximity of individual proteins to the particle surface is unknown.