Association of vaccinia virus fusion regulatory proteins with the multicomponent entry/fusion complex

The proteins encoded by the A56R and K2L genes of vaccinia virus form a heterodimer (A56/K2) and have a fusion regulatory role as deletion or mutation of either causes infected cells to form large syncytia spontaneously. Here, we showed that syncytia formation is dependent on proteins of the recently described entry fusion complex (EFC), which are also required for virus-cell fusion and low-pH-triggered cell-cell fusion. This finding led us to consider that A56/K2 might prevent fusion by direct or indirect interaction with the EFC. To test this hypothesis, we made a panel of recombinant vaccinia viruses that have a tandem affinity purification tag attached to A56, K2, or the A28 EFC protein. Interaction between A56/K2 and the EFC was demonstrated by their copurification from detergent-treated lysates of infected cells and identification by mass spectrometry or Western blotting. In addition, a purified soluble transmembrane-deleted form of A56/K2 was shown to interact with the EFC. Tagged A56 did not interact with the EFC in the absence of K2, nor did tagged K2 interact with the EFC in the absence of A56. The finding that both A56 and K2 are required for efficient binding to the EFC fits well with prior experiments showing that mutation of either A56 or K2 results in spontaneous fusion of infected cells. Because A56 and K2 are located on the surface of infected cells, they are in position to interact with the EFC of released progeny virions and prevent back-fusion and syncytia formation.     

Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously

A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research.     

Vaccinia mature virus fusion regulator A26 protein binds to A16 and G9 proteins of the viral entry fusion complex and dissociates from mature virions at low pH

Vaccinia mature virus enters cells through either endocytosis or plasma membrane fusion, depending on virus strain and cell type. Our previous results showed that vaccinia virus mature virions containing viral A26 protein enter HeLa cells preferentially through endocytosis, whereas mature virions lacking A26 protein enter through plasma membrane fusion, leading us to propose that A26 acts as an acid-sensitive fusion suppressor for mature virus (S. J. Chang, Y. X. Chang, R. Izmailyan R, Y. L. Tang, and W. Chang, J. Virol. 84:8422-8432, 2010). In the present study, we investigated the fusion suppression mechanism of A26 protein. We found that A26 protein was coimmunoprecipitated with multiple components of the viral entry-fusion complex (EFC) in infected HeLa cells. Transient expression of viral EFC components in HeLa cells revealed that vaccinia virus A26 protein interacted directly with A16 and G9 but not with G3, L5 and H2 proteins of the EFC components. Consistently, a glutathione S-transferase (GST)-A26 fusion protein, but not GST, pulled down A16 and G9 proteins individually in vitro. Together, our results supported the idea that A26 protein binds to A16 and G9 protein at neutral pH contributing to suppression of vaccinia virus-triggered membrane fusion from without. Since vaccinia virus extracellular envelope proteins A56/K2 were recently shown to bind to the A16/G9 subcomplex to suppress virus-induced fusion from within, our results also highlight an evolutionary convergence in which vaccinia viral fusion suppressor proteins regulate membrane fusion by targeting the A16 and G9 components of the viral EFC complex. Finally, we provide evidence that acid (pH 4.7) treatment induced A26 protein and A26-A27 protein complexes of 70 kDa and 90 kDa to dissociate from mature virions, suggesting that the structure of A26 protein is acid sensitive.     

Vaccinia virus A56/K2 fusion regulatory protein interacts with the A16 and G9 subunits of the entry fusion complex

Deletion of the A56R or K2L gene of vaccinia virus (VACV) results in the spontaneous fusion of infected cells to form large multinucleated syncytia. A56 and K2 polypeptides bind to one another (A56/K2) and together are required for interaction with the VACV entry fusion complex (EFC); this association has been proposed to prevent the fusion of infected cells. At least eight viral polypeptides comprise the EFC, but no information has been available regarding their interactions either with each other or with A56/K2. Utilizing a panel of recombinant VACVs designed to repress expression of individual EFC subunits, we demonstrated that A56/K2 interacted with two polypeptides: A16 and G9. Both A16 and G9 were required for the efficient binding of each to A56/K2, suggesting that the two polypeptides interact with each other within the EFC. Such an interaction was established by the copurification of A16 and G9 from infected cells under conditions in which a stable EFC complex failed to assemble and from detergent-treated lysates of uninfected cells that coexpressed A16 and G9. A recombinant VACV that expressed G9 modified with an N-terminal epitope tag induced the formation of syncytia, suggesting partial interference with the functional interaction of A56/K2 with the EFC during infection. These data suggest that A16 and G9 are physically associated within the EFC and that their interaction with A56/K2 suppresses spontaneous syncytium formation and possibly "fuse-back" superinfection of cells.     

Site-directed combinatorial construction of chimaeric genes: general method for optimizing assembly of gene fragments

Site-directed construction of chimaeric genes by in vitro recombination "mixes-and-matches" precise building blocks from multiple parent proteins, generating libraries of hybrids to be tested for structure-function relationships and/or screened for favorable properties and novel enzymatic activities. A direct annealing and ligation method can construct chimaeric genes without requiring sequence identity between parents, except for the short (approximately 3 nt) sequences of the fragment overhangs used for specific ligation. Careful planning of the assembly process is necessary, though, in order to ensure effective construction of desired fragment assemblies and to avoid undesired assemblies (e.g., repetition of fragments, fragments out of order). We develop algorithms for specific planned ligation of short overhangs (SPLISO) that efficiently explore possible assembly plans, varying the fragment overhangs and the order of ligation steps in the assembly pathway. While there is a combinatorial explosion in the number of possible assembly plans as the number of breakpoints and parent genes increases, we employ a dynamic programming approach to find globally optimal ones in low-order polynomial time (in practice, taking only seconds for basic assembly plans). We demonstrate the effectiveness of our algorithms in planning the assembly of hybrid libraries, under a variety of experimental options and restrictions, including flexibility in the position and amino acid sequence of breakpoints. Our method promises to enable more effective application of site-directed recombination to protein investigation and engineering.     

A modular assembly cloning technique (aided by the BIOF software tool) for seamless and error-free assembly of long DNA fragments

ABSTRACT: BACKGROUND: Molecular cloning of DNA fragments >5 kbp is still a complex task. When no genomic DNA library is available for the species of interest, and direct PCR amplification of the desired DNA fragment is unsuccessful or results in an incorrect sequence, molecular cloning of a PCR-amplified region of the target sequence and assembly of the cloned parts by restriction and ligation is an option. Assembled components of such DNA fragments can be connected together by ligating the compatible overhangs produced by different restriction endonucleases. However, designing the corresponding cloning scheme can be a complex task that requires a software tool to generate a list of potential connection sites. FINDINGS: The BIOF program presented here analyzes DNA fragments for all available restriction enzymes and provides a list of potential sites for ligation of DNA fragments with compatible overhangs. The cloning scheme, which is called modular assembly cloning (MAC), is aided by the BIOF program. MAC was tested on a practical dataset, namely, two non-coding fragments of the translation elongation factor 1 alpha gene from Chinese hamster ovary cells. The individual fragment lengths exceeded 5 kbp, and direct PCR amplification produced no amplicons. However, separation of the target fragments into smaller regions, with downstream assembly of the cloned modules, resulted in both target DNA fragments being obtained with few subsequent steps. CONCLUSIONS: Implementation of the MAC software tool and the experimental approach adopted here has great potential for simplifying the molecular cloning of long DNA fragments. This approach may be used to generate long artificial DNA fragments such as in vitro spliced cDNAs.     

Purification and immunogenicity of a recombinant Bordetella pertussis S1S3FHA fusion protein expressed by Streptococcus gordonii

Acellular pertussis vaccines typically consist of antigens isolated from Bordetella pertussis, and pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two prominent components. One of the disadvantages of a multiple-component vaccine is the cost associated with the production of the individual components. In this study, we constructed an in-frame fusion protein consisting of PT fragments (179 amino acids of PT subunit S1 and 180 amino acids of PT subunit S3) and a 456-amino-acid type I domain of FHA. The fusion protein was expressed by the commensal oral bacterium Streptococcus gordonii. The fusion protein was secreted into the culture medium as an expected 155-kDa protein, which was recognized by a polyclonal anti-PT antibody, a monoclonal anti-S1 antibody, and a monoclonal anti-FHA antibody. The fusion protein was purified from the culture supernatant by affinity and gel permeation chromatography. The immunogenicity of the purified fusion protein was assessed in BALB/c mice by performing parenteral and mucosal immunization experiments. When given parenterally, the fusion protein elicited a very strong antibody titer against the FHA type I domain, a moderate titer against native FHA, and a weak titer against PT. When given mucosally, it elicited a systemic response and a mucosal response to FHA and PT. In Western blots, the immune sera recognized the S1, S3, and S2 subunits of PT. These data collectively indicate that fragments of the pertussis vaccine components can be expressed in a single fusion protein by S. gordonii and that the fusion protein is immunogenic. This multivalent fusion protein approach may be used in designing a new generation of acellular pertussis vaccines.     

Expression of the A56 and K2 Proteins Is Sufficient To Inhibit Vaccinia Virus Entry and Cell Fusion

Many animal viruses induce cells to fuse and form syncytia. For vaccinia virus, this phenomenon is associated with mutations affecting the A56 and K2 proteins, which form a multimer (A56/K2) on the surface of infected cells. Recent evidence that A56/K2 interacts with the entry/fusion complex (EFC) and that the EFC is necessary for syncytium formation furnishes a strong connection between virus entry and cell fusion. Among the important remaining questions are whether A56/K2 can prevent virus entry as well as cell-cell fusion and whether these two viral proteins are sufficient as well as necessary for this. To answer these questions, we transiently and stably expressed A56 and K2 in uninfected cells. Uninfected cells expressing A56 and K2 exhibited resistance to fusing with A56 mutant virus-infected cells, whereas expression of A56 or K2 alone induced little or no resistance, which fits with the need for both proteins to bind the EFC. Furthermore, transient or stable expression of A56/K2 interfered with virus entry and replication as determined by inhibition of early expression of a luciferase reporter gene, virus production, and plaque formation. The specificity of this effect was demonstrated by restoring entry after enzymatically removing a chimeric glycophosphatidylinositol-anchored A56/K2 or by binding a monoclonal antibody to A56. Importantly, the antibody disrupted the interaction between A56/K2 and the EFC without disrupting the A56-K2 interaction itself. Thus, we have shown that A56/K2 is sufficient to prevent virus entry and fusion as well as formation of syncytia through interaction with the EFC.     

An alternative method for the synthesis of tailor-made genes

Oligonucleotide synthesis was coupled with amplification by the polymerase chain reaction to generate an exact translational fusion between a plant signal sequence and an animal structural gene. A synthetic 111-mer oligonucleotide representing less than two percent of the reaction products was successfully amplified by using short primers containing restriction sites designed for ease of cloning and providing in-frame fusion. The method overcomes the length-versus-yield dilemma in oligonucleotide synthesis, and is generally adaptable to the construction of a translationally competent coding sequence from any two DNA fragments.     

Novel green fluorescent protein (GFP) baculovirus expression vectors

Baculovirus expression vectors were constructed, which contained gfp as a reporter gene. Substitutions in the amino acid sequences were carried out to produce two mutant forms of GFP. One of these mutants produces blue color when excited by ultraviolet (UV) light. The other mutant produces a yellow color that can be visualized in regular daylight. The gene of interest cloned in-frame with the gfp open reading frame allows visualization of the produced GFP-fusion protein using UV light. The presence of an in-frame 6 x His tag between the gfp cDNA and the multiple cloning site allows purification of the fusion protein on a Ni-NTA (nickel-nitrilo-triacetic acid) agarose matrix.     

A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs

Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.     

Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis

Apoptotic cells in rat thymus were labeled in situ in paraffin-embedded and frozen tissue sections by ligation of double-stranded DNA fragments containing digoxigenin or Texas red. Two forms of double-stranded DNA fragments were prepared using the polymerase chain reaction: one was synthesized using Taq polymerase, which yields products with single- base 3' overhangs, and one using Pfu polymerase, which produces blunt- ended products. Both types of fragment could be ligated to apoptotic nuclei in thymus, indicating the presence in such nuclei of DNA double- strand breaks with single-base 3' overhangs as well as blunt ends. However, in nuclei with DNA damage resulting from a variety of nonapoptotic processes (necrosis, in vitro autolysis, peroxide damage, and heating) single-base 3' overhangs were either nondetectable or present at much lower concentrations than in apoptotic cells. Blunt DNA ends were present in such tissues, but at lower concentrations than in apoptotic cells. In contrast, in all of these forms of DNA damage, nuclei contained abundant 3'-hydroxyls accessible to labeling with terminal deoxynucleotidyl transferase. Thus, although single-base 3' overhangs and blunt ends are present in apoptotic nuclei, the specificity of the in situ ligation of 3'-overhang fragments to apoptotic nuclei indicates that apoptotic cells labeled in this way can readily be distinguished from cells with nonapoptotic DNA damage. These data are consistent with the involvement of an endonuclease similar to DNase I in apoptosis, which is predicted to leave short 3' overhangs as well as blunt ends in digestion of chromatin.     

Purification and Immunogenicity of a Recombinant Bordetella pertussis S1S3FHA Fusion Protein Expressed by Streptococcus gordonii

Acellular pertussis vaccines typically consist of antigens isolated from Bordetella pertussis, and pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two prominent components. One of the disadvantages of a multiple-component vaccine is the cost associated with the production of the individual components. In this study, we constructed an in-frame fusion protein consisting of PT fragments (179 amino acids of PT subunit S1 and 180 amino acids of PT subunit S3) and a 456-amino-acid type I domain of FHA. The fusion protein was expressed by the commensal oral bacterium Streptococcus gordonii. The fusion protein was secreted into the culture medium as an expected 155-kDa protein, which was recognized by a polyclonal anti-PT antibody, a monoclonal anti-S1 antibody, and a monoclonal anti-FHA antibody. The fusion protein was purified from the culture supernatant by affinity and gel permeation chromatography. The immunogenicity of the purified fusion protein was assessed in BALB/c mice by performing parenteral and mucosal immunization experiments. When given parenterally, the fusion protein elicited a very strong antibody titer against the FHA type I domain, a moderate titer against native FHA, and a weak titer against PT. When given mucosally, it elicited a systemic response and a mucosal response to FHA and PT. In Western blots, the immune sera recognized the S1, S3, and S2 subunits of PT. These data collectively indicate that fragments of the pertussis vaccine components can be expressed in a single fusion protein by S. gordonii and that the fusion protein is immunogenic. This multivalent fusion protein approach may be used in designing a new generation of acellular pertussis vaccines.     

Engineering isoflavone metabolism with an artificial bifunctional enzyme

Plant secondary metabolism has been a focus of research in recent years due to its significant roles in plant defense and in human medicine and nutrition. A protein engineering strategy was designed to more effectively manipulate plant secondary metabolite (isoflavonoid) biosynthesis. A bifunctional isoflavone synthase/chalcone isomerase (IFS/CHI) enzyme was constructed by in-frame gene fusion, and expressed in yeast and tobacco. The fusion protein was targeted to the endoplasmic reticulum (ER) membrane and the individual enzymatic functions of its component fragments were retained when assayed in yeast. Petals and young leaves of IFS/CHI transgenic tobacco plants produced higher levels of the isoflavone genistein and genistein glycosides as a ratio of total flavonoids produced than did plants transformed with IFS alone. Thus, through a combined molecular modeling, in vitro protein engineering and in planta metabolic engineering approach, it was possible to increase the potential for accumulation of isoflavonoid compounds in non-legume plants. Construction of bifunctional enzymes will simplify the transformation of plants with multiple pathway genes, and such enzymes may find broad uses for enzyme (e.g., cytochrome P450 family) and biochemical pathway engineering.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5′ end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3′ overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications.     

A modified plasmid vector pCMV–3Tag–LIC for rapid, reliable, ligation-independent cloning of polymerase chain reaction products

Here we present a modified vector pCMV–3Taq–LIC for a rapid, simple, and relatively cheap method to build expression constructs. After being digested by Nt.BspQI and EcoRV, a lineal vector with specific 11-base single overhangs is obtained. Polymerase chain reaction (PCR) products with complementary overhangs are created by building appropriate extensions into the primers and treating them with T4 DNA polymerase. The annealing of the insert and the vector is performed in the absence of ligase by simple mixing of the DNA fragments. This process is very specific because only the desired products can form. Using this vector, we successfully constructed hnRNP K full-length complementary DNA (cDNA) expression plasmid.