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PubMed1990

Engineering resistance to mixed virus infection in a commercial potato cultivar: resistance to potato virus X and potato virus Y in transgenic Russet Burbank.

Potato virus X (PVX) and potato virus Y (PVY) infection in potato may result in the loss of certification of seed potatoes and affect quality and yield of potatoes in commercial production. We transformed a major commercial cultivar of potato, Russet Burbank, with the coat protein genes of PVX and PVY. Transgenic plants that expressed both CP genes were resistant to infection by PVX and PVY by mechanical inoculation. One line was also resistant when PVY was inoculated with viruliferous green peach aphids. These experiments demonstrate that CP protection is effective against mixed infection by two different viruses and against mechanical and aphid transmission of PVY.

Amino Acid Sequence↗
PubMed2005

Selection of muscle-binding peptides from context-specific peptide-presenting phage libraries for adenoviral vector targeting.

Production of cell-targeting vectors in part involves the addition of new targeting ligands to the vector to mediate binding to the cells of interest. For viral vectors, the ideal approach is to genetically engineer new ligands into the capsid proteins of the virus to generate a single agent to mediate therapy. Although this is ideal, this insertion of an exogenous ligand from one structural context into the differing structural context of a capsid protein can ablate the function of the ligand or disrupt viral assembly and function. To address this context problem for adenoviral vectors, we have engineered a "context-specific" peptide-presenting phage library. We have displayed a 12-amino-acid (12-mer) random peptide library between the H and I sheets of the fiber protein of adenovirus type 5 on the pIII protein of fd bacteriophage. This library was used for peptide selection against C2C12 mouse skeletal muscle cells. Five rounds of selection combined with four rounds of clearing on nontarget cells selected one primary peptide designated 12.51, which bound target C2C12 cells approximately 100-fold better than the positive control RGD peptide. Translation of 12.51 back into the fiber protein produced a ligand-modified adenoviral vector that mediated 14-fold-better transduction of target C2C12 cells. These data suggest context-specific peptide-presenting libraries may allow selection of compatible peptide ligands for functional translation into viral vectors for retargeting.

Adenoviridae↗
PubMed1993

Genetically engineered resistance against grapevine chrome mosaic nepovirus.

Nepoviruses are a group of isometric plant viruses with a genome divided between two-single-stranded, positive-sense, RNA molecules. They are usually transmitted by nematodes and a number of them have significant economic impact, especially in perennial crops such as grapevine and fruit trees. Like all other picorna-like viruses, nepoviruses express their coat protein (CP) as part of a larger polyprotein which is further processed by a virus-encoded protease, a feature which poses specific problems when trying to express the viral coat protein in transgenic plants. A hybrid gene, driving the high-level expression of the CP of grapevine chrome mosaic nepovirus (GCMV) has been constructed and transferred to the genome of tobacco plants. Progeny of CP-expressing transformants show resistance against GCMV. When compared to control plants, fewer inoculated plants become infected and those that become infected accumulate reduced levels of viral RNAs. This protection was also shown to be efficient when plants are inoculated with purified viral RNA.

Capsid↗
PubMedDate not supplied

Genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene.

Three versions of the zucchini yellow mosaic virus (ZYMV) coat protein gene were engineered for expression in plants: the full-length coat protein sequence, the conserved core portion of the gene, and an antisense version. These constructs were introduced into muskmelon (Cucumis melo) and tobacco plants (Nicotiana tabacum) via Agrobacterium tumefaciens-mediated transformation; gene expression was verified by Northern and Western analysis. Transgenic R0 and R1 muskmelon plants expressing the full-length coat protein gene exhibited apparent immunity to ZYMV infection: There was a lack of symptom development during a 3-mo observation period and no measurable virus accumulation as determined by ELISA. Melon plants expressing the core or antisense constructs showed a several-day delay of systemic symptom development and reduction in virus titer. Furthermore, transgenic R1 tobacco plants expressing the full-length coat protein, core, or antisense constructs of ZYMV, a nonpathogen of tobacco, showed a short delay in symptom development and reduced virus titer when inoculated with the heterologous potyviruses, potato virus Y, and tobacco etch virus. The transgenic tobacco plants were not protected against the non-potyvirus, tobacco mosaic virus.

Base Sequence↗
PubMed1988

Resistance to TMV in transgenic plants results from interference with an early event in infection.

Constitutive expression of the tobacco mosaic virus (TMV) coat protein (CP) gene in transgenic tobacco plants results in inhibition of disease symptom development following inoculation with TMV. Evidence is presented here that this protection is also observed in leaf mesophyll protoplasts isolated from these plants. Protoplasts were resistant to infection by TMV at concentrations of 10 microgram/ml to 1 mg/ml when introduced by either electroporation or polyethylene glycol-mediated inoculation. There was little protection against infection by TMV RNA and the protection was lost as the concentration of TMV RNA in the inoculum increased. When virus was incubated briefly at pH 8.0 prior to inoculation, protection broke down in a manner similar to that observed following RNA inoculation. Analogous results were obtained in experiments with whole plants. Because virus treated in this manner has presumably lost little or no CP, these results suggest that expression of the TMV CP gene in transgenic plant cells prevents TMV from uncoating. A model is presented for the mechanism of this blockage which relates these results to early events in TMV infection.

Capsid↗
PubMed1999

Sequence-specific antibodies against human IgE isoforms induced by an epitope display system.

BACKGROUND: Unlike other immunoglobulin isotypes, the human C epsilon gene generates by alternative splicing two types of secretory and two types of membrane epsilon chains. The two secreted epsilon heavy chains, epsilon(S1) and epsilon(S2), differ only in the sequence of the last eight C-terminal amino acids, being epsilon(S2) six amino acids longer. The two types of membrane isoforms differ in the extracellular membrane proximal domain, with the longer variant, epsilon(mL), containing 52 extra amino acids which are absent in the shorter epsilon(mS) isoform. OBJECTIVES: We wished to produce quality antibody reagents that specifically detect epitopes that are epsilon isoform-specific. STUDY DESIGN: Short sequences of seven or ten amino acids were chosen as target epitopes and expressed as part of the highly immunogenic loops of deletion variants of engineered Flock House Virus capsid protein RNA2. Chimeric proteins were expressed in E. coli, and used to immunize rabbits. Antisera were screened by immunoblotting of purified IgE isoforms expressed by murine transfectomas. RESULTS: Chimeric proteins expressing epsilon isoform-specific epitopes proved to be strong immunogens in vivo and induced highly specific rabbit antisera. Two antisera so obtained recognize specifically the IgE-S2 isoform. A third one recognizes the long membrane variant m(L)IgE and a fourth one detects an epitope specific to m(S)IgE. CONCLUSION: Here we describe a simplified and efficient protocol of immunization which does not require peptide synthesis and conjugation to carrier protein. Our results show that short peptides of unknown immunogenicity, when genetically introduced into the modified Flock House Virus epitope display system, successfully induced IgE isoform-specific polyclonal antisera in rabbits. These are valuable tools to specifically identify secretory and membrane isoforms of human IgE, and the method is potentially applicable to other variant isoforms or mutants of a given protein.

Amino Acid Sequence↗
PubMed2005

Gutless adenovirus: last-generation adenovirus for gene therapy.

Last-generation adenovirus vectors, also called helper-dependent or gutless adenovirus, are very attractive for gene therapy because the associated in vivo immune response is highly reduced compared to first- and second-generation adenovirus vectors, while maintaining high transduction efficiency and tropism. Nowadays, gutless adenovirus is administered in different organs, such as the liver, muscle or the central nervous system achieving high-level and long-term transgene expression in rodents and primates. However, as devoid of all viral coding regions, gutless vectors require viral proteins supplied in trans by a helper virus. To remove contamination by a helper virus from the final preparation, different systems based on the excision of the helper-packaging signal have been generated. Among them, Cre-loxP system is mostly used, although contamination levels still are 0.1-1% too high to be used in clinical trials. Recently developed strategies to avoid/reduce helper contamination were reviewed.

Adenoviridae↗
PubMed2003

Virotherapeutics: conditionally replicative adenoviruses for viral oncolysis.

Viral oncolysis, or virotherapy, is an endeavor to use viruses as therapeutic agents in an effort to exploit their highly evolved qualities of host cell killing and simultaneous multiplication and spread. This review describes the concept of oncolytic adenoviruses, also called conditionally replicative adenoviruses (CRAds), and recent developments--inspired by early clinical results--that aim at the optimization of CRAd efficacy. Molecular strategies applied for the development of oncolytic adenoviruses include (i) the genetic manipulation of the expression and/or function of key regulatory viral proteins in order to restrict viral replication and spread to tumor cells, (ii) the engineering of the adenoviral capsid for efficient and tumor-targeted infection, and (iii) the incorporation of heterologous genes to facilitate combination therapies or tracking of the virus. Initial clinical trials have provided proof-of-concept for adenoviral oncolysis in patients and a favorable safety profile for oncolytic adenoviruses has been demonstrated. In conclusion, adenoviral oncolysis, with its distinct therapeutic mechanism, shows remarkable therapeutic potential. Advanced generations of virotherapeutics are currently in development.

Adenoviridae↗
PubMed2002

Engineering of adenovirus vectors containing heterologous peptide sequences in the C terminus of capsid protein IX.

The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.

Adenoviruses, Human↗
PubMed2006

Structurally mapping the diverse phenotype of adeno-associated virus serotype 4.

The adeno-associated viruses (AAVs) can package and deliver foreign DNA into cells for corrective gene delivery applications. The AAV serotypes have distinct cell binding, transduction, and antigenic characteristics that have been shown to be dictated by the capsid viral protein (VP) sequence. To understand the contribution of capsid structure to these properties, we have determined the crystal structure of AAV serotype 4 (AAV4), one of the most diverse serotypes with respect to capsid protein sequence and antigenic reactivity. Structural comparison of AAV4 to AAV2 shows conservation of the core beta strands (betaB to betaI) and helical (alphaA) secondary structure elements, which also exist in all other known parvovirus structures. However, surface loop variations (I to IX), some containing compensating structural insertions and deletions in adjacent regions, result in local topological differences on the capsid surface. These include AAV4 having a deeper twofold depression, wider and rounder protrusions surrounding the threefold axes, and a different topology at the top of the fivefold channel from that of AAV2. Also, the previously observed "valleys" between the threefold protrusions, containing AAV2's heparin binding residues, are narrower in AAV4. The observed differences in loop topologies at subunit interfaces are consistent with the inability of AAV2 and AAV4 VPs to combine for mosaic capsid formation in efforts to engineer novel tropisms. Significantly, all of the surface loop variations are associated with amino acids reported to affect receptor recognition, transduction, and anticapsid antibody reactivity for AAV2. This observation suggests that these capsid regions may also play similar roles in the other AAV serotypes.

Binding Sites↗
PubMed1988

Engineering a poliovirus type 2 antigenic site on a type 1 capsid results in a chimaeric virus which is neurovirulent for mice.

Poliovirus type 2 (PV-2) Lansing strain produces a fatal paralytic disease in mice after intracerebral injection, whereas poliovirus type 1 (PV-1) Mahoney strain causes disease only in primates. Atomic models derived from the three-dimensional crystal structure of the PV-1 Mahoney strain have been used to locate three antigenic sites on the surface of the virion. We report here the construction of type 1-type 2 chimaeric polioviruses in which antigenic site 1 from the PV-1 Mahoney strain was substituted by that of the PV-2 Lansing strain by nucleotide cassette exchange in a cloned PV-1 cDNA molecule. These chimaeras proved to have mosaic capsids with composite type 1 and type 2 antigenicity, and induced a neutralizing response against both PV-1 and PV-2 when injected into rabbits. Moreover, a six-amino-acid change in PV-1 antigenic site 1 was shown to be responsible for a remarkable host-range mutation in so far as one of the two type 1-type 2 chimaera was highly neurovirulent for mice.

Animals↗
PubMed1997

Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: implications for morphogenesis and organization of encapsidated RNA.

The capsid protein of hepatitis B virus, consisting of an "assembly" domain (residues 1-149) and an RNA-binding "protamine" domain (residues 150-183), assembles from dimers into icosahedral capsids of two different sizes. The C terminus of the assembly domain (residues 140-149) functions as a morphogenetic switch, longer C termini favoring a higher proportion of the larger capsids, it also connects the protamine domain to the capsid shell. We now have defined the location of this peptide in capsids assembled in vitro by engineering a mutant assembly domain with a single cysteine at its C terminus (residue 150), labeling it with a gold cluster and visualizing the cluster by cryo-electron microscopy. The labeled protein is unimpaired in its ability to form capsids. Our density map reveals a single undecagold cluster under each fivefold and quasi-sixfold vertex, connected to sites at either end of the undersides of the dimers. Considering the geometry of the vertices, the C termini must be more crowded at the fivefolds. Thus, a bulky C terminus would be expected to favor formation of the larger (T = 4) capsids, which have a greater proportion of quasi-sixfolds. Capsids assembled by expressing the full-length protein in Escherichia coli package bacterial RNAs in amounts equivalent to the viral pregenome. Our density map of these capsids reveals a distinct inner shell of density-the RNA. The RNA is connected to the protein shell via the C-terminal linkers and also makes contact around the dimer axes.

Capsid↗
PubMed1997

Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells.

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the "small-conductance" basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming bona fide IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.

Amino Acid Sequence↗
PubMed2026

Adenoviral Vectors in Gene Therapy: A Detailed Overview.

Adenoviral vectors (AdVs) represent one of the most extensively researched platforms in the realm of gene therapy, providing advantages such as high transduction efficiency, large transgene capacity, and broad tropism. This review provides a detailed and structured overview of AdVs, highlighting their biology, gene delivery mechanisms, clinical applications, and challenges limiting their broader therapeutic applicability. The study also explores recent progress in vector engineering, such as rare serotypes, capsid modifications, third-generation vectors, as well as strategies for immune modulation and toxicity reduction. AdVs are used in therapies for genetic disorders, oncology, and vaccinology, alongside innovations such as CRISPR-Cas9, nanotechnology, and artificial intelligence design. Nevertheless, persistent hurdles, including vector immunogenicity, hepatotoxicity, scalability, and the lack of durable expression, prevent widespread clinical use. This review consolidates current knowledge and presents a future perspective on how AdVs may evolve as powerful, adaptable, and precise tools in modern gene therapy. By contextualizing strengths and unresolved challenges, this work aims to give researchers and clinicians a balanced foundation for evaluating their future roles in translational medicine.

Humans↗
PubMed2006

Current developments in adenovirus-based cancer gene therapy.

Adenovirus (Ad)-based cancer gene therapy is a promising, novel approach for treating cancer resistant to established treatment modalities. Unfortunately, the efficacy of nonreplicative first generation Ads was low and data from clinical trials were disappointing. To address this problem, conditionally replicating Ads have been constructed. Infection of tumor cells with conditionally replicating Ads results in tumor-specific replication, subsequent oncolysis and release of the virus progeny. Recently, it has been suggested that the low expression of the coxsackie-Ad receptor is the rate-limiting factor for infectivity with serotype 5 (Ad5). Unfortunately, coxsackie-Ad receptor expression is highly variable and often low on many tumor types. Consequently, molecular strategies have been applied for the development of coxsackie-Ad receptor-independent oncolytic Ads. This review describes recent developments of Ad-based cancer gene therapy, including novel engineering techniques of the Ad capsid for efficient tumor targeting, as well as targeting techniques, to restrict transgene expression to cancer cells.

Adenoviridae↗
PubMed2006

Peptide display on Potato virus X: molecular features of the coat protein-fused peptide affecting cell-to-cell and phloem movement of chimeric virus particles.

The potexvirus Potato virus X (PVX) can be modified genetically to generate chimeric virus particles (CVPs) carrying heterologous peptides fused to coat protein (CP) subunits. A spontaneous PVX mutant expressing a truncated, but functional, form of the CP has been isolated. With the aim of exploiting this virus to display peptides useful for vaccine formulations, two novel viral expression vectors based on pPVX201 (bearing the wild-type PVX genome) were constructed encoding the truncated CP. Both vectors were able to produce infectious virus particles in planta and were used to insert a panel of sequences encoding peptides of biopharmaceutical interest as N-terminal fusions to the truncated cp gene. The analysis of infection progression induced by the different constructs enabled identification of two important structural features of the fused peptide, namely tryptophan content and isoelectric point, critically affecting the formation of PVX CVPs and virus movement through the plant. These results are discussed in view of the rising interest in engineered plant viruses for development of peptide-based epitope vaccines.

Capsid Proteins↗
PubMed2010

AAV's anatomy: roadmap for optimizing vectors for translational success.

Adeno-Associated Virus based vectors (rAAV) are advantageous for human gene therapy due to low inflammatory responses, lack of toxicity, natural persistence, and ability to transencapsidate the genome allowing large variations in vector biology and tropism. Over sixty clinical trials have been conducted using rAAV serotype 2 for gene delivery with a number demonstrating success in immunoprivileged sites, including the retina and the CNS. Furthermore, an increasing number of trials have been initiated utilizing other serotypes of AAV to exploit vector tropism, trafficking, and expression efficiency. While these trials have demonstrated success in safety with emerging success in clinical outcomes, one benefit has been identification of issues associated with vector administration in humans (e.g. the role of pre-existing antibody responses, loss of transgene expression in non-immunoprivileged sites, and low transgene expression levels). For these reasons, several strategies are being used to optimize rAAV vectors, ranging from addition of exogenous agents for immune evasion to optimization of the transgene cassette for enhanced therapeutic output. By far, the vast majority of approaches have focused on genetic manipulation of the viral capsid. These methods include rational mutagenesis, engineering of targeting peptides, generation of chimeric particles, library and directed evolution approaches, as well as immune evasion modifications. Overall, these modifications have created a new repertoire of AAV vectors with improved targeting, transgene expression, and immune evasion. Continued work in these areas should synergize strategies to improve capsids and transgene cassettes that will eventually lead to optimized vectors ideally suited for translational success.

Cystic Fibrosis↗
PubMed2005

In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: a strategy for efficient display of large full-length proteins.

An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc(-) phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.

Animals↗