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PubMed1991

Specificity of the polioviral proteinase 3C towards genetically engineered cleavage sites in the viral capsid.

In a study of the cleavage specificity of poliovirus proteinase 3Cpro, two mutant polioviruses were constructed to include putative 3Cpro cleavage sites in the BC loop of VP1. The BC loop of VP1 in the wild-type virus is the neutralization antigenic site IA, consisting of a continuous chain of nine amino acids (ASTTNKDKL). The first mutant, W1-1D-BC1, has four altered amino acids in the BC loop (ASTQGPGKL); the second mutant, W1-1D-BC2, has an insertion of nine amino acids in the BC loop (ASTGTAKVQGPGNKDKL). W1-1D-BC1 and W1-1D-BC2 were viable, grew to high titre and produced plaques of normal size. W1-1D-BC1 virions were resistant to proteolytic cleavage of the BC loop in vivo as well as upon incubation with a large excess of 3Cpro in vitro, although a synthetic decapeptide (PASTQGPGKL) containing the amino acids of the BC loop in W1-1D-BC1 was cleaved by 3Cpro. In contrast, W1-1D-BC2 yielded virus the VP1 of which was cleaved partially in vivo and completely when incubated with 3Cpro in vitro. Our results showed that an insertion of nine amino acids into the antigenic loop of poliovirus, representing a synthetic 3Cpro cleavage site, renders the loop susceptible to cleavage by proteinase 3Cpro, but that this cleavage is restricted if the loop is the length of that in the native virion. This result implies that, in this case, structural restrictions override sequence determinants for cleavage of the BC loop by 3Cpro.

3C Viral Proteases↗
PubMed2005

Engineering thermal stability in RNA phage capsids via disulphide bonds.

The RNA bacteriophages, a group that includes phages Qbeta and MS2, have a number of potential bionanotechnological applications, including cell specific drug delivery and as substrates for the formation of novel materials. Despite extensive sequence identity between their coat protein subunits, and an almost identical three-dimensional fold, Qbeta and MS2 capsids have dramatically different thermal stabilities. The increased stability of Qbeta has been correlated with the inter-subunit disulphide bonds present in that capsid and not present in MS2. We have tested this hypothesis directly using mass spectrometry. Analysis of the dissociated coat protein subunits suggests that inter-molecular disulphides are formed at the capsid five-fold but may not be at the three-fold axes. This conclusion has been tested by engineering disulphide cross-links into either the five-fold or three-fold positions of the recombinant MS2 capsid. Five-fold cross-linking results in a mutant with stability properties similar to those of Qbeta. Three-fold cross-linking results in a mutant unable to assemble T = 3 shells, implying that five-fold structures are on pathway to capsid assembly in these phages. The results demonstrate how it is possible to redesign the physical properties of phage shells and may be of general relevance to future applications of viruses and virus-like particles.

Allolevivirus↗
PubMed1989

A genetically engineered cell line that produces empty capsids of B19 (human) parvovirus.

B19 parvovirus is pathogenic in humans, causing the common childhood exanthem fifth disease and bone-marrow failure, both acute (transient aplastic crisis of hemolysis) and chronic (pure erythrocyte aplasia in immunodeficiency). The virus is tropic for a human red cell progenitor cell, and failure to culture B19 in a cell line has limited its clinical study. We cotransfected the right half of the cloned B19 genome and a minigene derived from the human dihydrofolate reductase gene (DHFR) into dhfr--Chinese hamster ovary cells and screened selected clones by RNA analysis; after amplification in methotrexate, clones were tested for capsid protein expression. A cell line, designated 3-11-5, stably expressed nearly full-length transcripts for the two capsid proteins. These cells produced the major and minor structural protein species in natural proportions that self-assembled into virion capsids. Capsids from 3-11-5 cells could be separated from virions by sucrose gradient sedimentation and had the density on cesium chloride isopycnic sedimentation of empty parvovirus capsids. Capsid protein was present in both nuclei and cytoplasm on immunofluorescence study but fractionated with the cytosol on purification. Empty capsid production was equal to or greater than virion production by infected bone-marrow cells, 1000-2000 capsids per cell, but cell growth was not diminished by capsid production. This cell line will be useful in developing practical assays for B19 parvovirus antibody and a vaccine for the virus, as well as potentially serving as a packaging cell line for gene therapy.

Animals↗
PubMed1999

RNA-controlled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein.

Repeated, specific interactions between capsid protein (CP) subunits direct virus capsid assembly and exemplify regulated protein-protein interactions. The results presented here reveal a striking in vivo switch in CP assembly. Using cryoelectron microscopy, three-dimensional image reconstruction, and molecular modeling, we show that brome mosaic virus (BMV) CP can assemble in vivo two remarkably distinct capsids that selectively package BMV-derived RNAs in the absence of BMV RNA replication: a 180-subunit capsid indistinguishable from virions produced in natural infections and a previously unobserved BMV capsid type with 120 subunits arranged as 60 CP dimers. Each such dimer contains two CPs in distinct, nonequivalent environments, in contrast to the quasi-equivalent CP environments throughout the 180-subunit capsid. This 120-subunit capsid utilizes most of the CP interactions of the 180-subunit capsid plus nonequivalent CP-CP interactions. Thus, the CP of BMV, and perhaps other viruses, can encode CP-CP interactions that are not apparent from mature virions and may function in assembly or disassembly. Shared structural features suggest that the 120- and 180-subunit capsids share assembly steps and that a common pentamer of CP dimers may be an important assembly intermediate. The ability of a single CP to switch between distinct capsids by means of alternate interactions also implies reduced evolutionary barriers between different capsid structures. The in vivo switch between alternate BMV capsids is controlled by the RNA packaged: a natural BMV genomic RNA was packaged in 180-subunit capsids, whereas an engineered mRNA containing only the BMV CP gene was packaged in 120-subunit capsids. RNA features can thus direct the assembly of a ribonucleoprotein complex between alternate structural pathways.

Bromovirus↗
PubMed2024

Suppression of CNS APOE4 Expression by miRNAs Delivered by the S2 AAVrh.10 Capsid-Modified AAV Vector.

The homozygous Apolipoprotein E (APOE4) genotype is the major risk factor for the development of early Alzheimer's disease. Genome engineering studies in mouse models of human APOE4-dependent pathology have established that reduction of APOE4 expression can rescue the phenotype. We hypothesized that APOE4 could be suppressed in the CNS of APOE4 homozygotes using adeno-associated virus (AAV) expression of microRNAs (miRNA) designed to hybridize to APOE mRNA. We screened nine different miRNAs targeting APOE following transfection in HEK293T and Huh7 cells. Optimal APOE suppression was obtained with mir2A (targeting coding region nt330-351) and mirN4 (3' untranslated region nt1142-1162). miRNA expression cassettes were designed with two copies of each of these two miRNAs co-expressed with a mCherry transgene. To optimize delivery of these miRNAs, an engineered AAVrh.10 variant was identified from a screen of multiple peptide insertions into capsid loop IV and substitutions in loop VIII. This led to identifying the AAV.S2 capsid with enhanced transduction of both neurons and glia and enhanced distribution in the brain. The engineered capsid was used to deliver the APOE miRNA suppression cassette to the hippocampus of TRE4 mice (human APOE4 knock-in replacement of the murine apoE locus). Two weeks after intra-hippocampus administration, regional expression of miRNA at the injection site was quantified at the mRNA level relative to an endogenous reference. The AAV.S2 capsid provided 2.31 &#xb1; 0.37-fold higher expression of miRNA over that provided by AAVrh.10 (p < 0.05). In the targeted region, a single intra-hippocampus AAV.S2 administration suppressed hippocampal APOE4 mRNA levels by 76.5 &#xb1; 3.9% compared with 41.3 &#xb1; 3.3% with the same cassette delivered by the wildtype AAVrh.10 capsid (p < 0.0001). We conclude that an expression cassette with two different miRNAs targeting APOE4 delivered by the AAV.S2 capsid will generate highly significant suppression of APOE4 in the CNS.

Dependovirus↗
PubMed2002

Tropism-modified adenoviral and adeno-associated viral vectors for gene therapy.

One of the most rapidly advancing areas of gene therapy is vector development. For the majority of gene therapy procedures, efficient and selective transduction would provide safe and more effective treatments at optimal vector doses. Advances in vector targeting strategies have been rapid within the field of DNA-based viruses, particularly adenovirus (Ad) and more recently adeno-associated virus (AAV) based vectors. Vector targeting at the level of virus: cell interaction can be achieved using both non-genetic and genetic methodology. Non-genetic approaches typically utilise bispecific antibodies that both neutralise wild-type virus tropism and provide a new cell binding capacity. For genetic targeting strategies, the virus capsid can be engineered to express foreign ligands that target selected receptors in the absence or presence of additional modification to ablate the virus' natural tropism. This review covers technological advances that have led to targeting of Ad and AAV and highlights the potential for these 'designer' viruses for future gene-based therapeutics.

Adenoviridae↗
PubMed2002

The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy.

The structure of the adeno-associated virus (AAV-2) has been determined to 3-A resolution by x-ray crystallography. AAV is being developed as a vector for gene therapy to treat diseases including hemophilia, cancer, and cystic fibrosis. As in the distantly related autonomous parvoviruses, the capsid protein has a beta-barrel fold, but long loops between the beta-strands share little structural homology with other parvoviruses, leading to unique surface features. Most prominent are groups of threefold-related peaks, each an intimate association of loops from two neighboring subunits. Mutations affecting cell entry and receptor binding are clustered near the positively charged side of each peak, implicating the region in attachment to the cellular receptor, heparan sulfate proteoglycan. Amino acids involved in antibody binding are in the same general vicinity. The structure will guide rational engineering of vector capsids to tailor cellular targeting and to avoid immediate neutralization by an immune system sensitized by prior exposure to AAV.

Amino Acid Sequence↗
PubMed2024

Chemical approaches to probe and engineer AAV vectors.

Adeno-associated virus (AAV) has emerged as the most promising vector for in vivo human gene therapy, with several therapeutic approvals in the last few years and countless more under development. Underlying this remarkable success are several attractive features that AAV offers, including lack of pathogenicity, low immunogenicity, long-term gene expression without genomic integration, the ability to infect both dividing and non-dividing cells, etc. However, the commonly used wild-type AAV capsids in therapeutic development present significant challenges, including inadequate tissue specificity and the need for large doses to attain therapeutic effectiveness, raising safety concerns. Additionally, significant preexisting adaptive immunity against most natural capsids, and the development of such anti-capsid immunity after the first treatment, represent major challenges. Strategies to engineer the AAV capsid are critically needed to address these challenges and unlock the full promise of AAV gene therapy. Chemical modification of the AAV capsid has recently emerged as a powerful new approach to engineer its properties. Unlike genetic strategies, which can be more disruptive to the delicate capsid assembly and packaging processes, "late-stage" chemical modification of the assembled capsid-whether at natural amino acid residues or site-specifically installed noncanonical amino acid residues-often enables a versatile approach to introducing new properties to the capsid. This review summarizes the significant recent progress in AAV capsid engineering strategies, with a particular focus on chemical modifications in advancing the next generation of AAV-based gene therapies.

Dependovirus↗
PubMed2022

AAV vectors: The Rubik's cube of human gene therapy.

Defective genes account for &#x223c;80% of the total of more than 7,000 diseases known to date. Gene therapy brings the promise of a one-time treatment option that will fix the errors in patient genetic coding. Recombinant viruses are highly efficient vehicles for in&#xa0;vivo gene delivery. Adeno-associated virus (AAV) vectors offer unique advantages, such as tissue tropism, specificity in transduction, eliciting of a relatively low immune responses, no incorporation into the host chromosome, and long-lasting delivered gene expression, making them the most popular viral gene delivery system in clinical trials, with three AAV-based gene therapy drugs already approved by the US Food and Drug Administration (FDA) or European Medicines Agency (EMA). Despite the success of AAV vectors, their usage in particular scenarios is still limited due to remaining challenges, such as poor transduction efficiency in certain tissues, low organ specificity, pre-existing humoral immunity to AAV capsids, and vector dose-dependent toxicity in patients. In the present review, we address the different approaches to improve AAV vectors for gene therapy with a focus on AAV capsid selection and engineering, strategies to overcome anti-AAV immune response, and vector genome design, ending with a glimpse at vector production methods and the current state of recombinant AAV (rAAV) at the clinical level.

United States↗
PubMed2005

Identification of sites in adenovirus hexon for foreign peptide incorporation.

Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His6) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His6 epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His6 epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His6 epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His6 epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His6 epitopes did not appear to mediate His6-dependent viral infection, as assessed in two His6 artificial receptor systems. Our study provided valuable information for studies involving hexon modification.

Adenoviridae↗
PubMed2003

Viral assembly of oriented quantum dot nanowires.

The highly organized structure of M13 bacteriophage was used as an evolved biological template for the nucleation and orientation of semiconductor nanowires. To create this organized template, peptides were selected by using a pIII phage display library for their ability to nucleate ZnS or CdS nanocrystals. The successful peptides were expressed as pVIII fusion proteins into the crystalline capsid of the virus. The engineered viruses were exposed to semiconductor precursor solutions, and the resultant nanocrystals that were templated along the viruses to form nanowires were extensively characterized by using high-resolution analytical electron microscopy and photoluminescence. ZnS nanocrystals were well crystallized on the viral capsid in a hexagonal wurtzite or a cubic zinc blende structure, depending on the peptide expressed on the viral capsid. Electron diffraction patterns showed single-crystal type behavior from a polynanocrystalline area of the nanowire formed, suggesting that the nanocrystals on the virus were preferentially oriented with their [001] perpendicular to the viral surface. Peptides that specifically directed CdS nanocrystal growth were also engineered into the viral capsid to create wurtzite CdS virus-based nanowires. Lastly, heterostructured nucleation was achieved with a dual-peptide virus engineered to express two distinct peptides within the same viral capsid. This work represents a genetically controlled biological synthesis route to a semiconductor nanoscale heterostructure.

Bacteriophage M13↗
PubMed2003

Antibody-mediated targeting of an adenovirus vector modified to contain a synthetic immunoglobulin g-binding domain in the capsid.

Adenovirus vectors have been targeted to different cell types by genetic modification of the capsid or by using recombinant or chemically engineered adaptor molecules. However, both genetic capsid modifications and bridging adaptors have to be specifically tailored for each particular targeting situation. Here, we present an efficient and versatile strategy allowing the direct use of monoclonal antibodies against cell surface antigens for targeting of adenovirus vectors. A synthetic 33-amino-acid immunoglobulin G (IgG)-binding domain (Z33) derived from staphylococcal protein A was inserted into the adenovirus fiber protein. The fiber retained the ability to assemble into trimers, bound IgG with high affinity (Kd = 2.4 nM), and was incorporated into vector particles. The transduction efficiency of the Z33-modified adenovirus vector in epidermal growth factor receptor (EGFR)-expressing cells was strongly and dose-dependently enhanced by combination with an EGFR-specific monoclonal antibody. The antibody-mediated increase in cellular transduction was abolished in the presence of competing protein A. In targeting experiments with differentiated primary human muscle cells, up to a 77-fold increase in reporter gene transfer was achieved by preincubation of the vector with monoclonal antibodies directed against neuronal cell adhesion molecule or integrin alpha(7), respectively. The IgG-binding adenovirus vector holds promise for directed gene transfer to a wide variety of cell types by simply changing the target-specific antibody.

Adenoviridae↗
PubMed2002

Advances in gene transfer into haematopoietic stem cells by adenoviral vectors.

Until recently, the cells of haematopoietic origin were not considered good adenoviral (Adv) targets, primarily because they lacked the specific Adv receptors required for productive and efficient Adv infections. In addition, because of limitations inherent in Adv infections, such as short-term expression and a non-integrating nature, their application has been precluded from haematopoietic stem cell (HSC) and bone marrow transduction protocols where long-term expression has been required. Therefore, limited research utilising Adv-mediated gene transfer into haematopoietic cells had been conducted. With recent insights into the critical interactions between adenovirus (Adv) and cells, new Adv-mediated gene transduction strategies have now been reported that may overcome these limitations. These new strategies include Adv possessing synthetic polymer coatings, genetically modified capsid proteins or antibody-redirected fibres that can efficiently redirect and retarget Adv to transfer genes into HSC. Additionally, new hybrid Advs, engineered with both modified capsid proteins and novel cis-acting integration sequences, are also being developed which can efficiently deliver and integrate Adv delivered genes into HSC. This is an area of research that is now rapidly gaining momentum in terms of techniques and applications. Here we review the current status of adenovirus-based vectors as a means to achieve high-level gene transfer into haematopoietic cell types.

Adenoviridae↗
PubMed2003

Complete alanine scanning of intersubunit interfaces in a foot-and-mouth disease virus capsid reveals critical contributions of many side chains to particle stability and viral function.

Spherical virus capsids are large, multimeric protein shells whose assembly and stability depend on the establishment of multiple non-covalent interactions between many polypeptide subunits. In a foot-and-mouth disease virus capsid, 42 amino acid side chains per protomer are involved in noncovalent interactions between pentameric subunits that function as assembly/disassembly intermediates. We have individually truncated to alanine these 42 side chains and assessed their relevance for completion of the virus life cycle and capsid stability. Most mutations provoked a drastic reduction in virus yields. Nearly all of these critical mutations led to virions whose thermal inactivation rates differed from that of the parent virus, and many affected also early steps in the viral cycle. Rapid selection of genotypic revertants or variants with forward or compensatory mutations that restored viability was occasionally detected. The results with this model virus indicate the following. (i). Most of the residues at the interfaces between capsid subunits are critically important for viral function, in part but not exclusively because of their involvement in intersubunit recognition. Each hydrogen bond and salt bridge buried at the subunit interfaces may be important for capsid stability. (ii). New mutations able to restore viability may arise frequently at the subunit interfaces during virus evolution. (iii). A few interfacial side chains are functionally tolerant to truncation and may provide adequate mutation sites for the engineering of a thermostable capsid, potentially useful as an improved vaccine.

Alanine↗
PubMed2000

Antiviral activity of an intracellularly expressed single-chain antibody fragment directed against the murine leukemia virus capsid protein.

We have addressed the possibility that intracellularly expressed miniantibodies directed against the viral capsid protein can be used as antiretroviral agents in gene transfer experiments. R187 is a rat monoclonal antibody that has been reported to recognize the MuLV p30gag capsid polypeptide. We report here that it also binds to the Pr65gag precursor polyprotein. R187 has been cloned and expressed in the form of a single-chain variable fragment (scFv) that shows the same binding specificity as the parental antibody. When expressed intracellularly, the R187 scFv favors the production of viral particles showing reduced infectivity. It, however, exerts no detectable protective effect against infection. This was observed both when using replication-incompetent MuLV-derived vector and replication-competent wild-type MuLV. Although the intimate mechanism of the inhibition is not clear, this work raises the possibility that gene engineering of anti-capsid protein scFvs may offer an additional lead for gene therapy of severe retrovirus-linked diseases.

Amino Acid Sequence↗
PubMed1989

Engineering virus resistance in agricultural crops.

Plant viral genomes are relatively small and in the past decade many have been characterized at the molecular level. This has prompted research into the development of virus resistance based on interference with the viral multiplication cycle by teh introduction of viral sequences into the plant genome. Several strategies have been tested. The most successful one so far involves the constitutive expression of the coat protein gene of the virus against which resistance is desired. In this review we describe progress made in engineering virus resistance into potato, an important agricultural crop. To this end the molecular structure of the potato viruses X and Y and leafroll is discussed as well as the introduction of resistance against potato virus X into potato. In addition, we address the question of preservation of cultivar-specific characteristics, an important prerequisite for commercial application. Finally, recent investigations for alternative forms of virus resistance are described against the background of the results of coat protein-mediated protection.

Capsid↗
PubMed1997

Expression patterns of vascular-specific promoters RolC and Sh in transgenic potatoes and their use in engineering PLRV-resistant plants.

The expression patterns of GUS fusion constructs driven by the Agrobacterium rhizogenes RolC and the maize Sh (Shrunken; sucrose synthase-1) promoters were examined in transgenic potatoes (cv. Atlantic). RolC drove high-level gene expression in phloem tissue, bundle sheath cells and vascular parenchyma, but not in xylem or non-vascular tissues. Sh expression was exclusively confined to phloem tissue. Potato leafroll luteovirus (PLRV) replicates only in phloem tissues, and we show that when RolC is used to drive expression of the PLRV coat protein gene, virus-resistant lines can be obtained. In contrast, no significant resistance was observed when the Sh promoter was used.

Animals↗