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At least 163 records · Page 9Linked to original sources
PubMed1999

Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2.

We demonstrate the rapid and reliable quantification of physical AAV-2 (adeno-associated virus type 2) particles via a novel ELISA based on a monoclonal antibody which selectively recognizes assembled AAV-2 capsids. Titration of a variety of recombinant AAV-2 (rAAV) preparations revealed that at least 80+percent of all particles were empty, compared with a maximum of 50percent in wild-type AAV-2 stocks, indicating that the recombinant genomes were less efficiently encapsidated. This finding was confirmed upon titration of CsCl gradient fractions from recombinant and wild-type AAV-2 stocks. ELISA-based measurement of capsid numbers revealed a large number of physical particles with low densities corresponding to empty capsids in the recombinant, but not in the wild-type AAV-2 preparations. Moreover, additional expression of VP proteins during rAAV production was found to result in an excessive capsid formation, whilst yielding only minor increases in DNA-containing or transducing rAAV particles. We conclude that encapsidation of viral genomes rather than capsid assembly can be limiting for rAAV production, provided that a critical level of VP expression is maintained. The feasibility of quantifying AAV-2 capsid numbers via the ELISA allows determination of physical to DNA-containing or infectious particle ratios. These are important parameters which should help to optimize and standardize the production and application of recombinant AAV-2.

Antibodies, Monoclonal↗
PubMed1994

RGD sequence of foot-and-mouth disease virus is essential for infecting cells via the natural receptor but can be bypassed by an antibody-dependent enhancement pathway.

Foot-and-mouth disease virus appears to initiate infection by binding to cells at an Arg-Gly-Asp (RGD) sequence found in the flexible beta G-beta H loop of the viral capsid protein VP1. The role of the RGD sequence in attachment of virus to cells was tested by using synthetic full-length viral RNAs mutated within or near the RGD sequence. Baby hamster kidney (BHK) cells transfected with three different RNAs carrying mutations bordering the RGD sequence produced infectious viruses with wild-type plaque morphology; however, one of these mutant viruses bound to cells less efficiently than wild type. BHK cells transfected with RNAs containing changes within the RGD sequence produced noninfectious particles indistinguishable from wild-type virus in terms of sedimentation coefficient, binding to monoclonal antibodies, and protein composition. These virus-like particles are defined as ads- viruses, since they were unable to adsorb to and infect BHK cells. These mutants were defective only in cell binding, since antibody-complexed ads- viruses were able to infect Chinese hamster ovary cells expressing an immunoglobulin Fc receptor. These results confirm the essential role of the RGD sequence in binding of foot-and-mouth disease virus to susceptible cells and demonstrate that the natural cellular receptor for the virus serves only to bind virus to the cell.

Adsorption↗
PubMed2002

Hexon gene switch strategy for the generation of chimeric recombinant adenovirus.

The usefulness of adenovirus as a vehicle for transgene delivery is limited greatly by the induction of neutralizing anti-adenoviral immunity following the initial administration, thereby resulting in shorter-term and reduced levels of transgene expression. In this paper, we outline a strategy for the generation of recombinant Ad5-based adenovectors that have undergone a complete hexon exchange in an effort to circumvent pre-existing anti-vector humoral immunity. Eighteen different chimeric adenoviral vectors (from subgroups A, B, C, D, and E) have been constructed using a combination of direct cloning and bacterial homologous recombination methods. However, only chimeric Ad5-based constructs in which the hexons from Ad1, Ad2, Ad6, and Ad12 are incorporated in place of the Ad5 hexon were successfully rescued into viruses. Despite several attempts, the remaining fourteen chimeric adenovectors were not rescuable. In vivo rodent studies using transgenes for human immunodeficiency virus type 1 (HIV-1) gag and secreted human alkaline phosphatase (SEAP) suggest that the Ad5/Ad6-gag chimera (wherein Ad5 hexon was replaced with that of Ad6) is able to evade neutralizing antibodies generated against Ad5 vector efficiently. However, it appears that cross-reactive cytotoxic T lymphocytes (CTL) may also play a role in controlling in vivo infectivity of Ad5/Ad6-gag chimera. The Ad5/Ad12 chimera was found to be extremely ineffective in the i.m. delivery and expression of HIV-1 gag in mice compared to the Ad5/Ad6 construct. Implications of these results will be discussed.

Adenoviruses, Human↗
PubMed2001

Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1.

The high-yield expression of a neutralizing epitope from human immunodeficiency virus type 1 (HIV-1) on the surface of a plant virus and its immunogenicity are presented. The highly conserved ELDKWA epitope from glycoprotein (gp) 41 was expressed as an N-terminal translational fusion with the potato virus X (PVX) coat protein. The resulting chimeric virus particles (CVPs), purified and used to immunize mice intraperitoneally or intranasally, were able to elicit high levels of HIV-1-specific immunoglobulin G (IgG) and IgA antibodies. Furthermore, the human immune response to CVPs was studied with severe combined immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID). hu-PBL-SCID mice immunized with CVP-pulsed autologous dendritic cells were able to mount a specific human primary antibody response against the gp41-derived epitope. Notably, sera from both normal and hu-PBL-SCID mice showed an anti-HIV-1-neutralizing activity. Thus, PVX-based CVPs carrying neutralizing epitopes can offer novel perspectives for the development of effective vaccines against HIV and, more generally, for the design of new vaccination strategies in humans.

AIDS Vaccines↗
PubMed2004

Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX.

The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amino acids in length. The extensions evaluated consist of alpha-helical spacers up to 75 A in length and to which peptide ligands were fused. The pIX variants were efficiently incorporated into the capsids of Ad particles. On intact particles, the MYC-tagged-pIX molecules were readily accessible to anti-MYC antibodies, as demonstrated by electron microscopic analyses of immunogold-labeled virus particles. The labeling efficiency improved with increasing spacer length, suggesting that the spacers lift and expose the ligand at the capsid surface. Furthermore, we found that the addition of an integrin-binding RGD motif to the pIX markedly stimulated the transduction of coxsackievirus group B and hAd receptor-deficient endothelioma cells, demonstrating the utility of pIX modification in gene transfer. Our data demonstrate that the minor capsid protein IX can be used as an anchor for the addition of polypeptide ligands to Ad particles.

Adenoviridae↗
PubMed2006

JAM-A-independent, antibody-mediated uptake of reovirus into cells leads to apoptosis.

Apoptosis plays a major role in the cytopathic effect induced by reovirus following infection of cultured cells and newborn mice. Strain-specific differences in the capacity of reovirus to induce apoptosis segregate with the S1 and M2 gene segments, which encode attachment protein sigma1 and membrane penetration protein mu1, respectively. Virus strains that bind to both junctional adhesion molecule-A (JAM-A) and sialic acid are the most potent inducers of apoptosis. In addition to receptor binding, events in reovirus replication that occur during or after viral disassembly but prior to initiation of viral RNA synthesis also are required for reovirus-induced apoptosis. To determine whether reovirus infection initiated in the absence of JAM-A and sialic acid results in apoptosis, Chinese hamster ovary (CHO) cells engineered to express Fc receptors were infected with reovirus using antibodies directed against viral outer-capsid proteins. Fc-mediated infection of CHO cells induced apoptosis in a sigma1-independent manner. Apoptosis following this uptake mechanism requires acid-dependent proteolytic disassembly, since treatment of cells with the weak base ammonium chloride diminished the apoptotic response. Analysis of T1L x T3D reassortant viruses revealed that the mu1-encoding M2 gene segment is the only viral determinant of the apoptosis-inducing capacity of reovirus when infection is initiated via Fc receptors. Additionally, a temperature-sensitive, membrane penetration-defective M2 mutant, tsA279.64, is an inefficient inducer of apoptosis. These data suggest that signaling pathways activated by binding of sigma1 to JAM-A and sialic acid are dispensable for reovirus-mediated apoptosis and that the mu1 protein plays an essential role in stimulating proapoptotic signaling.

Animals↗
PubMed1997

Advances in adenoviral vectors: from genetic engineering to their biology.

Ad2 and Ad5 belong to a group of human cytolytic viruses that target the respiratory airways for reproduction, whereas latent infections establish within other tissues. Signals therefore exist that control this dichotomic process in different cell types, perhaps including cis and/or trans elements of viral origin. Since 1993, Ad2- and Ad5-based adenoviruses lacking all or part of the E1 regulatory region have been undergoing evaluation in phase I trials that target cancer and cystic fibrosis. These viruses are extremely attenuated and actually do not reproduce in most human cells. However, they retain most of the virus genetic program and often promote a significant cytotoxicity after infection, emphasizing the need to further cripple the virus biology to extend the duration of transgene expression, if required. We will review the strategies currently followed to engineer a professional lytic virus for epithelial cells into an innocuous gene delivery vehicle. Potential effects on the transducing properties of the vector that may result from the inactivation of viral activities that normally allow/regulate extrachromosomal gene expression during wild-type infection are discussed.

Adenovirus E1 Proteins↗
PubMed1996

Propagation of an attenuated virus by design: engineering a novel receptor for a noninfectious foot-and-mouth disease virus.

To gain entry into cells, viruses utilize a variety of different cell-surface molecules. Foot-and-mouth disease virus (FMDV) binds to cell-surface integrin molecules via an arginine-glycine-aspartic acid (RGD) sequence in capsid protein VP1. Binding to this particular cell-surface molecule influences FMDV tropism, and virus/receptor interactions appear to be responsible, in part, for selection of antigenic variants. To study early events of virus-cell interaction, we engineered an alternative and novel receptor for FMDV. Specifically, we generated a new receptor by fusing a virus-binding, single-chain antibody (scAb) to intracellular adhesion molecule 1 (ICAM1). Cells that are normally not susceptible to FMDV infection became susceptible after being transfected with DNA encoding the scAb/ICAM1 protein. An escape mutant (B2PD.3), derived with the mAb used to generate the genetically engineered receptor, was restricted for growth on the scAb/ICAM1 cells, but a variant of B2PD.3 selected by propagation on scAb/ICAM1 cells grew well on these cells. This variant partially regained wild-type sequence in the epitope recognized by the mAb and also regained the ability to be neutralize by the mAb. Moreover, RGD-deleted virions that are noninfectious in animals and other cell types grew to high titers and were able to form plaques on scAb/ ICAM1 cells. These studies demonstrate the first production of a totally synthetic cell-surface receptor for a virus. This novel approach will be useful for studying virus reception and for the development of safer vaccines against viral pathogens of animals and humans.

Amino Acid Sequence↗
PubMed1993

Minus sense transcripts of brome mosaic virus RNA-3 intercistronic region interfere with viral replication.

Interference with virus replication through the use of defective viral sequences is providing new insight to replication strategies and novel approaches for induced resistance. Because replication of brome mosaic virus (BMV) is potentiated by the intercistronic region of RNA-3, we examined the effect of adding various (-)sense RNAs corresponding to this region in co-transfections with wild type BMV RNAs. Progeny accumulation in barley protoplasts transfected with RNAs 1+2 was decreased by 90% in the presence of (-)RNA-3 delta HindIII, the longest (-)sense transcript tested, and by 85% when RNA-3 was also present. This trans interference was concentration dependent, and the use of deletion derivatives of (-)RNA-3 delta HindIII revealed that previously identified regulatory sequences within the intercistronic region were responsible for the observed interference. These deletion mutants were found to be of differing stabilities and several served as effective substrates for host-encoded polymerase to yield complementary (+)strands. Indeed, it is possible that the copying of viral RNA by the host polymerase serves as a hybrid arrest mechanism for discriminating against viral RNA functions. However, neither the ability of these sequences to serve as templates for host polymerase nor their (+)strand products contributed to the interference phenomenon, which may provide a new approach for engineering resistance to viral infection.

Capsid↗
PubMed1982

Expression of the structural proteins of Semliki Forest virus from cloned cDNA microinjected into the nucleus of baby hamster kidney cells.

The three structural proteins of Semliki Forest virus--i.e., the capsid, p62, and E1 proteins--were expressed in baby hamster kidney cells from cloned DNA transcribed from the virus-specific 4.1-kilobase mRNA. The cDNA was engineered into an expression vector developed by others [Mulligan, R. C. & Berg, P. (1980) Science 209, 1422--1427] downstream from the simian virus 40 early promoter and was introduced into cell nuclei by microneedle injection. Immunofluorescence analysis of injected cells showed that the capsid protein was located in the cell cytoplasm, whereas the membrane proteins were associated with cellular membranes. The p62 protein was shown to be transported from the rough endoplasmic reticulum to the plasma membrane, whereas the E1 protein remained in the rough endoplasmic reticulum.

Animals↗
PubMed1992

Use of antisense RNA to confer bacteriophage resistance in dairy starter cultures.

The strategy and implementation of a unique system for engineering bacteriophage resistant starter cultures of Lactococcus lactis employing antisense RNA is reviewed. As a necessary prerequisite for developing this system, we have cloned and sequenced a number of bacteriophage genes coding for minor and major structural proteins. In addition, we have also identified a series of genes whose function(s) is not known but their sequences appear to be conserved in a vast number of isolates. One of these latter sequences, designated gp51C, codes for a 51-kDa protein which is extremely charged and shares some homology with yeast translation initiation factor. Resistance to a broad class of isometric bacteriophages has been achieved by expression of an antisense RNA targeted against, for example, gp51C. In the best case, expression of the antisense gp51C RNA results is a greater than 99% reduction in the total number of plaque forming units. Additional antisense RNA constructs directed against other bacteriophage genes, including the major capsid protein, also appear effective at inhibiting infection from 40-55% suggesting that this approach may prove useful for engineering a set of truly isogenic strains to be used in a starter culture rotation plan.

Bacteriophages↗
PubMed2003

[Relativity betweeen expression level of potato virus X coat protein and the codon usage of wobble codon].

The relationship between the quantities of rare codons and the expression levels of foreign genes is very important in gene engineering. But up to now, there is few experiment on it in multi-cellular eukaryotes, especially in plants. To study the effect of rare codons on the expression level of potato virus X (PVX) coat protein (CP) in transgenic tobacco, some preferred codons in PVX coat protein gene were substituted with synonymous rare codons without changing the encoded amino acid sequence using site-specific mutation. The modified coat protein gene (cpm) and non-modified coat protein gene (cpw) were inserted into binary vector under the control of CaMV35S promoter, and these two plant expression constructs were transferred into tobacco (Nicotiana tabacum cv. Xanthi) genomes via Agrobacterium mediated method and transgenic plants were generated. Western blot and ELISA of these transgenic tobaccos showed that the expression level of modified CP is about one third to one sixth of that of non-modified CP. Northern blot analysis of RNAs from some transformants indicated that the transcriptional levels of cpm are the same as that of cpw, which implicated that the quantity of rare codons in foreign gene was a possible limited factor in foreign gene expression process. Changing the quantity of rare codons maybe an effective method for organism to control gene expression.

Amino Acid Sequence↗
PubMed2000

Design and evolution of artificial M13 coat proteins.

Using simple design and selective pressure, we have evolved an artificial M13 bacteriophage coat protein. M13 coat proteins first reside in the bacterial inner membrane and subsequently surround the DNA core of the assembled virus. The artificial coat protein (ACP) was designed and evolved to mimic both functions of the natural M13 coat proteins, but with an inverted orientation. ACP is a non-functional coat protein because it is not required for the production of phage particles. Instead, it incorporates into a phage coat which still requires all the natural coat proteins for structural integrity. In contrast with other M13 coat proteins, which can display polypeptides as amino-terminal fusions, ACP permits the carboxy-terminal display of large polypeptides. The results suggest that viruses can co-opt host membrane proteins to acquire new coat proteins and thus new functions. In particular, M13 bacteriophage can be engineered for new functions, such as carboxy-terminal phage display.

Amino Acid Sequence↗
PubMed1996

Role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage.

Filamentous bacteriophage virions can be engineered to display small foreign peptides in the N-terminal regions of all 2700 copies of the major coat protein (pVIII), but larger peptides can be accommodated only in hybrid virions, in which modified and wild-type coat protein subunits are interspersed. The copy number of peptides accepted in hybrid virions is generally believed to be related to peptide size: the larger the insert, the lower the number of modified coat protein subunits in the assembled virion. However, we show here that some large peptides can be displayed at a much higher copy number than smaller ones and that some relatively small peptides are poorly displayed, if at all, in hybrid virions. X-ray diffraction studies of a recombinant virion together with model building experiments with peptide and protein epitopes of known structure demonstrated that it is feasible to accommodate much larger structures, without perturbation of the capsid protein packing, than it has proved possible to generate in vivo. We show further that the insertion of certain peptides greatly slowed or even prevented the processing of the pVIII pro-coat by leader peptidase at the inner membrane of the Escherichia coli cell. A good correlation was found between the effect of the insert on the rate of the processing of the pro-coat, an essential step in virus assembly, and the number of the mature but modified proteins in the subsequently assembled hybrid virion. These results have important implications for the design of peptide display systems based on filamentous bacteriophage.

Amino Acid Sequence↗
PubMed2001

Efficient and selective AAV2-mediated gene transfer directed to human vascular endothelial cells.

Gene therapy vectors based on adeno-associated virus-2 (AAV2) offer considerable promise for human gene therapy. Applications for AAV vectors are limited to tissues efficiently transduced by the vector due to its natural tropism, which is predominantly skeletal muscle, neurons, and hepatocytes. Tropism modification to elevate efficiency and/or selectivity to individual cell types would enhance the scope of AAV for disease therapies. The vascular endothelium is implicitly important in cardiovascular diseases and cancer, but is relatively poorly transduced by AAV vectors. We therefore genetically incorporated the peptide SIGYPLP, which targets endothelial cells (EC), into position I-587 of AAV capsids. SIGYPLP-modified AAV (AAVsig) showed enhanced transduction of human EC compared with AAV with a wild-type capsid (AAVwt), a phenotype independent of heparan sulphate proteoglycan (HSPG) binding. In contrast, AAVsig did not enhance transduction of primary human vascular smooth muscle cells or human hepatocytes, principal targets for AAV vectors in local or systemic gene delivery applications, respectively. Furthermore, infection of EC in the presence of bafilomycin A(2) indicated that intracellular trafficking of AAV particles was altered by targeting AAV by means of SIGYPLP. AAV vectors with enhanced tropism for EC will be useful for diverse gene therapeutics targeted at the vasculature.

Amino Acid Sequence↗
PubMed1986

Gene transfer into intact plant cells by electroinjection through cell walls and membranes.

Tobacco mosaic virus (TMV) RNA was introduced directly into mesophyll cells of Nicotiana tabacum var. Samsun using electric-field pulses (electroinjection). The injected gene was successfully expressed in the recipient cells as judged by the assay for the virus coat protein using immunofluorescence and by the virus infectivity assay of the homogenate of the electroinjected cells for local lesions on tobacco leaves. As much as 50% of the cells that survived 24 days after electroinjection showed immunofluorescent specks.

Capsid↗
PubMed2006

Improved cardiac gene transfer by transcriptional and transductional targeting of adeno-associated viral vectors.

OBJECTIVE: Vectors based on recombinant adeno-associated virus 2 (AAV-2) are a promising tool for cardiac gene transfer. However, potential therapeutic applications need to consider the predominant transduction of the liver once AAV-2 vectors enter the systemic circulation. We therefore aimed to increase efficiency and specificity of cardiac vector delivery by combining transcriptional and cell surface targeting. METHODS: For analysis of transcriptional targeting, recombinant AAV vectors were generated harboring a luciferase reporter gene under control of the cytomegalovirus (CMV) promoter or the 1.5-kb cardiac myosin light chain promoter fused to the CMV immediate-early enhancer (CMV(enh)/MLC1.5). Luciferase activities were determined in representative organs three weeks after intravenous injection of the vector into adult mice. Transductional targeting was studied using luciferase-reporter constructs crosspackaged into capsids of AAV serotypes 1 to 6 and modified AAV-2 capsids devoid of binding their primary receptor heparan sulfate proteoglycan. RESULTS: Intravenous injections of AAV-2 vectors harboring the CMV(enh)/MLC1.5 promoter enabled a specific and 50-fold higher reporter gene expression in left ventricular myocardium of adult mice compared to vectors containing the CMV promoter. Comparison of AAV-2 vector genomes crosspackaged into capsids of AAV-1 to -6 showed that AAV-1, -4, -5, and -6 capsids increased cardiac transduction efficiency by about 10-fold. However, transduction of other organs such as the liver was also increased after systemic administration. In contrast, AAV-2-based vectors with ablated binding to their primary receptor heparan sulfate proteoglycan enabled a significantly increased efficiency of cardiac gene transfer and reduced transduction of the liver. CONCLUSIONS: Combining transcriptional targeting by the CMV(enh)/MLC1.5 promoter and AAV vectors devoid of binding the AAV-2 primary receptor results in an efficient cardiac gene transfer with a significantly reduced hepatic transduction.

Animals↗
PubMed2006

TMV-peptide fusion vaccines induce cell-mediated immune responses and tumor protection in two murine models.

Fusion of peptides to viral carriers has proven an effective method for improving cellular immunity. In this study we explore the ability of a plant virus, Tobacco mosaic virus (TMV), to stimulate cellular immunity by interacting directly with immune cells. Fluorescently labeled TMV was incubated in vitro with murine spleen or lymph node cells, and near quantitative labeling of lymphocytes was achieved after 2 h, which persisted for up to 48 h. Direct TMV uptake and upregulation of the CD86 activation marker was measured in nearly all dendritic cells (DCs) by flow cytometry. To demonstrate that TMV can also provide functional antigen delivery and immune stimulation in vivo, two well-characterized T-cell epitopes that provide protection against tumor challenge in mice were fused to TMV coat protein by genetic manipulation, or by chemical conjugation. Vaccination of C57BL/6 mice elicited measurable cellular responses by interferon gamma (IFN gamma) ELISpot and resulted in significantly improved protection from tumor challenge in both the EG.7-Ova and B16 melanoma models. From these results we conclude that TMV was an effective antigen carrier for inducing cellular immune responses to less than 1 microg of peptide.

Animals↗