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PubMed1997

HIV-1 capsid mutants inhibit the replication of wild-type virus at both early and late infection phases.

In-frame mutations were introduced into various portions of the human immunodeficiency virus type 1 (HIV-1) gag gene, and potentials of the mutants to suppress the replication of wild-type HIV-1 were monitored. In contrast to results obtained with matrix and nucleocapsid mutants, almost all capsid mutants blocked HIV-1 replication completely in single-round replication assays. A capsid mutant designated C6b was demonstrated to be one of the most efficient inhibitors for HIV-1 reported to date, and to be effective at both early and late viral replication phases. T-cells, which are engineered to express the C6b Gag in response to HIV-1 infection, were perfectly resistant to HIV-1.

Blotting, Western↗
PubMed2002

Human l1 retrotransposition is associated with genetic instability in vivo.

Retrotransposons have shaped eukaryotic genomes for millions of years. To analyze the consequences of human L1 retrotransposition, we developed a genetic system to recover many new L1 insertions in somatic cells. Forty-two de novo integrants were recovered that faithfully mimic many aspects of L1s that accumulated since the primate radiation. Their structures experimentally demonstrate an association between L1 retrotransposition and various forms of genetic instability. Numerous L1 element inversions, extra nucleotide insertions, exon deletions, a chromosomal inversion, and flanking sequence comobilization (called 5' transduction) were identified. In a striking number of integrants, short identical sequences were shared between the donor and the target site's 3' end, suggesting a mechanistic model that helps explain the structure of L1 insertions.

5' Flanking Region↗
PubMed1997

Filamentous phage infection-mediated gene expression: construction and propagation of the gIII deletion mutant helper phage R408d3.

We describe the use of transcriptional fusions to the phage shock protein (psp) promoter. These fusions are expressed only when cells are infected by filamentous phage. In an application, the psp promoter was fused to the protein coding part of filamentous phage gene III (gIII). Protein III (pIII) is needed to complement mutant f1 phage containing a deletion of gIII, but its synthesis also renders cells resistant to infection. By inducing pIII production from psp-gIII only in the cells that are already infected with phage, it was possible to obtain plaques from phage in which gIII had been completely deleted. gIII was deleted from two helper phages: R408 and VCSM13, which were then propagated on cells containing the psp-gIII fusion. These two phages were tested for use in a phage display method that requires generation of noninfectious, phagemid-containing virion-like particles. Both helpers worked, but R408d3 was superior to VCSM13d3, because it generated about 1800-times fewer background infectious particles.

Capsid Proteins↗
PubMed2003

Genetic manipulations of adenovirus type 5 fiber resulting in liver tropism attenuation.

The development of genetically modified adenoviral vectors capable of specifically transducing a given cell population requires the addition and functional presentation of particular tropism determinants within the virus capsid, together with the abrogation of the molecular determinants that dictate their natural tropism in vivo. The human adenovirus serotype 5 (Ad5) first attaches to the cell surface following high-affinity binding of the C-terminal knob of the fiber capsid protein to the coxsackie and adenovirus receptor (CAR). Here we have assessed whether genetic shortening of the fiber shaft (virus BS1), or replacing the Ad5 fiber shaft and knob with their Ad3 counterparts (virus DB6), could cripple this interaction in vitro and in vivo. A 10-fold decrease in the binding of the modified capsids to soluble CAR was evidenced, which correlated with a similar reduction of their ability to transduce CAR-positive cells in vitro. The ability of BS1 to interact with cellular integrins was also impaired, suggesting that the penton base and the short-shafted fiber when embedded in the capsid preclude each other from efficiently interacting with their cognate cell surface receptors (CAR and integrins respectively). BS1 and DB6 intravenous injections in mice further supported a profound impairment of the ability of the capsid-modified viruses to transduce the liver as demonstrated by a 10-fold reduction of intracellular viral DNA and transgene expression. Interestingly enough, the host humoral response was also specifically weakened in BS1- and DB6-inoculated animals. Taken together, these observations indicate that (i) fiber shortening and (ii) pseudo-typing of Ad5-based vectors with the shaft and knob from non-CAR-binding serotypes constitute two promising strategies to successfully attenuate their native tropism in vitro and most importantly in vivo.

Animals↗
PubMed2000

Differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system.

Virus-like particles (VLPs) are being currently investigated in vaccines against viral infections in humans. There are different recombinant-protein-expression systems available for obtaining the necessary VLP preparation for vaccination. However, the differences in post-translational modifications of the recombinant proteins obtained and their differences in efficacy in eliciting an anti-viral response in vaccines are not well established. In this study we have compared the post-translational modifications of human papillomavirus type-6b major capsid protein L1 (HPV 6bL1) expressed using recombinant baculovirus (rBV) in Sf9 (Spodoptera frugiperda) insect cells, with the protein expressed using recombinant vaccinia virus (rVV) in CV-1 kidney epithelial cells. Two-dimensional gel electrophoresis of biosynthetically labelled rBV-expressed HPV 6bL1 showed several post-translationally modified variants of the protein, whereas rVV-expressed HPV 6bL1 showed only a few variants. Phosphorylations were detected at threonine and serine residues for the L1 expressed from rBV compared with phosphorylation at serine residues only for the L1 expressed from rVV. HPV 6bL1 expressed using rBV incorporated [(3)H]mannose and [(3)H]galactose, whereas HPV 6bL1 expressed using rVV incorporated only [(3)H]galactose. We conclude that post-translational modification of recombinant HPV 6bL1 can differ according to the system used for its expression. Since recombinant L1 protein is a potential human-vaccine candidate, the implication of the observed differences in post-translational modifications on immunogenicity of L1 VLPs warrants investigation.

Animals↗
PubMed2009

A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection.

To engineer gene vectors that target striated muscles after systemic delivery, we constructed a random library of adeno-associated virus (AAV) by shuffling the capsid genes of AAV serotypes 1 to 9, and screened for muscle-targeting capsids by direct in vivo panning after tail vein injection in mice. After 2 rounds of in vivo selection, a capsid gene named M41 was retrieved mainly based on its high frequency in the muscle and low frequency in the liver. Structural analyses revealed that the AAVM41 capsid is a recombinant of AAV1, 6, 7, and 8 with a mosaic capsid surface and a conserved capsid interior. AAVM41 was then subjected to a side-by-side comparison to AAV9, the most robust AAV for systemic heart and muscle gene delivery; to AAV6, a parental AAV with strong muscle tropism. After i.v. delivery of reporter genes, AAVM41 was found more efficient than AAV6 in the heart and muscle, and was similar to AAV9 in the heart but weaker in the muscle. In fact, the myocardium showed the highest gene expression among all tissues tested in mice and hamsters after systemic AAVM41 delivery. However, gene transfer in non-muscle tissues, mainly the liver, was dramatically reduced. AAVM41 was further tested in a genetic cardiomyopathy hamster model and achieved efficient long-term delta-sarcoglycan gene expression and rescue of cardiac functions. Thus, direct in vivo panning of capsid libraries is a simple tool for the de-targeting and retargeting of viral vector tissue tropisms facilitated by acquisition of desirable sequences and properties.

Animals↗
PubMed2004

Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda Red recombination.

There are extensive efforts to develop cell-targeting adenoviral vectors for gene therapy wherein endogenous cell-binding ligands are ablated and exogenous ligands are introduced by genetic means. Although current approaches can genetically manipulate the capsid genes of adenoviral vectors, these approaches can be time-consuming and require multiple steps to produce a modified viral genome. We present here the use of the bacteriophage lambda Red recombination system as a valuable tool for the easy and rapid construction of capsid-modified adenoviral genomes.

Adenoviridae↗
PubMed1993

Recombinant human hepatitis B virus reverse transcriptase is active in the absence of the nucleocapsid or the viral replication origin, DR1.

The double-stranded DNA genome of hepatitis B virus (HBV) is reverse transcribed from the viral pregenome RNA template by a virally encoded reverse transcriptase enzyme (RT) that possesses both priming and elongation activities. Prior efforts have failed to express an active form of HBV RT outside the nucleocapsid in animal cells or to release it from viral nucleocapsids, thus restricting the characterization of this important enzyme. Here, we have engineered epitope-tagged HBV RT proteins and expressed them in Xenopus oocytes via a synthetic RT mRNA which does not include the viral capsid protein or the known initiation site for viral DNA synthesis, DR1. We demonstrate the production of an immunoprecipitable 96-kDa HBV RT protein and show, using a simple in vitro RT assay, that oocyte lysates containing this protein possess an activity that (i) catalyzes an RNA-dependent deoxynucleotide triphosphate polymerization reaction by using an as-yet-unidentified RNA template and (ii) is sensitive to the RT inhibitors actinomycin D and phosphonoformate. Experiments with the chain terminator ddATP suggest that a significant amount of chain elongation occurs in our in vitro reaction. Electrophoretic analysis reveals a heterogeneous array of RT reaction products with sizes ranging from about 100 bases to far larger than that of the input RT mRNA. These products appear to contain covalently bound protein, consistent with the notion that the RT protein may have primed their synthesis. We conclude that HBV RT activity can be uncoupled from both the nucleocapsid and the replication origin, DR1. Our results raise the possibility that unless HBV employs novel mechanisms to regulate its constitutively active RT, cellular RNAs may be reverse transcribed during HBV infection, with potential implications for the development of HBV-related liver cancer. The use of the oocyte system should facilitate studies of HBV RT, including the development of HBV RT inhibitors for antiviral therapy.

Amino Acid Sequence↗
PubMed2004

Targeted retroviral transduction of c-kit+ hematopoietic cells using novel ligand display technology.

Gene therapy for a wide variety of disorders would be greatly enhanced by the development of vectors that could be targeted for gene delivery to specific populations of cells. We describe here high-efficiency targeted transduction based on a novel targeting strategy that exploits the ability of retroviruses to incorporate host cell proteins into the surface of the viral particle as they bud through the plasma membrane. Ecotropic retroviral particles produced in cells engineered to express the membrane-bound form of stem cell factor (mbSCF) transduce both human cell lines and primary cells with high efficiency in a strictly c-kit (SCF receptor)-dependent fashion. The availability of efficient targeted vectors provides a platform for the development of a new generation of therapies using in vivo gene delivery.

Capsid Proteins↗
PubMed2006

Metabolic biotinylation provides a unique platform for the purification and targeting of multiple AAV vector serotypes.

The development of rationally designed targeted gene delivery vectors is an important focus for gene therapy. While genetic modification of AAV can produce vectors with modified tropism, incorporation of targeting peptides into the structural context of the AAV virion often results in loss of function or loss of virion integrity. To address this issue, we have developed a targeting system using metabolically biotinylated AAV. We generated serotype 1, 2, 3, 4, and 5 AAV capsids with small peptide insertions that are metabolically biotinylated in packaging cells during vector production by coexpression of the Escherichia coli BirA, biotin ligase, gene. Biotin moieties are exposed on the surface of assembled AAV particles and can interact with avidin. Metabolically biotinylated AAV vectors produced in this manner maintained endogenous titer and tissue tropism, could be purified on monomeric avidin resin, and could be retargeted to cells engineered to express an artificial avidin-biotin receptor. This technology provides not only a single platform for the purification of multiple AAV vector serotypes, but also a means for the development of multiple targeted AAV vectors utilizing a single capsid modification via straightforward avidin-biotin ligand coupling.

Amino Acid Sequence↗
PubMed2004

Intracellular expression of engineered RNase P ribozymes effectively blocks gene expression and replication of human cytomegalovirus.

A ribozyme (M1GS RNA) constructed from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping region of two human cytomegalovirus (HCMV) mRNAs, which encode for the viral essential protease (PR) and capsid assembly proteins (AP), respectively. The results show a reduction of >80% in the expression levels of PR and AP and an inhibition of approximately 2000-fold of viral growth in cells that stably expressed the ribozyme. In comparison, <10% reduction in the expression of the targets and viral growth was found in cells that either did not express the ribozyme or produced a "disabled" ribozyme carrying mutations that abolished its catalytic activity. Examination of replication of the virus in the ribozyme-expressing cells indicates that packaging of the viral genomic DNA into capsids is blocked, and suggests that the antiviral effects are because the ribozyme specifically inhibits the AP and PR expression and, consequently, abolishes viral capsid formation and growth. Our results show that RNase P ribozymes are highly effective in blocking HCMV growth by targeting the PR and AP mRNAs and demonstrate the feasibility to use these ribozymes in gene therapy for antiviral applications.

Cytomegalovirus↗
PubMed2006

Expression of Aedes trypsin-modulating oostatic factor on the virion of TMV: A potential larvicide.

We report the engineering of the surface of the tobacco mosaic virus (TMV) virion with a mosquito decapeptide hormone, trypsin-modulating oostatic factor (TMOF). The TMV coat protein (CP) was fused to TMOF at the C terminus by using a read-through, leaky stop codon that facilitated expression of CP and chimeric CP-TMOF (20:1 ratio) that were coassembled into virus particles in infected Nicotiana tabacum. Plants that were infected with the hybrid TMV RNA accumulated TMOF to levels of 1.3% of total soluble protein. Infected tobacco leaf discs that were fed to Heliothis virescens fourth-instar larvae stunted their growth and inhibited trypsin and chymotrypsin activity in their midgut. Purified CP-TMOF virions fed to mosquito larvae stopped larval growth and caused death. Because TMV has a wide host range, expressing TMV-TMOF in plants can be used as a general method to protect them against agricultural insect pests and to control vector mosquitoes.

Aedes↗
PubMed1999

BHV-1: new molecular approaches to control a common and widespread infection.

BACKGROUND: Herpesviruses are widespread viruses, causing severe infections in both humans and animals. Eradication of herpesviruses is extremely difficult because of their ability to establish latent and life-long infections. However, latency is only one tool that has evolved in herpesviruses to successfully infect their hosts; such viruses display a wide (and still incompletely known) panoply of genes and proteins that are able to counteract immune responses of their hosts. Envelope glycoproteins and cytokine inhibitors are two examples of such weapons. All of these factors make it difficult to develop diagnostics and vaccines, unless they are based on molecular techniques. MATERIALS AND METHODS: Animal herpesviruses, because of their striking similarity to human ones, are suitable models to study the molecular biology of herpesviruses and develop strategies aimed at designing neurotropic live vectors for gene therapy as well as engineered attenuated vaccines. RESULTS: BHV-1 is a neurotropic herpesvirus causing infectious rhinotracheitis (IBR) in cattle. It is a major plague in zootechnics and commercial trade, because of its ability to spread through asymptomatic carrier animals, frozen semen, and embryos. Such portals of infections are also important for human herpesviruses, which mainly cause systemic, eye, and genital tract infections, leading even to the development of cancer. CONCLUSIONS: This review covers both the genetics and molecular biology of BHV-1 and its related herpesviruses. Epidemiology and diagnostic approaches to herpesvirus infections are presented. The role of herpesviruses in gene therapy and a broad introduction to classic and engineered vaccines against herpesviruses are also provided. http://link.springer-ny. com/link/service/journals/00020/bibs/5n5p261.html

Animals↗
PubMed2003

Evaluation of genetically engineered derivatives of a Chinese strain of foot-and-mouth disease virus reveals a novel cell-binding site which functions in cell culture and in animals.

Adaptation of field isolates of foot-and-mouth disease virus (FMDV) to grow in cells in culture can result in changes in viral properties that include acquisition of the ability to bind to cell surface heparan sulfate (HS). After 13 passages on BHK cells to produce a vaccine, a Cathay topotype isolate of FMDV serotype O from China (O/CHA/90) extended its cell culture host range and bound to heparin-Sepharose, although it did not require cell surface HS as a receptor molecule. To understand these phenomena, we constructed chimeric viruses by using a type A(12) infectious cDNA and the capsid protein-coding regions of O/CHA/90 and its cell culture-adapted derivative (vac-O/CHA/90). Using a set of viruses derived from these chimeras by exchanging portions of the capsid-coding regions, we discovered that a group of amino acid residues that surround the fivefold axis of the icosahedral virion determine host range in cell culture and influence pathogenicity in pigs. These residues included aromatic amino acids at positions 108 and 174 and positively charged residues at positions 83 and 172 in protein 1D. To test if these residues participated in non-integrin-dependent cell binding, the integrin-binding RGD sequence in protein 1D was changed to KGE in two different chimeras. Evaluation of these KGE viruses indicated that growth in cell culture was not dependent on HS. One of these viruses was tested in pigs, where it produced a mild disease and maintained its KGE sequence. These results are discussed in terms of receptor utilization and pathogenesis of this important pathogen.

Amino Acid Sequence↗
PubMed2001

Modifying the specificity of an RNA backbone contact.

The interaction between the MS2 bacteriophage coat protein homodimer and its cognate RNA hairpin is facilitated by 21 different RNA-protein contacts. In one of these contacts, the 2'-hydroxyl group at ribose -5 of the RNA acts as a hydrogen bond donor to Glu63 in one subunit of the protein. Previous experiments showed that substitution of ribose -5 with deoxyribose resulted in a 24-fold decrease in binding affinity between RNA and protein. Using a protein where the two MS2 monomers were fused to increase stability, the contribution of this contact to the overall binding affinity was investigated by site-directed mutagenesis. When Glu63 was substituted with glutamine, aspartate, or alanine, the binding affinity of the hairpin for the protein was weakened by 12 to 100-fold, similar to that observed with deoxyribose at position -5. However, the specificity of the three mutant proteins for RNAs with various modifications at the 2'-position of ribose -5 differed dramatically. While the Glu63Asp protein resembled the wild-type protein in preferring the 2'-hydroxyl group over a proton or a bulky 2'-substituent, both the Glu63Ala and Glu63Gln proteins preferred bulky 2'-substituents over the 2'-hydroxyl group by more than 100-fold. These experiments emphasize the ease with which the specificity of a protein-nucleic acid interaction can be changed at thermodynamically important sites.

Binding Sites↗
PubMed1989

Adenoviruses as vectors for the transfer of genetic information and for the construction of new type vaccines.

At present many types of corpuscular nondefective, conditional-defective and helper-dependent expressing adenoviral vectors are available which can be used in constructing gene-engineered live or inactivated viral vaccines. In particular, promising results have been obtained with live recombinant human adenoviruses expressing the S antigen of hepatitis B virus, capsid protein of rotaviruses and gB protein of herpes virus. These recombinants are proper candidates for testing as corresponding vaccine strains, a good alternative to well-known recombinant vaccine virus.

Adenoviridae↗
PubMed2002

Functional selection of phage displayed peptides for facilitated design of fusion tags improving aqueous two-phase partitioning of recombinant proteins.

Aqueous two-phase systems allow for the unequal distribution of proteins and other molecules in water-rich solutions containing phase separating polymers or surfactants. One approach to improve the partitioning properties of recombinant proteins is to produce the proteins as fused to certain peptide tags. However, the rational design of such tags has proven difficult since it involves a compromise between multivariate parameters such as partitioning properties, solvent accessibility and production/secretion efficiency. In this work, a novel approach for the identification of suitable peptide tag extensions has been investigated. Using the principles of selection, rather than design, peptide sequences contributing to an improved partitioning have been identified using phage display technology. A 40 million member phagemid library of random nona-peptides, displayed as fusion to the major coat protein pVIII of the filamentous phage M13, was employed in the selection of top-phase partitioning phage particles in a PEG/sodium phosphate system. After multiple cycles of selection by partitioning, peptides with high frequencies of both tyrosine and proline residues were found to be over represented in selected clones. The identified peptide sequences, or derivatives thereof, were subsequently individually analyzed for their partitioning behavior as displayed on phage, as free synthetic peptides and as genetically fused to a recombinant model target protein. The results showed that novel peptide sequences capable of enhancing top-phase partitioning without interfering with protein production and secretion indeed could be identified for the aqueous two-phase system investigated.

Amino Acid Sequence↗
PubMed2006

In planta production of two peptides of the Classical Swine Fever Virus (CSFV) E2 glycoprotein fused to the coat protein of potato virus X.

BACKGROUND: Classical Swine Fever (CSFV) is one of the most important viral infectious diseases affecting wild boars and domestic pigs. The etiological agent of the disease is the CSF virus, a single stranded RNA virus belonging to the family Flaviviridae. All preventive measures in domestic pigs have been focused in interrupting the chain of infection and in avoiding the spread of CSFV within wild boars as well as interrupting transmission from wild boars to domestic pigs. The use of plant based vaccine against CSFV would be advantageous as plant organs can be distributed without the need of particular treatments such as refrigeration and therefore large areas, populated by wild animals, could be easily covered. RESULTS: We report the in planta production of peptides of the classical swine fever (CSF) E2 glycoprotein fused to the coat protein of potato virus X. RT-PCR studies demonstrated that the peptide encoding sequences are correctly retained in the PVX construct after three sequential passage in Nicotiana benthamiana plants. Sequence analysis of RT-PCR products confirmed that the epitope coding sequences are replicated with high fidelity during PVX infection. Partially purified virions were able to induce an immune response in rabbits. CONCLUSION: Previous reports have demonstrated that E2 synthetic peptides can efficiently induce an immunoprotective response in immunogenized animals. In this work we have showed that E2 peptides can be expressed in planta by using a modified PVX vector. These results are particularly promising for designing strategies for disease containment in areas inhabited by wild boars.

Animals↗