Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Packaging”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 919 records · Page 51Linked to original sources
PubMed2001

Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome.

Herpes simplex virus type 1 (HSV-1) amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis-acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 "helper" functions, amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of amplicon vectors are still contaminated with replication-competent helper virus at a frequency of 10(-4)-10(-6). To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli, either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVDeltapacDelta27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 "stuffer" sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected amplicon DNA without generating replication-competent helper virus (<1 helper virus per 10(8) TU amplicon vectors), and (iii) the resulting amplicon stocks have titers of up to 3-10 x 10(8) TU/ml after concentration. Elimination of replication-competent helper virus from HSV-1 amplicon vector stocks further improves safety in gene transfer applications.

Animals↗
PubMed2001

Generation of a flexible cell line with regulatable, high-level expression of HIV Gag/Pol particles capable of packaging HIV-derived vectors.

HIV-derived vectors are of potential clinical relevance due to their ability to transduce nondividing cells in vitro and in vivo. However, the generation of cell lines stably and reproducibly expressing high amounts of defined subviral particles, capable of packaging and transducing HIV-derived vectors, has been hampered by the cytotoxicity of some of the required gene products, in particular of the HIV-1 protease. The successful use of regulatable gene expression systems to overcome this problem requires that the remaining basally expressed gene product activity is below the threshold for cytotoxicity. To try to achieve this, we have consecutively introduced appropriate plasmids, encoding HIV rev and HIV gag/pol gene products, each under the control of separate ecdysone-inducible promoters, into human 293 cells. Using a protocol in which a specific HIV protease inhibitor, Saquinavir, was continuously present in the culture medium during selection, we could generate stable cell lines inducibly expressing high amounts of subviral particles. A cell line, termed 293-Rev/Gag/Pol(i), which has been characterized in more detail, inducibly releases, within 48 h postinduction, high amounts of HIV Gag/Pol particles (about 10 microg CA/ml). These HIV Gag/Pol particles can package and transduce third-generation HIV vectors to high titers. Thus, in addition to other applications, the 293-Rev/Gag/Pol(i) cell line represents a "founder" packaging cell line which, depending on the requirement, can be further modified to include specific transgene-encoding vector and targeting glycoprotein genes.

Cell Line↗
PubMed2001

Development of a competitive PCR method for physical titration of recombinant EBV vector in a helper-dependent packaging system.

Epstein-Barr virus (EBV) is a gamma-herpesvirus with B lymphotropism and a double-stranded DNA genome of 172 kb that is episomally maintained in permissive cells during latency. EBV-based vectors containing minimal cis elements for replication, amplification, and helper-dependent packaging in a producer cell line HH514 have been developed to deliver therapeutic/suicide transgenes as infectious viral particles (miniEBV) to EBV-transformed B lymphoblastoid cells or B lymphoma cells. A quantitative, competitive PCR-based assay was developed to determine the relative packaging efficiencies of miniEBV and helper P3HR1 coproduced in HH514 cells. This provides a rapid and accurate quantitation of the physical titer of the virus preparation, which helps preserve the biological titer of the virus preparation and increase the efficiency of transgene delivery by miniEBV infection. In addition, it provides a sensitive and accurate way to evaluate future development of a helper-free packaging system by detecting any possible helper virus contamination.

DNA Primers↗
PubMed2001

A novel approach to cancer therapy using an oncolytic herpes virus to package amplicons containing cytokine genes.

There are two promising herpes viral-based anticancer strategies: one involves replication-defective viruses to transfer therapeutic transgenes, and the other involves replication-conditional oncolytic viruses, which selectively infect and destroy cancer cells directly. This study examines a novel dual herpesvirus preparation, which combines the immunostimulatory effects of amplicon-mediated IL2 expression with direct viral-induced oncolysis. The oncolytic virus G207 was used as the helper virus to package a herpes simplex virus (HSV)-amplicon vector carrying the gene IL2 (HSV-IL2), yielding a single preparation with two complementary modes of action. In vivo comparison was carried out in a syngeneic squamous cell carcinoma flank tumor model. We directly injected established tumors with HSV-IL2, G207, G207 mixed with HSV-IL2, or G207-packaged HSV-amplicon carrying the IL2 transgene (G207[IL2]). Significant inhibition of tumor growth was seen at 2 weeks in the G207[IL2]-treated tumors relative to controls (0.57+/-0.44 cm(3) versus 39.45+/-5.13 cm(3), P<0.00001), HSV-IL2 (20.97+/-4.60 cm(3)), and the G207 group (7.71+/-2.10 cm(3)). This unique use of a replication-conditional, oncolytic virus to package a replication-incompetent amplicon vector demonstrates impressive efficacy in vitro and in vivo, and avoids the theoretical concerns of recombination with reversion to wild type.

Animals↗
PubMed2001

Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison.

Recombinant adeno-associated (rAAV) viral vectors hold great therapeutic potential for human diseases. However, a relatively small packaging capacity (less than 5 kb) has limited the application of rAAV for certain diseases such as cystic fibrosis and Duchenne muscular dystrophy. Here we compared two mechanistically distinct approaches to overcome packaging restraints with rAAV vectors. The trans-splicing approach reconstitutes gene expression from two independent rAAV vectors, each encoding unique, nonoverlapping halves of a transgene. This process requires intermolecular concatamerization and subsequent splicing between independent vectors. A distinct overlapping vector approach uses homologous recombination between overlapping regions in two independent vectors. Using the beta-galactosidase gene as template, trans-splicing approaches were threefold (in primary fibroblasts) and 12-fold (in muscle tissue) more effective in generating full-length transgene products than the overlapping vector approach. Nevertheless, the efficiency of trans-splicing remained moderate at approximately 4.3% (for muscle) and 7% (for fibroblasts) of that seen with a single vector encoding the full-length transgene. The efficiency of trans-splicing was augmented 1185-fold by adenoviral E4, but not E2a, gene products. This augmentation was much less pronounced with the overlapping vectoring approach (12-fold). Trans-splicing and overlapping vector approaches are two viable alternatives to expand rAAV packaging capacity.

Adenovirus E2 Proteins↗
PubMed1993

Protein folding studies in vivo with a bacteriophage T4 expression-packaging-processing vector that delivers encapsidated fusion proteins into bacteria.

A cloned phage T4 gene which expresses the nonessential capsid scaffold protein IPIII was modified to permit construction and packaging of protein fusions within the capsid. IPIII deletion phage packaged IPIII-beta-galactosidase, IPIII-beta-globin, and IPIII-beta-globin-beta-galactosidase fusion proteins; the latter protein fusion was specifically processed by the T4 gene 21 head morphogenetic proteinase in vivo at a consensus leu(ile)-P1-glu* cleavage site to regenerate beta-galactosidase. Phage inject IPIII-beta-galactosidase protein into bacteria, but less activity is recovered in infections of Escherichia coli dnaK or groEL mutants, suggesting that these host molecular chaperones are required for beta-galactosidase intracellular folding. This expression-packaging-processing (EPP) vector directs protein fusions into capsids for easy detection and purification and permits study of protein delivery and folding in bacteria.

Bacteriophage T4↗
PubMed1995

Defective retroviral endogenous RNA is efficiently transmitted by infectious particles produced on an avian retroviral vector packaging cell line.

This report describes the contamination of "helper-free" stocks of defective retroviral vector with particles bearing retroviral endogenous RNA. An avian leukosis virus-based packaging cell line was developed from LMH cells that bear the ev1, ev3, and ev6 retroviral endogenous loci. The results show that an endogenous retroviral transcript (ev3) was packaged into virions produced by this packaging cell line and was efficiently transferred along with the vector to target cells. The titer of the ev contaminant particles was estimated at 50-100 CA-p27gag-expressing units/ml of supernatant.

Animals↗
PubMed1995

Circularly permuted viral pRNA active and specific in the packaging of bacteriophage phi 29 DNA.

A viral-encoded 120-base pRNA has been shown to have an essential role in the packaging of bacteriophage phi 29 DNA. The finding that both the 5'- and 3'-termini of the pRNA are proximate and crucial for biological function (C. Zhang, C. Lee, and P. Guo, 1994, Virology, 201, 77-85) prompted investigation of the activity of circularly permuted pRNAs (cpRNA) and of the expandability and essentiality of bases extending from the termini. A 117-base pRNA with a deletion of three bases downstream of the proximal terminus was active in DNA packaging. Concatemeric DNAs containing two tandem pRNA genes separated by a short or a long loop sequence were constructed. The cpRNAs from these DNA templates were transcribed in vitro and shown to be active in phi 29 DNA packaging, with activity comparable to the parental (noncircularly permuted) pRNA, indicating that neither of the loops tested affected the activity and folding of the cpRNA. As few as four bases were sufficient to serve as a loop for the terminal 180 degree turn, and a loop as long as 27 bases did not affect the cpRNA structure and function. Eight cpRNAs were constructed to assess the effect of openings within the wild-type pRNA structure. Opening of the bulge at residue 38 did not affect cpRNA activity, but opening the bulge at residue 55 greatly reduced it. Although the sequence of the 5',3'-terminal loop was not important for the folding and activity of the cpRNA, the activities of cpRNAs with openings at individual bulges or hairpins were different, indicating that each region plays a different role in pRNA folding and function. Our results indicate that it is possible to generate active circularly permuted pRNA by assigning internal sites of the pRNA as new 3'- and 5'-termini. The creation of new variable ends makes the labeling of internal bases of the pRNA molecule possible and will facilitate the analysis of pRNA secondary and tertiary structure.

Bacillus Phages↗
PubMed1995

Inhibition of phage phi 29 assembly by antisense oligonucleotides targeting viral pRNA essential for DNA packaging.

A sensitive and efficient system for the functional assay of antisense oligonucleotides (oligos) was developed based on an in vitro viral assembly system. A 120-base RNA (pRNA), which indispensably participates in bacteriophage phi 29 DNA packaging, was the target for antisense action. Antisense oligos bound to pRNA, as revealed by a slower electrophoretic mobility of pRNA/oligo complexes in comparison with native pRNA. Infectious viruses were assembled in vitro with synthetic pRNA and DNA, as well as with viral proteins produced from cloned genes. Up to 10(7) plaque-forming units per milliliter were obtained in the absence of antisense oligos, while as few as zero plaques were detected in the presence of certain antisense oligos. A 1-base mismatch greatly influenced the inhibitory effect of the antisense oligos, but this 1-based mismatch was not important when the mismatch was placed at the end of the oligo. Five oligos did not bind pRNA or inhibit the assembly of the virion, suggesting that the RNA sequences complementary to these oligos are nonessential or buried internally in the RNA. Viral assembly was strongly inhibited by antisense oligos P15 and P10, targeting either the 5'- or the 3'-end of the pRNA, respectively. Viral assembly was also strongly inhibited by oligo P6, targeting an internal region, residues 75-91, of pRNA. Oligo P6 inhibited DNA packaging activity by blocking the binding of pRNA to the procapsid, while P10 and P15 inhibited DNA packaging activity but did not block the binding of pRNA to the procapsid, suggesting that in addition to the reported internal domain for procapsid binding, pRNA contains another domain at the paired 5'/3'-ends with a yet to be defined role in DNA translocation.

Bacillus Phages↗
PubMed1995

5' regions of HIV-1 RNAs are not sufficient for encapsidation: implications for the HIV-1 packaging signal.

The location and nature of the HIV-1 packaging signal are largely unknown, despite several genetic and biochemical mutational analyses. In this report we present our attempts to define a minimal HIV-1 packaging signal through the generation of test RNAs containing small blocks of HIV-1 sequences. We constructed RNAs differing in the position and identity of the HIV-1 sequence and the segments of heterologous sequences. However, none of the vectors were efficiently, encapsidated by wild-type HIV-1 virions. These results contrast those of Moloney murine leukemia virus and Rous sarcoma virus, where small viral segments mediate the efficient encapsidation of heterologous RNAs. The results suggest that the HIV-1 packaging signal may be extremely dispersed or heavily context-dependent.

Animals↗
PubMed1996

Increased transcomplementation properties of plasmids carrying HSV-1 origin of replication and packaging signals.

This report describes a very simple method that facilitates the analysis of the functional activity of essential genes of herpes simplex virus type 1 (HSV-1) in cell culture. The method, which depends on the use of complementing plasmids containing a virus origin of replication (ori s) and a packaging signal (amplicon plasmids), was tested for its ability to detect complementation of the defective HSV-1 Cgal delta 42 virus, a UL42 null mutant, by amplicon plasmids carrying either wild-type (pA-UL42) or mutated alleles (pA-delta UL42) of the HSV-1 UL42 gene. In nonpermissive Vero cells transfected with amplicon plasmid pA-UL42 and superinfected with the defective Cgal delta 42 virus, both the plasmid and the helper genomes were amplified and packaged, giving rise to a self-complementing amplicon/helper virus population able to disseminate and to form lytic plaques in cell culture. These plaques were due to complementation and not to recombination between the defective virus and the amplicon plasmid as confirmed by Southern blot analysis of individual plaques. No complementation was observed by superinfection of cells transfected with the noncomplementing pA-delta UL42 amplicon plasmid. Instead, small foci were observed in cells transfected with plasmids which express wild-type UL42 but were unable to amplify or to become packaged, and the few true lytic plaques that were observed in these systems resulted from the spread of competent recombinant viruses. Results presented in this work indicate that (i) self-complementation between a defective virus and an amplicon plasmid provides a strong and very sensitive method to assess function activity of an HSV-1 essential gene in a one-step experiment and (ii) oris-carrying plasmids could be instrumental in the production and selection of recombinant HSV-1 vectors.

Animals↗
PubMed1997

Identification of a specific interaction between the coronavirus mouse hepatitis virus A59 nucleocapsid protein and packaging signal.

The coronavirus mouse hepatitis virus (MHV) is an enveloped positive stranded RNA virus. In infected cells MHV produces a 3' coterminal nested set of subgenomic messenger RNAs. Only the genomic RNA, however, is encapsidated by the nucleocapsid protein and incorporated in infectious MHV virions. It is believed that an RNA packaging signal (Ps), present only in the genomic RNA, is responsible for this selectivity. Earlier studies mapped this signal to a 69-nt stem-loop structure positioned in the 3' end of ORF1b. The selective encapsidation mechanism probably initiates by specific interaction of the packaging signal with the nucleocapsid protein. In this study we demonstrate the in vitro interaction of the MHV-A59 nucleocapsid protein with the packaging signal of MHV using gel retardation and UV cross-linking assays. This interaction was observed not only with the nucleocapsid protein from infected cells but also with that from purified virions and from cells expressing a recombinant nucleocapsid protein. The specificity of the interaction was demonstrated by competition experiments with nonlabeled Ps containing RNAs, tRNA, and total cytoplasmic RNA. The results indicated that no virus specific modification of the N-protein or the presence of other viral proteins are required for this in vitro intervention. The assays described in this report provide us with a powerful tool for studying encapsidation (initiation) in more detail.

Animals↗
PubMed1998

Mutational analysis of stem-loops in the RNA packaging signal of the Moloney murine leukemia virus.

The retroviral RNA genome is targeted for incorporation into the nascent virion particle by the psi region, a specific block of RNA sequences near the 5' end. A number of deletions and linker insertion mutations were introduced into the psi region of cloned DNA of the Moloney murine leukemia virus, and the mutants were introduced into cells in culture and tested for their ability to direct the assembly of virions and the packaging of viral RNA. Only a small portion of the psi region was important for packaging, containing the so-called stem-loops C and D. Additional mutants were used to demonstrate that the base pairing of stem D, and the sequence of loop D, were essential for normal packaging of the RNA. Two mutants with alterations near the 5' splice donor were also replication-defective, probably due to effects on gene expression. The results allow a high-resolution definition of the RNA structures required during virus replication in culture.

3T3 Cells↗
PubMed1998

Regulated lentiviral packaging cell line devoid of most viral cis-acting sequences.

Packaging cell lines derived from human immunodeficiency virus-1 (HIV-1) are promising tools for in vivo somatic cell gene therapy protocols due to the ability of lentiviruses to infect nondividing cells. We describe here the generation of a safe, stable HIV-1 packaging cell line capable of expressing all of the HIV-1 structural, enzymatic, and regulatory proteins but lacking majority of the cis-acting sequences. The use of an inducible expression system circumvented the cytotoxic and cytostatic effects associated with the expression of some of the HIV-1 viral proteins. Reverse transcriptase activity was detectable in the supernatant from the stable packaging line 1 day after induction, while vector titers peaked 5 days postinduction. Vector titers of up to 3.5 x 10(4) infectious units/ml (IU/ml) were maintained through 8 months following the establishment of the cell line. Lineage-specific gene delivery can be achieved from this established cell line as viral stocks obtained specifically infect CD4(+) target cells. Moreover, this cell line provides a safe and easy to use system for screening of drugs that inhibit HIV-1 replication.

Gene Expression Regulation, Viral↗
PubMed1998

The herpes simplex virus 1 UL 17 gene is required for localization of capsids and major and minor capsid proteins to intranuclear sites where viral DNA is cleaved and packaged.

In nuclei of cells infected with herpes simplex virus (HSV), synthesized viral DNA accumulates as concatamers that are cleaved into genomic lengths and inserted into preformed capsids. Whereas newly replicated DNA and enzymes required for DNA synthesis accumulate in sites of infected cell nuclei termed replication compartments, the intranuclear site of DNA cleavage and packaging is currently controversial. DNA packaging requires the UL6, UL15, UL17, UL25, UL28, UL32, and UL33 genes in addition to the major capsid proteins. Using confocal immunofluorescence microscopy, it was observed that in > 95% of HEp-2 cells fixed at late times after infection with wild-type HSV-1, capsids, major capsid proteins ICP5 and ICP35, and the UL6-encoded minor capsid protein localized in DNA replication compartments. These data support the hypothesis that capsid assembly and DNA cleavage/packaging normally occur in HEp-2 cell replication compartments. In contrast, cells infected with a viral mutant lacking functional UL17 contained antigenically dense nuclear aggregates that stained with ICP35, ICP5, and capsid specific antibodies. Cells infected with the UL17 mutant virus also displayed UL6-specific fluorescence in a diffuse pattern at the nuclear periphery in regions not containing ICP35 and ICP5. Displacement of ICP35 from replication compartments was not observed in cells infected with cleavage/packaging mutants lacking UL28 and UL33. We conclude that the UL17 gene is required for correct targeting of capsids and major and minor capsid proteins to the DNA replication compartment of HEp-2 cells and deduce that this targeting reflects one functional role of UL17 in viral DNA cleavage and packaging.

Animals↗
PubMed2000

Molecular studies on bromovirus capsid protein. VII. Selective packaging on BMV RNA4 by specific N-terminal arginine residuals.

An arginine-rich RNA-binding motif (ARM) found at the N-proximal region of Brome mosaic virus (BMV) coat protein (CP) adopts alpha-helical conformation and shares homology with CPs of plant and insect RNA viruses, HIV-Rev and Tat proteins, bacterial antiterminators, and ribosomal splicing factors. The ARM of BMV CP, consisting of amino acids 9 through 21 with six arginine residues, is essential for RNA binding and subsequent packaging. In this study analysis of the alpha-helical contents of wild-type and mutant peptides by circular dichroism spectra identified protein determinants required for such conformation. Electrophoretic mobility-shift assays between viral RNA and BMV CP peptides with either proline or alanine substitutions revealed that the interaction is nonspecific. Expression in vivo of mature full-length BMV CP subunits, having the same substitutions for each arginine within the ARM, derived from biologically active clones was found to be competent to assemble into infectious virions and cause visible symptom phenotypes in whole plants. However, analysis of virion progeny RNA profiles of CP variants and subsequent in vitro reassembly assays between mutant CP and four BMV RNAs unveiled the ability of arginine residues at positions 10, 13, or 14 of the ARM to confer selective packaging of BMV RNA4. Thus, BMV CP contains determinants that specifically interact with RNA4 to ensure selective packaging.

Amino Acid Motifs↗
PubMed2001

An inducible packaging cell system for safe, efficient lentiviral vector production in the absence of HIV-1 accessory proteins.

Lentiviral vectors based on human immunodeficiency virus type 1 (HIV-1) possess the ability to deliver exogenous genes to both dividing and nondividing cells and to subsequently establish a stable provirus in these target cells, which can allow long-term expression of the transferred gene. Herein we describe a stable packaging cell line that is devoid of HIV-1 tat, vif, vpr, vpu, and nef. In order to avoid any risk of cytotoxicity associated with constitutive expression of HIV-1 protease or the VSV-G envelope protein, transcription of the packaging and envelope constructs was tightly controlled by employing the ecdysone-inducible system. Using this cell line, we have been able to consistently generate concentrated pseudotyped vector virus stocks with titers in the range of 10(8) IU/ml, which can efficiently transduce actively dividing and growth-arrested cells in vitro. This novel packaging cell line for lentiviral vectors facilitates the production of high-titer virus stocks in the absence of replication-competent virus and provides us with an important tool for use in future gene transfer studies.

Cell Line↗
PubMed2003

Analysis of RNA packaging in wild-type and mosaic protein capsids of flock house virus using recombinant baculovirus vectors.

Flock house virus (FHV) is a small icosahedral insect virus of the family Nodaviridae. Its genome consists of two positive-sense RNA molecules, RNA1 (replicase gene) and RNA2 (coat protein gene), which are encapsidated into a single virion. Expression of coat protein in Sf21 cells using a baculovirus vector results in formation of virus-like particles (VLPs) whose capsids are structurally indistinguishable from native virions. However, RNA packaging is not specific for RNA2, the coat protein message. Using ribonuclease protection assays, we showed that the fraction of RNA2 in VLPs is 19% relative to the amount present in a population of native virions. To investigate possible reasons for the reduced level of RNA2, we generated two new baculovirus vectors, AcR1delta and AcR2delta, expressing the replicase gene and the coat protein gene, respectively. The inserted genes carried the self-cleaving hepatitis delta ribozyme sequence at the 3' end to allow for synthesis of RNA1 and RNA2 transcripts with authentic 3' ends. Infection of Sf21 cells with AcR2delta yielded VLPs that contained 66% RNA2 relative to native virions. Coinfection of Sf21 cells with AcR1delta and AcR2delta launched self-directed FHV replication and resulted in formation of particles most of which contained RNA1 and RNA2. However, a small fraction of particles containing cellular RNA was detected as well. The latter particles could be eliminated by infecting Sf21 cells with AcR1delta followed by transfection with in vitro synthesized transcripts of RNA2. We have further utilized this system to show that two coat protein deletion mutants with distinct RNA packaging defects form mosaic virus capsids but do not complement each other to rescue specific packaging of FHV RNAs.

Animals↗