In vitro synthesis of infectious venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant.
TL;DR: A molecular clone of Venezuelan equine encephalitis virus (VEE) was constructed from four cDNAs that were synthesized using the viral RNA genome as template.
About: This article is published in Virology.The article was published on 1989-07-01. It has received 285 citations till now. The article focuses on the topics: Clone (cell biology) & Venezuelan equine encephalitis virus.
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TL;DR: This article corrects the article on p. 496 in vol.
1,986 citations
TL;DR: The VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.
531 citations
TL;DR: The cDNA clone of SFV was used to study what effects a deletion of the 6,000-molecular-weight membrane protein (6K membrane protein) had on virus replication, and conclusively show that the 6K protein is not needed for the heterodimerization of the p62 and E1 spike membrane proteins in the endoplasmic reticulum, nor is it needed for their transport out to the cell surface.
Abstract: We report on the construction of a full-length cDNA clone of Semliki Forest virus (SFV). By placing the cDNA under the SP6 promoter, infectious RNA can be produced in vitro and used to transfect cells to initiate virus infection. To achieve efficient transfections, a new protocol for electroporation of RNA was developed. This method gave up to 500-fold improvement over the traditional DEAE-dextran transfection procedure. Since virtually 100% of the cells can be transfected by electroporation, this method is a useful tool for detailed biochemical studies of null mutations of SFV that abolish production of infections virus particles. We used the cDNA clone of SFV to study what effects a deletion of the 6,000-molecular-weight membrane protein (6K membrane protein) had on virus replication. The small 6K protein is part of the structural precursor molecule (C-p62-6K-E1) of the virus. Our results conclusively show that the 6K protein is not needed for the heterodimerization of the p62 and E1 spike membrane proteins in the endoplasmic reticulum, nor is it needed for their transport out to the cell surface. The absence of the 6K protein did, however, result in a dramatic reduction in virus release, suggesting that the protein exerts its function late in the assembly pathway, possibly during virus budding.
474 citations
TL;DR: This review addresses important issues of the current status of viral vector design and discusses their key features as delivery systems in gene therapy of human inherited and acquired diseases at the level of laboratory developments and of clinical applications.
Abstract: The efficient delivery of therapeutic genes and appropriate gene expression are the crucial issues for clinically relevant gene therapy. Viruses are naturally evolved vehicles which efficiently transfer their genes into host cells. This ability made them desirable for engineering virus vector systems for the delivery of therapeutic genes. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses, adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Among these vector systems, retrovirus vectors represent the most prominent delivery system, since these vectors have high gene transfer efficiency and mediate high expression of therapeutic genes. Members of the DNA virus family such as adenovirus-, adeno-associated virus or herpesvirus have also become attractive for efficient gene delivery as reflected by the fast growing number of clinical trials using these vectors. The first clinical trials were designed to test the feasibility and safety of viral vectors. Numerous viral vector systems have been developed for ex vivo and in vivo applications. More recently, increasing efforts have been made to improve infectivity, viral targeting, cell type specific expression and the duration of expression. These features are essential for higher efficacy and safety of RNA- and DNA-virus vectors. From the beginning of development and utilisation of viral vectors it was apparent that they harbour risks such as toxicities, immunoresponses towards viral antigens or potential viral recombination, which limit their clinical use. However, many achievements have been made in vector safety, the retargeting of virus vectors and improving the expression properties by refining vector design and virus production. This review addresses important issues of the current status of viral vector design and discusses their key features as delivery systems in gene therapy of human inherited and acquired diseases at the level of laboratory developments and of clinical applications.
465 citations
TL;DR: The construction of a conditional lethal helper system has now largely overcome the problem of biosafety, and should further increase the utility of these types of vector in animal cell systems.
380 citations
References
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TL;DR: The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine, which is applied to mutations introduced via both oligonucleotides and error-prone polymerization.
Abstract: Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.
8,474 citations
TL;DR: Using the fully sequenced 1300 nucleotide-long bovine preproenkephalin mRNA, it is established by sequencing that the method yields faithful full-length transcripts.
4,214 citations
TL;DR: In this article, a dynamic programming algorithm was proposed to fold an RNA molecule that finds a conformation of minimum free energy using published values of stacking and destabilizing energies, based on applied mathematics.
Abstract: This paper presents a new computer method for folding an RNA molecule that finds a conformation of minimum free energy using published values of stacking and destabilizing energies. It is based on a dynamic programming algorithm from applied mathematics, and is much more efficient, faster, and can fold larger molecules than procedures which have appeared up to now in the biological literature. Its power is demonstrated in the folding of a 459 nucleotide immunoglobulin gamma 1 heavy chain messenger RNA fragment. We go beyond the basic method to show how to incorporate additional information into the algorithm. This includes data on chemical reactivity and enzyme susceptibility. We illustrate this with the folding of two large fragments from the 16S ribosomal RNA of Escherichia coli.
3,202 citations
TL;DR: A simple and rapid method for preparing plasmids for restriction enzyme analysis has been developed and can be readily adapted for the preparation of plasmid from liter cultures with quantitative yields.
3,110 citations
TL;DR: In the chapter, the IG, which has the potential to form five hairpin structures, is represented schematically and important regions designated and the origin of replication of the (+) strand is stated most important to the functioning of M 13KO7.
Abstract: Publisher Summary This chapter discusses the production of single-stranded plasmid DNA. The chapter focuses on M13KO7 and its uses. The chapter reviews M13 biology; M13 mutants play a vital role in the functioning of M13KO7. M13 is a phage that contains a circular single-stranded DNA (ssDNA) molecule of 6407 bases packaged in a filamentous virion, which is extruded from the cell without lysis. It can infect only cells having F-pili to which it binds for entering the cell. The phage genome consists of nine genes encoding 10 proteins and contains an intergenic region of 508 bases. Phage replication consists of three phases: (1) ss-double strand (ds), (2) ds-ds, and (3) ds-ss. The intergenic region (IG) structure contains regions important for four phage processes: (1) the sequences necessary for the recognition of an ssDNA by phage proteins for its efficient packaging into viral particles; (2) the site of synthesis of an RNA primer that is used to initiate strand synthesis; (3) the initiation; and (4) the termination of (+) strand synthesis. In the chapter, the IG, which has the potential to form five hairpin structures, is represented schematically and important regions designated. The origin of replication of the (+) strand is stated most important to the functioning of M 13KO7.
2,491 citations