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PubMed2003

In vitro and in ovo expression of chicken gamma interferon by a defective RNA of avian coronavirus infectious bronchitis virus.

Coronavirus defective RNAs (D-RNAs) have been used for site-directed mutagenesis of coronavirus genomes and for expression of heterologous genes. D-RNA CD-61 derived from the avian coronavirus infectious bronchitis virus (IBV) was used as an RNA vector for the expression of chicken gamma interferon (chIFN-gamma). D-RNAs expressing chIFN-gamma were shown to be capable of rescue, replication, and packaging into virions in a helper virus-dependent system following electroporation of in vitro-derived T7 RNA transcripts into IBV-infected cells. Secreted chIFN-gamma, under the control of an IBV transcription-associated sequence derived from gene 5 of the Beaudette strain, was expressed from two different positions within CD-61 and shown to be biologically active. In addition, following infection of 10-day-old chicken embryos with IBV containing D-RNAs expressing chIFN-gamma, the allantoic fluid was shown to contain biologically active chIFN-gamma, demonstrating that IBV D-RNAs can express heterologous genes in vivo.

Animals↗
PubMed1981

Inhibition of T7 development at high concentrations of the phage.

Escherichia coli B exposed to high doses of bacteriophage T7 did not lyse. A similar effect was observed when the high dose was added within the first 7 min after primary infection. No viable phage was formed. DNA synthesis was inhibited rapidly and the nucleoid structure was absent. Protein synthesis was in general markedly reduced and so were the activities of the phage-specific enzymes endolysin and DNA polymerase. However, phage genes were transcribed both by the host RNA polymerase and by the phage-specific enzyme. We suggest that inhibition of phage development is due to structural alterations occurring in the cell wall/membrane such that replication is inhibited.

Bacteriolysis↗
PubMed1996

RNA replication by a respiratory syncytial virus RNA analog does not obey the rule of six and retains a nonviral trinucleotide extension at the leader end.

Genome analogs ("minigenomes") of Sendai and measles viruses replicate efficiently only if their nucleotide length is an even multiple of six, a requirement called the rule of six (P. Calain and L. Roux, J. Virol. 67:4822-4830, 1993; M. S. Sidhu, J. Chan, K. Kaelin, P. Spielhofer, F. Radecke, H. Schneider, M. Masurekar, P. C. Dowling, M. A. Billeter, and S. A. Udem, Virology 208:800-807, 1995). The existence of a comparable requirement was tested for respiratory syncytial virus (RSV), which also is a member of family Paramyxoviridae and whose natural genome length also is a multiple of six. An internally truncated analog of RSV positive-sense replicative intermediate RNA (antigenome) bearing the chloramphenicol acetyltransferase gene as a reporter was synthesized from cDNA in vitro. This RNA was transfected into cells which were infected with RSV as a helper. Miniantigenomic RNA was indistinguishable from previously studied negative-sense minigenome RNA in its ability to participate in transcription, RNA replication, and incorporation into transmissible particles. Sixteen miniantigenomes which were of slightly different lengths and which in aggregate represented multiples of a wide range of integers including 1 to 15 were constructed. During transfection and two serial passages, the various miniantigenomes were essentially indistinguishable with regard to the efficiency of transcription, RNA replication, and packaging into transmissible particles. Progeny minigenomes of six different mutants were recovered postpassage, copied into cDNA, cloned, and sequenced completely. The length of each of these RNAs was found to have remained unchanged during replication and passage. Thus, RSV transcription and replication appear to lack the requirement that the template length be an even multiple of an integer such as six, which for Sendai and measles viruses is obligatory for nucleocapsid function. Each of the in vitro-synthesized miniantigenomes used in transfection contained a nonviral extension of three nucleotides, GGG, on the 5' (leader) end contributed by the T7 promoter. The termini of the recovered minigenomes were examined for five mutants by RNA circularization followed by cDNA synthesis, amplification, cloning, and sequencing. Unexpectedly, each recovered minigenome contained the complement of this nonviral extension on the 3' (leader) end, showing that it had been faithfully copied and maintained during RNA replication and passage. The nonviral trinucleotide did not appear to affect the activity of the template.

DNA, Complementary↗
PubMed1997

Ribonuclease H renaturation gel assay using a fluorescent-labeled substrate.

Ribonucleases H (RNases H) are enzymes that specifically degrade the RNA of RNA-DNA hybrids. These enzymes are involved in DNA replication, reverse transcription (RT) and antisense oligodeoxyribonucleotide-mediated arrest of translation. One of the most valuable tools for assaying RNase H activity is the renaturation gel assay with which such activities can be detected using purified protein preparations or crude extracts. Radioactive substrates [32P labeled poly(rA)-poly(dT) hybrid] are commonly used with exposure of the gel to X-ray film; this is possible at any time without disturbing the renaturation-degradation process. Here, we describe a method using fluorescent-labeled substrates. RNA-DNA substrates are synthesized by first transcribing DNA with T7 RNA polymerase using Bodipy-TR-14-UTP and the four normal nucleoside triphosphates. The run-off transcript is annealed to a short oligomeric DNA complementary to the 3'-end of the transcript, and the DNA portion of the hybrid is formed by RT. This RNA-DNA is added to the polyacrylamide mixture before polymerization, and SDS-PAGE is performed as usual. After various periods of renaturation, the gel is scanned to detect fluorescent substrate using the red-excited laser of a fluorescence scanner. This fluorescence method has all of the advantages of using radio-labeled substrates and none of its disadvantages, and the sensitivities of the two methods are comparable. In addition, we show that the sensitivity of this procedure can be increased if damaging chemicals remaining in the gel after polymerization are eliminated by simultaneous electrophoresis of the RNase H and a protein with higher mobility.

DNA↗
PubMed2004

Interferon alpha-2b inhibits negative-strand RNA and protein expression from full-length HCV1a infectious clone.

We have established a T7-based model system for hepatitis C virus (HCV) 1a strain, which involves the use of a replication-defective adenovirus that carries the gene for T7 RNA polymerase and a transcription plasmid containing full-length HCV cDNA clone. To facilitate high-level expression of HCV, sub-confluent Huh7 cells were first infected with adenovirus containing the gene for the T7 RNA polymerase and then transfected with the transcription plasmid. As a negative control, part of NS5B gene of this clone was deleted which abolishes the HCV RNA-dependent RNA polymerase and prevents replication of viral RNA. This model produces high levels of structural (core, E1, E2) and nonstructural proteins (NS5), which were detected by Western blot analysis and immunofluorescence assay. Negative-strand HCV RNA was detected only in the wild-type clone in the presence of actinomycin D, and no RNA was detected with the NS5B deleted mutant control. As a practical validation of this model, we showed that IFN alpha-2b selectively inhibits negative-strand RNA synthesis by blocking at the level of protein translation. The inhibitory effect of IFN alpha-2b is not due reduction of transcription by T7 polymerase or due to intracellular degradation of HCV RNA. This in vitro model provides an efficient and reliable means of assaying negative-strand RNA, protein processing, and testing the antiviral properties of interferon.

Animals↗
PubMed1989

In vitro construction of poliovirus defective interfering particles.

To construct poliovirus defective interfering (DI) particles in vitro, we synthesized an RNA from a cloned poliovirus cDNA, pSM1(T7)1, which carried a deletion in the genome region corresponding to nucleotide positions 1663 to 2478 encoding viral capsid proteins, by using bacteriophage T7 RNA polymerase. The RNA was designed to retain the correct reading frame in nucleotide sequence downstream of the deletion. HeLa S3 monolayer cells were transfected with the deletion RNA and then superinfected with standard virus as a helper. The DI RNA was observed in the infected cells after three passages at high multiplicity of infection. The sequence analysis of RNA extracted from the purified DI particle clearly showed that this DI RNA had the same deletion in size and location as that in the RNA used for the transfection. Thus, we succeeded in construction of a poliovirus DI particle in vitro. To gain insight into the mechanism for DI generation, we constructed poliovirus cDNAs pSM1(T7)1a and pSM1(T7)1b that, in addition to the same deletion as that in pSM1(T7)1, had insertion sequences of 4 bases and 12 bases, respectively, at the corresponding nucleotide position, 2978. The RNA transcribed from pSM1(T7)1a was not a template for synthesis of poliovirus nonstructural proteins and therefore was inactive as an RNA replicon. On the other hand, the RNA from pSM1(T7)1b replicated properly in the transfected cells. Superinfection of the transfected cells with standard virus resulted in production of DI particles derived from pSM1(T7)1b and not from pSM1(T7)1a. These observations indicate that deletion RNAs that are inactive replicons have little or no possibility of being genomes of DI particles suggesting the existence of a nonstructural protein(s) that has an inclination to function as a cis-acting protein(s). The method described here will provide a useful technique to investigate genetic information essential for poliovirus replication.

Amino Acid Sequence↗
PubMed1992

A versatile shuttle cosmid vector for use in Escherichia coli and actinomycetes.

A shuttle cosmid vector has been constructed for Escherichia coli and actinomycetes. This vector, pFD666, utilizes the origin of replication (ori) of the broad-host-range plasmid, pJV1, from Streptomyces phaeochromogenes, for replication in actinomycetes and is compatible with vectors derived from pIJ101. The pFD666 vector employs the neomycin phosphotransferase-encoding gene (neo) from transposon Tn5 as the selective marker. To achieve this, the native promoter of neo was replaced by one optimized for expression in both hosts. The polylinker used for cloning has nine unique sites flanked by the promoters for T7 and SP6 RNA polymerase for the production of specific RNA probes. Terminators on both sides of the polylinker protect the vector from transcription originating from cloned inserts. An M13 ori allows the production of single-stranded DNA.

Actinomycetales↗
PubMed2003

Genome of Xanthomonas oryzae bacteriophage Xp10: an odd T-odd phage.

Xp10 is a lytic bacteriophage of the phytopathogenic bacterium Xanthomonas oryzae. Though morphologically Xp10 belongs to the Syphoviridae family, it encodes its own single-subunit RNA polymerase characteristic of T7-like phages of the Podoviridae family. Here, we report the determination and analysis of the 44,373 bp sequence of the Xp10 genome. The genome is a linear, double-stranded DNA molecule with 3' cohesive overhangs and no terminal repeats or redundancies. Half of the Xp10 genome contains genes coding for structural proteins and host lysis functions in an arrangement typical for temperate dairy phages that are related to the Escherichia coli lambda phage. The other half of the Xp10 genome contains genes coding for factors of host gene expression shut-off, enzymes of viral genome replication and expression. The two groups of genes are transcribed divergently and separated by a regulatory region, which contains divergent promoters recognized by the host RNA polymerase. Xp10 has apparently arisen through a recombination between genomes of widely different phages. Further evidence of extensive gene flux in the evolution of Xp10 includes a high fraction (10%) of genes derived from an HNH-family endonuclease, and a DNA-dependent DNA polymerase that is closer to a homolog from Leishmania than to DNA polymerases from other phages or bacteria.

Amino Acid Sequence↗
PubMed1976

Genetic recombination of bacteriophage T7 DNA in vitro.

We have demonstrated recombination of bacteriophage T7 DNA in vitro. An extract of Escherichia coli B cells infected with wild-type T7(T7+) is incubated with mature DNA extracted from T7 phage. Packaging of the exogenous DNA within the phage head appears to be preceded by recombination of exogenously added DNA with DNA present in the extracts. In order to detect the recombination, we used an exogenous DNA bearing a marker (ss-) such that progeny phage which have packaged this marker are able to plate on Shigella sonnei D2 571-48, whereas T7+ phage present in the extracts do not. The recombinational process bears many of the characteristics of in vivo recombination. The exogenous DNA is not packaged intact but undergoes fragmentation to a length of about 3000 base pairs before being incorporated into a mature DNA molecule. If ss- DNA bearing an amber mutation is used in the assay, the frequency of amber+ progeny produced varies with the distance of the amber marker from the ss- marker. When DNA bearing three mutations is used in the reaction, phage heterozygous for the unselected marker are readily detected. Finally the products of phage genes 4 (DNA replication protein), 5(DNA polymerase), and 6(exonuclease), genes previously implicated in recombination in vivo, are required for the in vitro reaction.

Coliphages↗
PubMed1995

O6-methylguanine-induced replication blocks.

The ability of Klenow polymerase I, phage T7 polymerase (Sequenase), human polymerase alpha, and human polymerase beta to synthesize past (bypass) O6-methylguanine (O6-meG) lesions was studied in the presence of MgCl2 and MnCl2. An end-labeled 16-mer primer was annealed to the 3' end of gel-purified oligodeoxyribonucleotide templates (45-mers), each containing a single O6-meG in place of one G in the sequence -G1G2CG3G4T-. Extension products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. A fraction of the products extended by Klenow fragment terminated either opposite or one base before O6-meG located at sites 1 and 3. Termination occurred primarily one base before O6-meG located at sites 2 and 4. The remaining fractions that bypassed the lesions represented full-length product. In control reactions, the O6-meG-containing templates were annealed with complementary 45-mers, repaired with O6-alkylguanine DNA-alkyltransferase, annealed with an excess of labeled primer, and extended by Klenow fragment. Full-length extension of > 90% was observed with each template. Primer extension past O6-meG by DNA polymerase alpha and Sequenase was partially blocked in a manner which varied with the site of O6-meG in the template while primer extension by DNA polymerase beta was completely blocked (< 2% full length extension) with O6-meG at sites 1-4. Substitution of MnCl2 for MgCl2 in the reaction mixture greatly increased the bypass of O6-meG by Klenow fragment and DNA polymerase alpha but not Sequenase or DNA polymerase beta. The increased ability of Klenow fragment to bypass O6-meG in the presence of MnCl2 was found to result from an increased incorporation of G (O6-meG at sites 1 and 2) and A (O6-meG at sites 1, 2, and 3) opposite the lesion. The results indicate that O6-meG can block in vitro polymerization by several DNA polymerases and are consistent with the observed cytotoxic effects of methylating agents on mammalian cells.

Base Sequence↗
PubMed1975

Biological functions of the bacteriophage T3 SAMase gene.

Certain differences between phage T3 on the one hand and T3sam- and T7 on the other hand indicate that the T3-coded SAMase function is responsible (i) for the development of the pseudolysogenic state by preventing T3 DNA methylation, and (ii) for the partial protection of the phage DNA against restriction by the P system.

Coliphages↗
PubMed1992

Cellular expression of a functional nodavirus RNA replicon from vaccinia virus vectors.

RNA replication provides a powerful means for the amplification of RNA, but to date it has been found to occur naturally only among RNA viruses. In an attempt to harness this process for the amplification of heterologous mRNAs, both an RNA replicase and its corresponding RNA templates have been expressed in functional form, using vaccinia virus-bacteriophage T7 RNA polymerase vectors. Plasmids were constructed which contained in 5'-to-3' order (i) a bacteriophage T7 promoter; (ii) a full-length cDNA encoding either the RNA replicase (RNA 1) or the coat protein (RNA 2) of flock house virus (FHV), (iii) a cDNA sequence that encoded the self-cleaving ribozyme of satellite tobacco ringspot virus, and (iv) a T7 transcriptional terminator. Both in vitro and in vivo, circular plasmids of this structure were transcribed by T7 RNA polymerase to produce RNAs with sizes that closely resembled those of the two authentic FHV genomic RNAs, RNA 1 and RNA 2. In baby hamster kidney cells that expressed authentic FHV RNA replicase, the RNA 2 (coat protein) transcripts were accurately replicated. Moreover, the RNA 1 (replicase) transcripts directed the synthesis of an enzyme that could replicate not only authentic virion-derived FHV RNA but also the plasmid-derived transcripts themselves. Under the latter conditions, replicative amplification of the RNA transcripts ensued and resulted in a high rate of synthesis of the encoded proteins. This successful expression from a DNA vector of the complex biological process of RNA replication will greatly facilitate studies of its mechanism and is a major step towards the goal of harnessing RNA replication for mRNA amplification.

Animals↗
PubMed1979

The purification and properties of a double-stranded DNA-binding protein encoded by the gene D5 of bacteriophage T5.

We have purified a DNA-binding protein from bacteriophage T5-infected cells. The protein is the product of the T5 gene D5 and is produced in quantities ultimately exceeding 2% of the total cell protein. The protein has no tendency to aggregate and exists in solution as a monomer of about 29,000 daltons. Although the protein binds with a high affinity to both single-stranded (fd, T7) and duplex DNA (T5, T7), there is a preferred binding to duplex DNA. The binding of the gene D5 protein to duplex DNA is cooperative with an estimated dissociation constant of 6.27 x 10(-10) M and leads to the production of a more condensed form of the DNA. In contrast, binding of the gene D5 protein to single-stranded DNA is noncooperative with an estimated dissociation constant of only 1.85 x 10(-8) M and produces a sedimentation shift in the DNA proportional to the added mass of the protein. The protein product of the gene D5 of bacteriophage T5 is known to be required for both T5 DNA replication and for the inhibition of early phage gene expression. We also show here that the protein is required to initiate transcription of the late region of the T5 genome.

Amino Acids↗
PubMed2000

Improved technique for transient expression and negative strand virus rescue using fowlpox T7 recombinant virus in mammalian cells.

The suitability of recombinant T7 polymerase produced using either the highly attenuated MVA strain of vaccinia (MVA-T7) or fowlpox virus (FP-T7) for transient expression and negative strand virus rescue was compared in two mammalian cell lines (MDBK and Vero) and in primary cells of bovine, ovine and caprine origin. Such primary cells are more permissive for the growth of wild type strains of morbilliviruses, such as Rinderpest virus and Peste des petits ruminants virus. MVA-T7 was found to be highly cytopathic in the primary cells, multiplying rapidly and killing the cells within 3-5 days of infection, even when very low multiplicities of infection (MOI) were used. In contrast, FP-T7, which appeared to express similar amounts of T7 polymerase, was found to be non-cytopathic in a variety of primary and established cell lines of mammalian origin and was suitable for use in virus rescue experiments. MDBK cells and primary cells, unlike Vero cells, could not be efficiently transfected and so were unsuitable for virus rescue. Optimal conditions for rinderpest virus rescue in Vero cells were established using FP-T7 in place of MVA-T7. This system will be suitable for rescuing other viruses which grow in Vero cells.

Animals↗
PubMed2004

RNA displacement and resolution of the transcription bubble during transcription by T7 RNA polymerase.

Unlike DNA polymerases, RNA polymerases (RNAPs) must displace the nascent product from the template and restore the DNA to duplex form after passage of the transcription complex. To accomplish this, RNAPs establish a locally denatured "bubble" that encloses a short RNA:DNA hybrid. As the polymerase advances along the template, the RNA is displaced at the trailing edge of the bubble and the two DNA strands are reannealed. Structural analyses have revealed a number of elements that are likely to be involved in this process in T7 RNAP. In this work, we used genetic and biochemical methods to explore the roles of these elements during the transition from an initiation complex to an elongation complex. The results indicate that the transition is a multistep process and reveal a critical role for the nontemplate strand of the DNA.

Base Sequence↗
PubMed2001

The X philes: structure-specific endonucleases that resolve Holliday junctions.

Genetic recombination is a critical cellular process that promotes evolutionary diversity, facilitates DNA repair and underpins genome duplication. It entails the reciprocal exchange of single strands between homologous DNA duplexes to form a four-way branched intermediate commonly referred to as the Holliday junction. DNA molecules interlinked in this way have to be separated in order to allow normal chromosome transmission at cell division. This resolution reaction is mediated by structure-specific endonucleases that catalyse dual-strand incision across the point of strand cross-over. Holliday junctions can also arise at stalled replication forks by reversing the direction of fork progression and annealing of nascent strands. Resolution of junctions in this instance generates a DNA break and thus serves to initiate rather than terminate recombination. Junction resolvases are generally small, homodimeric endonucleases with a high specificity for branched DNA. They use a metal-binding pocket to co-ordinate an activated water molecule for phosphodiester bond hydrolysis. In addition, most junction endonucleases modulate the structure of the junction upon binding, and some display a preference for cleavage at specific nucleotide target sequences. Holliday junction resolvases with distinct properties have been characterized from bacteriophages (T4 endo VII, T7 endo I, RusA and Rap), Bacteria (RuvC), Archaea (Hjc and Hje), yeast (CCE1) and poxviruses (A22R). Recent studies have brought about a reappraisal of the origins of junction-specific endonucleases with the discovery that RuvC, CCE1 and A22R share a common catalytic core.

Archaea↗
PubMed1975

The sex-factor-dependent exclusion of coli virus T7.

The cause of T7 exclusion by the F episome was investigated. Extracts from neither normal nor infected F+ cells contained an inhibitor of gene expression in vitro. The protein synthesizing systems prepared in vitro from these cells supported T7 early and late protein synthesis with normal efficiency. The content of translational initiation factors in F- and F+ cells, both noninfected and infected, was almost identical. The episome-dependent block of T7 gene expression was observed only in intact cells and detailed kinetics of gene expression in vivo revealed a stop of all transcription and translation at or just before 11 min after T7 infection. The mechanism of F+-dependent T7 exclusion involves both episomal and viral gene products. The data indicate that a T7-induced membrane alteration of the F+ cell membrane leads to cessation of T7 development as well as to the death of the host cell ('suicide').

Cell Survival↗
PubMed2025

Establishment of reverse genetics systems for Colorado tick fever virus.

The Colorado tick fever virus (CTFV), which has 12-segmented double-stranded RNA genomes, is a pathogenic arbovirus that causes severe diseases in humans. However, little progress has been made in the analysis of replication mechanisms and pathogenicity. This virological constraint is due to the absence of a reverse genetics system for CTFV; therefore, we aimed to establish the system. Initially, the efficacy of CTFV replication was investigated in various cell lines. CTFV was found to grow in many cell types derived from different hosts and organs. Subsequently, BHK-T7 cells stably expressing T7 RNA polymerase were transfected with plasmids encoding each of the 12 CTFV gene segments, expression plasmids encoding all CTFV proteins, and a vaccinia virus RNA-capping enzyme. Following transfection, the cells were co-cultured with Vero or HeLa cells. Using this system, we rescued monoreassortants and recombinant viruses harboring peptide-tagged viral proteins. Furthermore, an improved system using Expi293F cells expressing T7 RNA polymerase was established, which enabled the generation of recombinant reporter CTFVs. In conclusion, these reverse genetics systems for CTFV will greatly contribute to the understanding of viral replication mechanisms, pathogenesis, and transmission, ultimately facilitating the development of rational treatments and candidate vaccines.

Animals↗