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At least 703 records · Page 39Linked to original sources
PubMed2003

Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids.

La Crosse virus (LACV), a member of the family Bunyaviridae, is the primary cause of paediatric encephalitis in the United States. In this study, a functional RNA polymerase (L) gene of LACV was cloned and a reverse genetics system established. A reporter minireplicon mimicking the viral genome was constructed by flanking the Renilla luciferase gene with the 3' and 5' noncoding regions of the genomic M segment. These noncoding regions serve as promoters for the viral polymerase. Both L and nucleocapsid (N) genes were expressed by means of T7 RNA polymerase, which was provided by the recombinant T7-expressing modified vaccinia virus Ankara. Renilla reporter activity in transfected cells reflected reconstitution of recombinant nucleocapsids by functional L and N gene products. Time-course experiments revealed a rapid increase in minireplicon activity from 10 to 18 h after the onset of L and N expression. Minireplicon activity was found to be dependent on the correct ratio of L to N plasmids, with too much of either construct resulting in downregulation. Furthermore, a specific inhibitory effect of LACV NSs protein on minireplicon activity was found. In passaging experiments using parental helper virions, it was demonstrated that the recombinant nucleocapsids are a useful model for transcription, replication and packaging of LACV.

Animals↗
PubMed2000

Evidence that the 5'-end cap structure is essential for encapsidation of hepatitis B virus pregenomic RNA.

Hepatitis B virus (HBV) replicates by reverse transcription of an RNA intermediate, the pregenomic RNA. The first step of HBV genome replication is the encapsidation of the pregenomic RNA encoding the encapsidation signal, termed epsilon, into the core particles, which is preceded by recognition and binding of HBV DNA polymerase to epsilon. The pregenomic RNA contains two identical epsilon elements due to its terminal redundancy: one near the 5' end and another near the 3' end. Despite the fact that both epsilon elements have an identical sequence, only the 5' epsilon, but not the 3' epsilon, is functional for encapsidation. To understand the molecular nature of this position effect, we made a series of lacZ RNA expression plasmids which contain the epsilon element at various positions from the 5' end of the transcripts. Following transfection, the lacZ RNAs in cytoplasmic core particles were measured by RNase protection assay for encapsidation. The results indicated that the lacZ RNAs with epsilon positioned up to 65 nucleotides from the 5' end were encapsidated, whereas the lacZ RNAs with epsilon positioned further downstream were not. Interestingly, the cap-free lacZ RNA transcribed by T7 RNA polymerase was not encapsidated, implying that the 5' cap structure is required for encapsidation of the pregenomic RNA. We hypothesized that HBV DNA polymerase must somehow recognize the cap structure and/or its associated factors, as well as the 5' epsilon, for encapsidation to occur.

Base Sequence↗
PubMed1993

Production of infectious in vitro transcripts from a full-length clover yellow mosaic virus cDNA clone.

A full-length cDNA copy of clover yellow mosaic virus (CYMV) RNA was constructed from two smaller cDNA clones. In vitro transcription of linearized plasmid with T7 RNA polymerase produced genomic-sized RNA. These transcripts caused symptoms typical of CYMV infection when used to inoculate both a systemic host (Vicia faba) and a local lesion host (Gomphrena globosa). Electron microscopy of extracts from individual local lesions revealed virus particles identical to native CYMV. Increasing the length of the poly(A) tail from 23 residues to 80 or to 135 residues increased the infectivity rate from 12% to 17% or to 35% that of native CYMV RNA, respectively.

Base Sequence↗
PubMed1976

Effects of the phage P1 restriction system on coliphage phi W: degradation and complex formation of phage phi W DNA.

Growth of phages phi W and T7 was restricted in Escherichia coli lysogenic for phage P1. Only a fraction of the infected cells gave burst of phages. Cells permitting phage growth gave normal burst size. Host strains carrying P1 mutants with defective endonuclease gave no restriction of phages T7 and phi 3, the latter a host-range mutant of phi W. Degradation but not modification of parental phage DNA could be demonstrated. Although no DNA, RNA or protein was synthesized in phi W infected P1 lysogenic cells, the parental phage DNA was found in increasingly larger complexes during the course of infection. At early times after infection, parental phage DNA was found to sediment about twice as fast as mature phage DNA. At later times during the infection the parental phage DNA was recovered as a very rapidly sedimenting material. Such material was also found in alkaline sucrose gradient centrifugation after treatment of the cell extract with sodium dodecyl sulphate, pronase digestion and phenol extractions.

Coliphages↗
PubMed1995

Synthesis of a full-length infectious cDNA clone of cucurbit aphid-borne yellows virus and its use in gene exchange experiments with structural proteins from other luteoviruses.

A full-length cDNA of cucurbit aphid-borne yellows virus (CABYV) has been constructed and expressed either as an in vitro transcript, under control of a bacteriophage T7 RNA polymerase promoter, or in vivo, under control of the cauliflower mosaic virus 35S promoter in an agroinfection vector. The biological activity of the cloned cDNA was demonstrated by the ability of its in vitro transcript to replicate in protoplasts and of the agroinfection vector to infect agroinoculated plants. Virus in the agroinfected plants cold be transmitted by the aphid vectors Myzus persicae and Aphis gossypii. The specificity of luteovirus RNA packaging was investigated by replacing (1) the CABYV coat protein gene (and the overlapping ORF5) by the corresponding region of potato leafroll luteovirus or (2) the CABYV readthrough domain by the readthrough domain of beet western yellows luteovirus. The resulting chimeric transcripts replicated in protoplasts and produced virions.

Animals↗
PubMed1994

Translesional synthesis on DNA templates containing site-specifically placed deoxyadenosine and deoxyguanosine adducts formed by the plant carcinogen aristolochic acid.

Synthetic oligonucleotides (18-mers) containing either a single deoxyadenosine residue or a single deoxyguanosine residue were treated with aristolochic acid I (AAI) or aristolochic acid II (AAII), the main components of the plant carcinogen aristolochic acid (AA). These reactions resulted in the formation of site-specifically adducted oligonucleotides containing the two known AAI-DNA adducts (dA-AAI, dG-AAI) or the two known AAII-DNA adducts (dA-AAII, dG-AAII) at position 15 from the 3' end. Using HPLC chromatography, the oligonucleotides were purified and subsequently shown to contain the adducts of interest by 32P-postlabelling. The adducted oligonucleotides were used as templates in primer (11-mer) extension reactions catalysed by modified bacteriophage T7 DNA polymerase (Sequenase). Regardless of the type of DNA adduct examined, DNA synthesis was blocked predominantly (80-90%) at the nucleotide 3' to each adduct, although primer extension to the full length of the template was noted with unmodified control templates. However, 15 nucleotide products, indicating blocking of DNA synthesis after incorporation of a nucleotide opposite the adduct and translesional synthesis products were formed in all cases in different amounts, depending on the adduct structure. When a 14-mer primer together with high dNTP concentrations was used to examine nucleotide incorporation directly across from the four different purine adducts we found that the deoxyadenosine adducts (dA-AAI and dA-AAII) allowed incorporation of dAMP and dTMP equally well, whereas the deoxyguanosine adducts (dG-AAI and dG-AAII) allowed preferential incorporation of dCMP. Molecular dynamic simulations showed that the aristolactam moiety of all adducts exhibit a strong stacking, with the adenine residue at the 3' end of the 14-mer primer. These studies demonstrate that all AA purine adducts provide severe blocks to DNA replication and that the guanine adducts may not be very efficient mutagenic lesions. In contrast, the translesional bypass past adenine adducts of the aristolochic acids suggests a mutagenic potential resulting from dAMP incorporation by polymerase. AT-->TA transversion mutations would be the mutagenic consequences of AA adenine adducts, which are consistent with the activating mutations of c-ras genes found in AA-induced tumours of rodents.

Aristolochic Acids↗
PubMed2006

mRNA expression, functional profiling and multivariate classification of colon biopsy specimen by cDNA overall glass microarray.

AIM: To understand the local pathophysiological alterations and gene ontology-based functional classification of colonic biopsies into inflammatory and neoplastic diseases. METHODS: Total RNA was extracted from frozen biopsies and amplified by T7-method. Expression profile was evaluated by Atlas Glass 1K microarrays. After microarray quality control, applicable data were available from 10 adenomas, 6 colorectal adenocarcinomas (CRCs), and 6 inflammatory bowel diseases (IBDs). Multivariate statistical and cell functional analyses were performed. Real-time RT-PCR and immunohistochemistry were used for validation. RESULTS: Discriminant analysis of selected genes, could correctly reclassify all 22 samples using 4 parameters (heat shock transcription factor-1, bystin-like, calgranulin-A, TRAIL receptor 3). IBD samples were characterized by overregulated chemokine (C-X-C motif) ligand 13, replication protein A1, E74-like factor 2 and downregulated TNF receptor-associated factor 6, BCL2-interacting killer genes. In adenomas upregulation of TNF receptor-associated factor 6, replication protein A1, E74-like factor 2 and underexpression of BCL2-associated X protein, calgranulin-A genes were found. CRC cases had significantly increased epidermal growth factor receptor, topoisomerase-1, v-jun, TNF receptor-associated factor 6 and TRAIL receptor 3, and decreased RAD51 and RAD52 DNA repair gene, protein phosphatase-2A and BCL2-interacting killer mRNA levels. Epidermal growth factor receptor RT-PCR and immunohistochemistry, topoisomerase-1 RT-PCR confirmed the chip results. CONCLUSION: Different histological alterations can be reclassified by functional, multivariate analysis using cDNA microarrays. Further studies with expanded sample number are needed for subclassification of pathological alterations.

Adenocarcinoma↗
PubMed1980

Gene-directed mutagenesis in bacteriophage T7 provided by polyalkylating RNAs complementary to selected DNA sites.

Bacteriophage T7 early transcripts were used as carriers of alkylating groups to affect complementary T7 DNA sites for inducing mutations in preselected genes. A heterofunctional polyalkylating agent N,N,N'-tri-(beta-chloroethyl)-N'-(p-formylphenyl)propylene diamine-1,3 was attached to 3-5% of the transcript nucleotides. The controlled alkylating groups carried on RNA were activated after RNA . DNA hybridization. The modified transcripts were shown to hybridize only with the complementary H strand and to form covalently bound R loops in the appropriate T7 sites. The T7 DNA molecules locally alkylated by the modified transcripts of gene 1.3 coding for T7 ligase were packaged into T7 proteins and used to infect Escherichia coli B. As judged by plating efficiency and the ligase-deficient E. coli BL2 strain, 4 of 140 plaques obtained after infection contained mutants defective in gene 1.3 in amounts of 0.7-1.5%. The T7 DNA locally alkylated by the modified transcripts of gene 0.3 and 1.1 were used for the transfection of E. coli C1757. Analysis of 24 plaques produced by transfection indicated that 3 contained mutants defective in gene 0.3 in amounts of 2-10%. The mutants had also a second unidentified mutation. Complementation analysis data suggest that the second mutation is due to a defect in gene 1.1. The results obtained demonstrate the efficiency of the approach developed for gene-directed mutagenesis.

Alkylation↗
PubMed2004

The structural basis of the transition from initiation to elongation phases of transcription, as well as translocation and strand separation, by T7 RNA polymerase.

The RNA polymerase from phage T7 is a 99kDa single polypeptide that is unrelated to the multisubunit cellular RNA polymerases, but exhibits nearly all of their properties. Six separate crystal structures have enhanced our understanding of promoter DNA recognition, duplex DNA opening, and the transition from the abortive initiation phase to the elongation phase. A major conformational change in the N-terminal domain removes the promoter-binding site, accounting for promoter clearance, and creates a tunnel through which the transcript passes, accounting for the processivity of the elongation phase. Structures of substrate and product complexes show that a rotational conformational change of the fingers domain is associated with translocation and downstream strand separation. The rotation that results in translocation is powered by the release of the pyrophosphate product.

DNA Replication↗
PubMed2005

Replication and packaging of Norwalk virus RNA in cultured mammalian cells.

Human noroviruses, the most common cause of nonbacterial gastroenteritis, are characterized by high infectivity rate, low infectious dose, and unusually high stability outside the host. However, human norovirus research is hindered by the lack of a cell culture system and a small animal model of infection. Norwalk virus (NV) is the prototype strain of human noroviruses. We report here replication of NV viral RNA and its packaging into virus particles in mammalian cells by intracellular expression of native forms of NV viral RNA devoid of extraneous nucleotide sequences derived from the expression vector by the use of replication-deficient vaccinia virus MVA encoding the bacteriophage T7 RNA polymerase (MVA/T7). Expressed genomic RNA was found to replicate; NV subgenomic RNA was transcribed from genomic RNA by use of NV nonstructural proteins expressed from genomic RNA and was subsequently translated into NV capsid protein VP1. Viral genomic RNA was packaged into virus particles generated in mammalian cells. The cesium chloride (CsCl) density gradient profile of virus particles containing genomic RNA was similar to that of NV purified from stool. These observations indicate that the NV cDNA constructed here is a biologically infectious clone, and that mammalian cells have the ability to replicate NV genomic RNA. This work establishes a mammalian cell-based system for analysis of human norovirus replication and, thus, makes it feasible to investigate antiviral agents in mammalian cells.

Animals↗
PubMed1995

RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA.

Previously, a cDNA was constructed so that transcription by T7 RNA polymerase yielded a approximately 1-kb negative-sense analog of genomic RNA of human respiratory syncytial virus (RSV) containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative RSV transcription motifs and flanked by the RSV genomic termini. When transfected into RSV-infected cells, this minigenome was "rescued," as evidenced by high levels of CAT expression and the production of transmissible particles which propagated and expressed high levels of CAT expression during serial passage (P.L. Collins, M. A. Mink, and D. S. Stec, Proc. Natl. Acad. Sci. USA, 88:9663-9667, 1991). Here, this cDNA, together with a second one designed to yield an exact-copy positive-sense RSV-CAT RNA antigenome, were each modified to contain a self-cleaving hammerhead ribozyme for the generation of a nearly exact 3' end. Each cDNA was transfected into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase, together with plasmids encoding the RSV N, P, and L proteins, each under the control of a T7 promoter. When the plasmid-supplied template was the mini-antigenome, the minigenome was produced. When the plasmid-supplied template was the minigenome, the products were mini-antigenome, subgenomic polyadenylated mRNA and progeny minigenome. Identification of progeny minigenome made from the plasmid-supplied minigenome template indicates that the full RSV RNA replication cycle occurred. RNA synthesis required all three RSV proteins, N, P, and L, and was ablated completely by the substitution of Asn for Asp at position 989 in the L protein. Thus, the N, P, and L proteins were sufficient for the synthesis of correct minigenome and antigenome, but this was not the case for subgenomic mRNA, indicating that the requirements for RNA replication and transcription are not identical. Complementation with N, P, and L alone yielded an mRNA pattern containing a large fraction of molecules of incomplete, heterogeneous size. In contrast, complementation with RSV (supplying all of the RSV gene products) yielded a single discrete mRNA band. Superinfection with RSV of cells staging N/P/L-based RNA synthesis yielded the single discrete mRNA species. Some additional factor supplied by RSV superinfection appeared to be involved in transcription, the most obvious possibility being one or more additional RSV gene products.

Bacteriophage T7↗
PubMed2004

Mechanisms of chromium-induced suppression of RNA synthesis in cellular and cell-free systems: relationship to RNA polymerase arrest.

Chromium(VI) (Cr(VI)) can suppress both DNA replication and transcription as a result of chromium (Cr)-induced DNA damage. While progress has been made in the characterization of Cr-induced DNA polymerase arresting lesions, very little information is available on the inhibition of transcription by this metal. The aim of the present study was to identify the molecular mechanisms involved in the reduction of RNA synthesis by Cr. Following treatment with a moderately cytotoxic dose (approximately LC50) of Cr(VI) (150 microM for 2 h), total RNA synthesis was initially suppressed in CHO cells and recovered to control levels within 72 h post-treatment. In vitro nuclear run-on transcription assays of nuclei isolated from Cr(VI)-treated cells showed a similar amount of RNA synthesis suppression as observed in intact cells. Qualitative analysis of nascent transcripts revealed a general, concentration-dependent reduction in size suggesting that transcriptional elongation was inhibited following Cr-treatment. Transcriptional initiation in these nuclei was also reduced. To better determine whether transcriptional suppression was related to Cr-induced DNA damage we examined the transcriptional activity of T7 RNA polymerase on Cr(III)-treated plasmid DNA. Treatment of pGEM3Z-TS DNA with Cr(III) resulted in transcriptional arrest which occurred primarily at GC-rich and palindromic regions. However, in contrast to the cellular data, transcriptional initiation was unaffected in the in vitro transcription arrest assays. Taken together, these results suggest that the suppression of RNA synthesis by Cr is related to chromium-induced template DNA damage which prevents elongation leading to premature RNA polymerase arrest.

Animals↗
PubMed2000

Production of recombinant snakehead rhabdovirus: the NV protein is not required for viral replication.

Snakehead rhabdovirus (SHRV) affects warm water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its nonvirion gene (NV). Because SHRV grows best at temperatures between 28 and 31 degrees C, we were able to use the T7 expression system to produce viable recombinant SHRV from a cloned cDNA copy of the viral genome. Expression of a positive-strand RNA copy of the 11, 550-nucleotide SHRV genome along with the viral nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins resulted in the generation of infectious SHRV in cells preinfected with a vaccinia virus vector for T7 polymerase expression. Recombinant virus production was verified by detection of a unique restriction site engineered into the SHRV genome between the NV and L genes. Since we were now able to begin examining the function of the NV gene, we constructed a recombinant virus containing a nonsense mutation located 22 codons into the coding sequence of the NV protein. The NV knockout virus was produced at a concentration as high as that of wild-type virus in cultured fish cells, and the resulting virions appeared to be identical to the wild-type virions in electron micrographs. These initial studies suggest that NV has no critical function in SHRV replication in cultured fish cells.

Amino Acid Sequence↗
PubMed2004

Construction strategy for an internal amplification control for real-time diagnostic assays using nucleic Acid sequence-based amplification: development and clinical application.

An important analytical control in molecular amplification-based methods is an internal amplification control (IAC), which should be included in each reaction mixture. An IAC is a nontarget nucleic acid sequence which is coamplified simultaneously with the target sequence. With negative results for the target nucleic acid, the absence of an IAC signal indicates that amplification has failed. A general strategy for the construction of an IAC for inclusion in molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assays is presented. Construction proceeds in two phases. In the first phase, a double-stranded DNA molecule that contains nontarget sequences flanked by target sequences complementary to the NASBA primers is produced. At the 5' end of this DNA molecule is a T7 RNA polymerase binding sequence. In the second phase of construction, RNA transcripts are produced from the DNA by T7 RNA polymerase. This RNA is the IAC; it is amplified by the target NASBA primers and is detected by a molecular beacon probe complementary to the internal nontarget sequences. As a practical example, an IAC for use in an assay for the detection of Mycobacterium avium subsp. paratuberculosis is described, its incorporation and optimization within the assay are detailed, and its application to spiked and natural clinical samples is shown to illustrate the correct interpretation of the diagnostic results.

Animals↗
PubMed1991

A domain at the 3' end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs.

Two murine hepatitis virus strain A59 defective interfering (DI) RNAs were generated by undiluted virus passages. The DI RNAs were encapsidated efficiently. The smallest DI particle, DI-a, contained a 5.5-kb RNA consisting of the following three noncontiguous regions from the MHV-A59 genome, which were joined in frame: the 5'-terminal 3.9 kb, a 798-nucleotide fragment from the 3' end of the polymerase gene, and the 3'-terminal 805 nucleotides. A full-length cDNA clone of the DI-a genome was constructed and cloned downstream of the bacteriophage T7 promoter. Transcripts derived from this clone, pMIDI, were used for transfection of MHV-A59-infected cells and found to be amplified and packaged. Deletion analysis of pMIDI allowed us to identify a 650-nucleotide region derived from the 3' end of the second open reading frame of the polymerase gene that was required for efficient encapsidation.

Amino Acid Sequence↗
PubMed1991

Interaction of HIV-1 reverse transcriptase with a synthetic form of its replication primer, tRNA(Lys,3).

Using synthetic oligonucleotides, a gene encoding the HIV-1 replication primer, tRNA(Lys,3), was constructed and placed downstream from a bacteriophage T7 promoter. In vitro transcription of this gene yielded a form of tRNA(Lys,3) which lacks the modified bases characteristic of the natural species and the 3' -C-A-dinucleotide. Synthetic tRNA(Lys,3) annealed to a pbs-HIV1 RNA template can prime cDNA synthesis catalysed by recombinant HIV-1 reverse transcriptase. Trans-DDP crosslinking indicates that this synthetic tRNA is still capable of interacting with HIV-1 RT via a 12-nucleotide portion encompassing the anticodon domain. Gel-mobility shift and competition analyses imply that the affinity of synthetic tRNA for RT is reduced. In contrast to earlier observations, synthetic tRNA is readily competed from RT by natural tRNA(Pro). The reduced affinity of synthetic tRNA(Lys,3) for RT is not appreciably affected by mutations in positions within the loop of the anticodon domain. These results would imply that the overall structure of the anticodon domain of tRNA(Lys,3) is an important factor in its recognition by HIV-1 RT. In addition, modified bases within this, although not absolutely required, would appear to make a significant contribution to the enhanced stability of the ribonucleoprotein complex.

Base Sequence↗
PubMed1988

Induction of the heat shock regulon of Escherichia coli markedly increases production of bacterial viruses at high temperatures.

Production of bacteriophages T2, T4, and T6 at 42.8 to 44 degrees C was increased from 8- to 260-fold by adapting the Escherichia coli host (grown at 30 degrees C) to growth at the high temperature for 8 min before infection; this increase was abolished if the host htpR (rpoH) gene was inactive. Others have shown that the htpR protein increases or activates the synthesis of at least 17 E. coli heat shock proteins upon raising the growth temperature above a certain level. At 43.8 to 44 degrees C in T4-infected, unadapted cells, the rates of RNA, DNA, and protein synthesis were about 100, 70, and 70%, respectively, of those in T4-infected, adapted cells. Production of the major processed capsid protein, gp23, was reduced significantly more than that of most other T4 proteins in unadapted cells relative to adapted cells. Only 4.6% of the T4 DNA made in unadapted cells was resistant to micrococcal nuclease, versus 50% in adapted cells. Thus, defective maturation of T4 heads appears to explain the failure of phage production in unadapted cells. Overproduction of the heat shock protein GroEL from plasmids restored T4 production in unadapted cells to about 50% of that seen in adapted cells. T4-infected, adapted E. coli B at around 44 degrees C exhibited a partial tryptophan deficiency; this correlated with reduced uptake of uracil that is probably caused by partial induction of stringency. Production of bacteriophage T7 at 44 degrees C was increased two- to fourfold by adapting the host to 44 degrees C before infection; evidence against involvement of the htpR (rpoH) gene is presented. This work and recent work with bacteriophage lambda (C. Waghorne and C.R. Fuerst, Virology 141:51-64, 1985) appear to represent the first demonstrations for any virus that expression of the heat shock regulon of a host is necessary for virus production at high temperature.

Adaptation, Physiological↗
PubMed1992

The 9-kDa hydrophobic protein encoded at the 3' end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated.

The open reading frame potentially encoding a 78 amino acid, 9101 Da hydrophobic protein (HP) and, mapping at the 3' end of the porcine transmissible gastroenteritis coronavirus (TGEV) genome, was shown to be expressed during virus replication. The cloned HP gene was placed in a plasmid under control of the T7 RNA polymerase promoter and in vitro translation of transcripts generated in vitro yielded a 9.1-kDa protein that was immunoprecipitable with porcine hyperimmune anti-TGEV serum. Antiserum raised in rabbits against a 31 amino acid synthetic polypeptide that represented the central hydrophilic region of HP specifically immunoprecipitated HP from TGEV-infected cells. HP was further shown to become associated with microsomal membranes during synthesis in vitro and was found to be closely associated with the endoplasmic reticulum and cell surface membranes in infected cells. The intracellular location of HP suggests that it may play a role in the membrane association of replication complexes or in virion assembly.

Amino Acid Sequence↗