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PubMed1977

Use of specific radioactive probes to study transcription and replication of the influenza virus genome.

Specific radioactive probes have been obtained for both influenza virion RNA (vRNA) and for its complement (complementary RNA or cRNA): 32P-labeled complementary DNA (cDNA) synthesized with the avian sarcoma virus reverse transcriptase, and [125I]vRNA, respectively. From the kinetics of annealing of these two probes to RNA from canine kidney cells infected with the WSN strain of influenza virus, we have determined the average number of cRNA and vRNA sequences in the nucleus and cytoplasm as a function of time after infection. Immediately after infection, a small amount of vRNA is detected, presumably from the inoculum virus. As expected, the amount of cRNA is insignificant. During the first 1.75 h of infection, the most significant increase observed is in cRNA sequences. Most of these cRNA sequences are found in the cytoplasm, but a significant amount (30%) is found in the nucleus. During this time, a small but significant increase in vRNA is also detected in the nucleus and cytoplasm. From 1.75 to 2.75 h, the absolute amounts of both cRNA and vRNA increase, predominantly in the cytoplasm, with cRNA remaining as the majority species. Subsequently, the amount of vRNA increases with respect to cRNA and becomes the majority species. At 3.75 h, 95% of both cRNA and vRNA are found in the cytoplasm. Addition of actinomycin D at 1.75 h completely suppresses the subsequent ninefold increase in cRNA and does not have a significant effect on the subsequent 14-fold increase in cytoplasmic vRNA. This assay is also able to detect the cRNA produced as a result of primary transcription, operationally defined as the cRNA produced in the presence of 100 mug of cycloheximide per ml added at zero time of infection. Increases in cRNA in the presence of cycloheximide are detectable in both the nucleus and the cytoplasm. Addition of actinomycin D as well as cycloheximide at zero time completely suppresses the appearance of cRNA in the cytoplasm, whereas a large fraction (50%) of the increase in nuclear cRNA still occurs.

Cell Line↗

PubMed1994

Use of complementary peptides and their antibodies in B-cell-mediated autoimmune disease: prevention of experimental autoimmune myasthenia gravis with a peptide vaccine.

We have developed and describe a new method of altering B-cell-mediated autoimmune diseases by induction of anti-idiotypic (Id) antibodies (Abs) by immunization with complementary peptides. Specifically, a peptide denoted RhCA 67-16 encoded by RNA complementary to RNA for the Torpedo acetylcholine receptor (AChR) main immunogenic region, AChR 61-76, was tested in the Lewis rat model of experimental autoimmune myasthenia gravis (EAMG). Immunization with RhCA 67-16 induced monoclonal and polyclonal anti-Id Ab against Abs to Torpedo AChR 61-76. RhCA 67-16 antisera inhibited AChR binding by AChR-specific Abs. In addition, a mAb to RhCA 67-16 (denoted TCM 240) recognized two well known EAMG-causing mAbs, 6 and 35. TCM 240, but not a control mAb F28C, inhibited mAb 6 binding to Torpedo AChR 67-76 peptide. mAb 35 binding to TCM 240 was inhibited by native Torpedo AChR as well as by RhCA 67-16. In in vivo experiments, immunization with a RhCA 67-16 keyhole limpet hemacyanin (KLH) conjugate blocked the development of EAMG after challenge with native Torpedo AChR (25% disease incidence versus 90% in the controls). This new approach may provide a novel therapy for MG and perhaps other B-cell-mediated autoimmune disorders through the induction of anti-Id Abs with complementary peptide antigens.

Amino Acid Sequence↗

PubMed1981

Determination of cellular RNA concentrations by electron microscopy of R loop-containing DNA.

R loop hybridizations and electron microscopy have been used to determine cellular RNA concentrations for cloned genes. In plasmid DNA sequence excess, all the complementary RNA is driven into R loop structures that can be assayed by electron microscopy. To determine the concentration of a particular poly(A)+ RNA, plasmid DNA crosslinked once every 2000-5000 base pairs with trioxsalen and UV light is hybridized in DNA sequence excess to various known amounts of total poly(A)+ RNA, and the R loops are stabilized by treatment with glyoxal. If necessary, excess nonhybridized RNA is removed by Sepharose 2B chromatography, which enables the visualization of less abundant transcripts. Reconstruction experiments demonstrated that electron microscopic determination of the fraction of plasmid DNA molecules containing specific RNA loops gives accurate values of specific RNA weight fractions or concentrations in the total poly(A)+ RNA populations. These methods were also used to determine the concentrations of five RNA species complementary to sequences on TRT3, a recombinant DNA plasmid containing yeast histone 2A and 2B genes and three other nonhistone genes. The methods described allow one to visualize the R loop structures for both abundant and nonabundant transcripts and to estimate concentrations of these RNA species simply by determining the fraction of DNA containing R loops.

Animals↗

PubMed1993

RNA-directed RNA polymerase from tomato leaves. II. Catalytic in vitro properties.

The catalytic properties of electrophoretically homogeneous RNA-directed RNA polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue were studied with the aid of oligonucleotides of defined sequence. It was found that RdRP catalyzes in vitro the transcription of short single-stranded RNA and DNA molecules into precisely complementary RNA copies up to the full length of these templates. The transcription of RNA- and DNA-oligonucleotide templates was equally effective. Differences in transcription efficiency were found to depend on nucleotide sequence rather than on the RNA or DNA nature of the single-stranded nucleic acid. Double-stranded nucleic acids such as poly(A).poly(U) and a double-stranded DNA 14-mer were not transcribed. The RdRP-directed transcription could be primed because RNA and DNA dinucleotides and trinucleotides complementary to the 3'-terminal nucleotides of the template were extended by the enzyme. The unprimed transcription was shown to start preferentially at the 3'-terminal nucleotides of the template. RdRP is capable of adding a single noncomplementary nucleotide to the 3' terminus of about 50% of the runoff transcripts. AMP was preferred over GMP, whereas CMP and UMP were terminally added at very low frequency.

Base Sequence↗

PubMed1975

Transcription of simian virus 40. V. Regulattion of simian virus 40 gene expression.

RNA "exhaustion type" hybridization was used to measure the complementarity of nuclear and cytoplasmic viral RNA to the early (E) and late (L) simian virus 40 (SV40) DNA strands. This type of hybridization measures the amount of labeled RNA complementary to each of the two DNA strands, rather than the fraction of each SV40 DNA strand that is homologous to SV40 RNA. At 48 h after infection, about 5% of the nuclear newly synthesized viral RNA was complementary to the E-strand (- strand) and 95% was complementary to the L-strand (+ strand). This proportion was independent of the labeling time, indicating similar accumulation of the E- and L-RNA transcripts in the nucleus. The nuclear E- and L-viral RNA transcripts sedimented in a similar manner on sucrose gradients. Of the cytoplasmic viral RNA only about 1% was complementary to the E-strand, these molecules sedimenting at 19S, whereas 99% were complementary to the L-strand and sedimented at 19S and 16S. The abundance of E-RNA transcripts in nuclei of cells infected with serially passaged virus was about four times higher than that in nuclei of cells infected with plaque-purified virus; however, the size and proportion of the corresponding cytoplasmic E- and L-RNA transcripts was independent of the type of virus used to infect the cells. According to these results at least two control mechanisms regulate viral gene expression in productively infected cells, one operates at the trnascriptional level and the second at the post-transcriptional level.

Cell Line↗

PubMed1994

Multimeric non-radioactive cRNA probes improve detection of potato spindle tuber viroid (PSTVd).

Experimental data showed that multimeric, complementary RNA (cRNA) probes, labelled with non-radioactive digoxigenin (DIG), improved sensitivity of detection of the potato spindle tuber viroid (PSTVd) RNA by 2- to 30-fold as compared with corresponding multimeric cDNA probes. The degree of PSTVd detectability improvement depended upon the type of alkaline phosphatase substrate (colorimetric vs. chemiluminescent) used. Use of hexameric DIG-labelled cRNA probes in combination with chemiluminescent (Lumi-Phos 530) substrate resulted in detection of 0.48 pg of PSTVd RNA. The size of the synthesized cRNA probes corresponded to the size of the respective PSTVd cDNA templates. Interestingly, there was no relationship between the size of the synthesized, DIG-labelled DNA probes and that of the PSTVd cDNA template. This type of anomaly was not observed with other plant viral cDNA templates. Monomeric or multimeric cDNA probes detected both a mild and a severe PSTVd strain in viroid-infected potato leaf extracts diluted 1024 to 2048 times. In comparison, cRNA probes exhibited a much greater dilution end point; PSTVd RNA was detectable in viroid-infected potato leaf tissue diluted up to 16,384 times. Comparable levels of PSTVd sensitivity of detection were obtained with viroid-infected potato tuber tissue.

Alkaline Phosphatase↗

PubMed1975

Strand-selective transcription of globin genes in rabbit erythroid cells and chromatin.

In order to investigate the symmetry of globin gene transcription, complementary RNA (cRNA) was synthesized using rabbit globin complementary DNA (cDNA) as a template for Escherichia coli DNA-dependent RNA polymerase (RNA nucleotidyltransferase). The cRNA hybridized specifically to its own cDNA template but not to sheep cDNA, rabbit globin mRNA, or poly(dT). Hybridization studies with cRNA demonstrated that RNA sequences transcribed from the DNA strand complementary to the globin gene region (anti-strand) were not present in cellular, total nuclear, or fractionated nuclear RNA from rabbit marrow. Such sequences were detected in RNA transcribed from rabbit marrow chromatin by E. coli or sheep liver RNA polymerases, but amounted to less than 50% of the globin mRNA sequences present in the same transcript. The evidence indicates that globin mRNA transcription is predominantly DNA strand specific.

Animals↗

PubMed1975

Rous sarcoma virus activates embryonic globin genes in chicken fibroblasts.

Complementary DNA (cDNA) specific for chick globin mRNA sequences fails to hybridize to total RNA extracted from chicken fibroblasts. After infection by Rous sarcoma virus, RNA complementary to globin cDNA is detectable in 100-500 copies per cell. Infection of fibroblasts with the transformation defective (td) deletion mutant of Rous sarcoma virus leads to normal virus production, but not to host cell transformation or accumulation of RNA sequences complementary to globin cDNA. Our evidence shows that the globin genes activated by Rous sarcoma virus are those specified by embryonic chick red cells; adult-specific globin sequences were not detected.

Age Factors↗

PubMed1986

Inhibition of replication and expression of human T-cell lymphotropic virus type III in cultured cells by exogenous synthetic oligonucleotides complementary to viral RNA.

The possibility of using oligodeoxynucleotides complementary to viral RNA or proviral DNA to inhibit the replication of human T-cell lymphotropic virus type III (HTLV-III) [the etiological agent of acquired immunodeficiency syndrome (AIDS)] in cultured human cells was addressed by studying the association of 32P-labeled oligodeoxynucleotides with mammalian cellular components. The results indicated that exogenous oligodeoxynucleotides at 20 microM became associated with the membrane/cytosol fractions of the cell in amounts approximating 1.5 microM. Oligodeoxynucleotides complementary to a region close to the tRNALys primer binding site on HTLV-III RNA and others complementary to HTLV-III mRNA donor or acceptor splice sites inhibited viral replication (assayed as reverse transcriptase) and gene expression (assayed as virus-encoded proteins p15 and p24) by as much as 95%. Use of control (random) oligodeoxynucleotides suggests that the antiviral effects were specific. Although these results pertain to HTLV-III-infected cells in tissue culture, rather than to AIDS patients, they nevertheless point to a therapeutic potential of the complementary oligodeoxynucleotide ("hybridization competition" or "hybridon") approach in the treatment of patients with AIDS and AIDS-related complex.

Base Sequence↗

PubMed1979

Characterization of virus-like particles produced by an influenza A virus.

The influenza strain 413 1,1 segregated as a stable recombinant during passage of the isolate 19/N which was obtained after double infection of chick embryo fibroblasts by virus N and the fowl plague virus (FPV) mutant ts 19. Its gene constellation was determined by molecular hybridization. Upon infection of chick embryo cells by this recombinant strain, two particle populations of high (H) and low (L) buoyant densities were produced. By biological and biochemical parameters, the H-population (delta = 1.22 g/cm3) cannot be distinguished from standard infectious influenza virus. In contrast, the noninfectious L-particles (delta = 1.14 g/cm3) lack all virus-specific glycoproteins (HA, NA) as well as the matrix protein M and are visualized by electron microscopy as spikeless particles. Significant changes in the quantitative composition of the phospholipid bilayer are evident as compared to the H-particles. In addition to the previously characterized eight genes both populations contain a variety of smaller RNA fragments which hybridize with complementary RNA and presumably represent degradation products of full-length genes.

Animals↗

PubMed1987

In vitro synthesis of influenza viral RNA: characterization of an isolated nuclear system that supports transcription of influenza viral RNA.

An in vitro system for the synthesis of influenza viral RNA was developed using isolated nuclei prepared from influenza virus-infected HeLa cells. In this system, two species of positive-sense RNA, i.e., mRNA and cRNA (complementary RNA to vRNA), were found to be synthesized when analyzed by RNA-RNA hybridization using a minus-strand RNA probe and high resolution gel electrophoresis. The in vitro RNA synthesis required Mg2+, GTP, CTP, UTP, a high concentration of ATP, and an ATP regenerating system. Neither actinomycin D nor alpha-amanitin, potent inhibitors for cellular DNA-dependent RNA polymerases, inhibited the RNA synthesis. Addition of ApG or capped RNA, well-known primers for virion-associated RNA polymerase, markedly enhanced the extent of RNA synthesis. The maximum activity was observed with nuclei isolated from cells at 5 h after infection. This system is useful for the purification and characterization of factors involved in the transcription of these two species positive-sense RNA.

Adenosine Triphosphate↗

PubMed1980

Interferon induction by viruses. IV. Sindbis virus: early passage defective-interfering particles induce interferon.

We have shown that a single defective-interfering (DI) particle of early (5th) passage Sindbis virus induces maximal amounts of interferon in an 'aged' primary chick embryo cell. The capacity of such DI particles to induce interferon is inactivated by small amounts of u.v. radiation (i/e dose = 232 ergs/mm2). The I/e dose for inactivation of the interferon-inducing capacity of infectious virus particles is 399 ergs/mm2 and for infectivity is 101 ergs/mm2. Pre-treatment with interferon blocks formation of interferon in response to either DI or infectious virus particles. Our results suggest that Sindbis virus genes must be expressed to form the interferon inducer, which is presumably a molecule of double-stranded (ds)RNA. We postulate that for interferon induction, the genomic RNA which codes for genes G and A must be translated into products whose concerted action produces a dsRNA molecule upon synthesis of a segment of RNA complementary to the genome. The RNA from early passage DI particles is sufficiently large (25S, 1.6 x 10(6) mol. wt.) to accommodate these genes, whereas the RNA from the late passage DI particles (20S, 1.0 x 10(6) mol. wt.) is not. Late (15th) passage DI particles do not induce interferon formation.

Animals↗

PubMed1980

Short-lived minus-strand polymerase for Semliki Forest virus.

Semliki Forest virus (SFV)-infected BHK-21, Vero, and HeLa cells incorporated [3H]uridine into 42S and 26S plus-strand RNA and into viral minus-strand RNA (complementary to the 42S virion RNA) early in the infectious cycle. Between 3 and 4 h postinfection, the synthesis of minus-strand RNA ceased in these cultures, although the synthesis of plus-strand RNA continued at a maximal rate. At the time of cessation of minus-strand RNA synthesis, two changes in the pattern of viral protein synthesis were detected: a decrease in the translation of nonstructural proteins and an increase in the translation of the viral structural proteins. Addition of cycloheximide and puromycin to cultures of SFV-infected BHK cells actively synthesizing both viral plus- and minus-strand RNA resulted within 15 to 30 min in the selective shutoff of minus-strand RNA synthesis. Removal of the cycloheximide-containing medium led to the resumption of minus-strand synthesis and to an increased rate of viral RNA synthesis. We conclude that the minus-strand polymerase regulates the rate of SFV plus-strand RNA synthesis by determining the number of minus-strand templates and that the synthesis of the minus-strand templates is regulated at the level of translation by a mechanism which utilizes one or more short-lived polymerase proteins.

Animals↗

PubMed1996

Saccharomyces cerevisiae L-BC double-stranded RNA virus replicase recognizes the L-A positive-strand RNA 3' end.

L-A and L-BC are two double-stranded RNA viruses present in almost all strains of Saccharomyces cerevisiae. L-A, the major species, has been extensively characterized with in vitro systems established, but little is known about L-BC. Here we report in vitro template-dependent transcription, replication, and RNA recognition activities of L-BC. The L-BC replicase activity converts positive, single-stranded RNA to double-stranded RNA by synthesis of the complementary RNA strand. Although L-A and L-BC do not interact in vivo, in vitro L-BC virions can replicate the positive, single-stranded RNA of L-A and its satellite, M1, with the same 3' end sequence and stem-loop requirements shown by L-A virions for its own template. However, the L-BC virions do not recognize the internal replication enhancer of the L-A positive strand. In a direct comparison of L-A and L-BC virions, each preferentially recognizes its own RNA for binding, replication, and transcription. These results suggest a close evolutionary relation of these two viruses, consistent with their RNA-dependent RNA polymerase sequence similarities.

Base Sequence↗

PubMed2000

Activation of the interferon-inducible 2'-5'-oligoadenylate synthetase gene by hepatitis C virus core protein.

The effects of hepatitis C virus (HCV) proteins on several signal transduction pathways in human nonneoplastic hepatocyte PH5CH8 cells were investigated using expression vectors encoding HCV proteins derived from HCV-infected human nonneoplastic cultured T-lymphocyte and hepatocyte cells (MT-2C and PH5CH7), which could support HCV replication. The amino acid sequences of HCV proteins obtained from HCV-infected human cells were identical or very close to the consensus sequences of the proteins derived from the original inoculum used for HCV infection. During the course of the study, we found that HCV core protein specifically activated the 40/46-kDa 2'-5'-oligoadenylate synthetase (2'-5'-OAS) gene promoter in a dose-dependent manner in different human hepatocyte cell lines (PH5CH8, HepG2, and PLC/PRF/5). We also found that the activation by core protein was further enhanced in the cells treated with alpha interferon. The expression of E1 or E2 envelope protein or nonstructural NS5A protein did not activate the 2'-5'-OAS gene promoter. We demonstrated that the activation by core protein in the hepatocyte cells was suppressed by antisense RNA complementary to core-encoding RNA. Deletion mutant analysis of core protein and deletion analysis of the 2'-5'-OAS gene promoter have been performed. Finally, we demonstrated that the activation of the 2'-5'-OAS gene occurred at the transcriptional level and furthermore demonstrated that the endogenous 2'-5'-OAS gene was also activated by core protein. This is the first report to show that a viral protein activated the 2'-5'-OAS gene.

2',5'-Oligoadenylate Synthetase↗

PubMed2003

Reverse genetics for crimean-congo hemorrhagic fever virus.

The widespread geographical distribution of Crimean-Congo hemorrhagic fever (CCHF) virus (more than 30 countries) and its ability to produce severe human disease with high mortality rates (up to 60%) make CCHF a major public health concern worldwide. We describe here the successful establishment of a reverse genetics technology for CCHF virus, a member of the genus Nairovirus, family BUNYAVIRIDAE: The RNA polymerase I (pol I) system was used to generate artificial viral RNA genome segments (minigenomes), which contained different reporter genes in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of the CCHF virus S segment. Reporter gene expression was observed in different eukaryotic cell lines following transfection and subsequent superinfection with CCHF virus, confirming encapsidation, transcription, and replication of the pol I-derived minigenomes. The successful transfer of reporter gene activity to fresh cells demonstrated the generation of recombinant CCHF viruses, thereby confirming the packaging of the pol I-derived minigenomes into progeny viruses. The system offers a unique opportunity to study the biology of nairoviruses and to develop therapeutic and prophylactic measures against CCHF infections. In addition, we demonstrated for the first time that the human pol I system can be used to develop reverse genetics approaches for viruses in the family BUNYAVIRIDAE: This is important since it might facilitate the manipulation of bunyaviruses with cell and host tropisms restricted to primates.

Animals↗

PubMed1985

Dysdifferentiative nature of aging: passage number dependency of globin gene expression in normal human diploid cells grown in tissue culture.

Aging may be a result of cells drifting away from their proper state of differentiation. This process has been called dysdifferentiation. Normal diploid cells grown in tissue culture conditions undergo numerous biochemical and morphological changes and have a finite division potential. These changes could be a result of such a dysdifferentiation process. Changes in the differentiated state of a cell are frequently manifested by the expression of genes that are normally repressed. Previous studies have shown about a two-fold age-dependent increase of alpha and beta globin-like RNA in mouse brain and liver tissues. Therefore, the possible presence and increase of globin RNA was investigated in the nonerythroid human diploid strain WI-38 grown in tissue culture as a function of population doublings. A DNA X RNA hybridization technique using specific complementary DNA (cDNA) to alpha and beta human globin was used to detect possible complementary RNA sequences in total cellular RNA preparations extracted from cells at population doublings of 26.4 and 46. No globin-like RNA sequences could be detected above background noise levels for either of these two passage numbers. Thus, the globin RNA genes appear to be highly repressed and this degree of repression maintained as the culture approaches its characteristic population doubling limit.

Aging↗

PubMed2004

Genetically targeted cancer therapy: tumor destruction by PKR activation.

The is a double-stranded RNA-activated protein kinase (PKR) has been largely investigated for its key role in viral host defense. Although best characterized by its function in mediating the antiviral and antiproliferative effects of interferon (IFN), PKR is also implicated in transcriptional regulation, cell differentiation, signal transduction, and tumor suppression. However, recent findings identifying PKR as an important effector of apoptosis have led to an increased interest in PKR modulation as an antitumor strategy. PKR can either be up-regulated through direct induction by the transcription factor E2F-1, or it can be activated through direct protein-protein interactions with the melanoma differentiation-associated gene-7 (MDA7, IL-24). Additionally, the intracellular formation of double-stranded RNA by transfection with antisense RNA complementary to tumor-specific RNA sequences can induce PKR activation and apoptosis selective to these tumor cells. The growing application of viral vector-based gene therapies and oncolytic, replicating viruses that must elude viral defense in order to be effective, has also drawn attention to PKR. Oncolytic viruses, like the attenuated herpes simplex virus R3616, the vesicular stomatitis virus, or reovirus, specifically replicate in tumor cells only because the viral host defense in the permissive cells is suppressed. In this article we review the role of PKR as an effector of apoptosis and a target for tumor treatment strategies and discuss the potential of PKR-modifying agents to treat patients with cancer. Targeted gene therapy against cancer can be approached by activation of PKR with the down-regulation of protein synthesis and induction of apoptosis, or by suppression of PKR with the propagation of oncolytic virus. Since the PKR pathway can be modified by many routes, antitumor therapies combining oncolytic virus, gene therapies, and chemotherapy with PKR modifiers are likely to emerge in the near future as therapeutic options in the treatment of patients with cancer.

Animals↗