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Physical properties of Rous Sarcoma Virus RNA
... As might be expected, the long extrapolation to Sendai virus RNA showed poor reproducibility and a poor fit. The value obtained by extrapolation, 5.5 million daltons, was lower than estimates of 6.3 to 7.5 million daltons, assuming comparable molecular weights for Sendai and Newcastle disease virus RNA species (7,20). ...
... The procedure was applicable as a micropreparative technique (employing both 6-and 12-mmdiameter gels) wherein RNA recovered from gels was subjected to further treatment and re-electrophoresis. The same approach served to demonstrate a problem that may arise in the use of DMSO, a reagent valuable for splitting doublestranded RNA (13) and disaggregating highmolecular-weight RNA from tumor viruses (7). The distribution of RNA after treatment was quite heterogeneous when compared with the untreated preparation, but relative mobilities of the major single-stranded components were not altered. ...
The electrophoretic mobilities of ribosomal ribonucleic acids (RNA) from cultured mammalian (HeLa, Vero, MDBK), avian (chick embryo), and bacterial (Escherichia coli) cells, and RNA species extracted from selected viruses (Sindbis, polio, tobacco mosaic, Sendai) were compared, employing a simple, inexpensive technique for slicing low-concentration polyacrylamide gels. The procedure provides for rapid fractionation of gels used for characterization of RNA, incorporating extrusion and serial sectioning of frozen gels. Among 28S ribosomal RNA species, Vero and MDBK were indistinguishable, whereas HeLA RNA had a slightly lower mobility (higher apparent molecular weight) and chick RNA had a higher mobility (lower apparent molecular weight). The 18S ribosomal RNA species of the three mammalian sources were indistinguishable, but chick 18S RNA had a slightly lower apparent molecular weight. The inverse relation between mobility and log-molecular weight among the ribosomal and viral RNA species, though not highly precise, demonstrates the applicability of the technique to the study of molecular weights of viral RNA species.
... The first evidence for the existence of a dimeric genome came from ultracentrifugation sedimentation analysis of gRNA extracted Rous sarcoma virus (RSV) (Duesberg, 1968;Mangel et al., 1974), even though a tetrameric organization of the genome had also been proposed (Montagnier et al., 1969). Subsequent sedimentation and electron microscopy analyses supported the dimeric organization of the genome and extended this observation to other retroviruses families such as alpharetroviruses, gammaretroviruses, and lentiviruses (Kung et al., 1976;Bender et al., 1978;Maisel et al., 1978;Murti et al., 1981;Höglund et al., 1997), thus revealing the conservation and the importance of gRNA dimerization in the life cycle of retroviruses. ...
... Even though the first evidence for retroviral RNA dimerization came from sedimentation and electron microscopy studies of alpha-retroviruses such as RSV (Duesberg, 1968;Canaani et al., 1973;Mangel et al., 1974), the precise mechanisms underlying this process remains surprisingly poorly defined in comparison with other model retroviruses such as HIV-1 and MuLV. Strikingly, while most DLS/DIS are found within the 5 -untranslated region (UTR) of gRNA, electron micrographs of gRNA dimers extracted from RSV virions located the DLS within the gag gene, around position 480-540 from the 5 end (Figure 2A) (Murti et al., 1981). ...
The genome of the retroviruses is a dimer composed by two homologous copies of genomic RNA (gRNA) molecules of positive polarity. The dimerization process allows two gRNA molecules to be non-covalently linked together through intermolecular base-pairing. This step is critical for the viral life cycle and is highly conserved among retroviruses with the exception of spumaretroviruses. Furthermore, packaging of two gRNA copies into viral particles presents an important evolutionary advantage for immune system evasion and drug resistance. Recent studies reported RNA switches models regulating not only gRNA dimerization, but also translation and packaging, and a spatio-temporal characterization of viral gRNA dimerization within cells are now at hand. This review summarizes our current understanding on the structural features of the dimerization signals for a variety of retroviruses (HIVs, MLV, RSV, BLV, MMTV, MPMV…), the mechanisms of RNA dimer formation and functional implications in the retroviral cycle.
... Although' C-type particles appear to contain several 4 species of cellular RNA (Bauer, 1966;Bonar et a.z., 1967;Imai et al., 1966;Gay et 'al., 1970;Erikson et al., 1973), the bulk of evidence suggests that the viral genome is a 60-70S single-stranded RNA with a molecular weight of 10-12 x 10 6 (Robinson and Baluda, 1965;Duesberg and Robinson, 1966). Upon denaturation, this molecule j dissociates into 355 and 45 subunits (Duesberg, 1968;Bader and Steck, 1969;Erikson, ~969;Montagnier et aZ., 1969;Manning et aL, 1972;Erikson and Erikson, 1971), and it has since been proposed that the viral genome by Bolognesi 'f.! aL, 1974and, also Ikeda et aL, 1975Ihle et aZ., 1975 (Baltimore, 1970;Temin and Mitzutani, 1970). The first characterization of virionassociated reverse transcriptase demonstrated its 5 6 capacity to use endogenous 60-705 viral RNA as template for the synthesis of complementary DNA (Garapin et aZ., 1970;Rokutanda et aZ., 1970;Spiegelman et aZ., 1970;Manly et aZ., 1971). ...
... Retroviruses are RNA viruses that replicate through a DNA phase, in which viral DNA is integrated into the host genome to form a provirus [1]. Retroviral genomes in virions are dimers, consisting of two copies of full-length, unspliced RNA, each of which encodes all of the genetic information needed for virus replication [2,3,4,5,6]. Packaging of the retroviral genome is mediated by interactions between the viral structural protein Gag and the cis-acting element(s), collectively called the packaging signal, in the viral RNA [7,8,9,10,11,12,13,14]. ...
How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses.
... A retrovirus is a RNA virus with a diameter of 80-100 nm. Viral RNA sediments as a complex of 60-70 Svedberg (S) units (corresponding to about 20-30 kb of RNA) [83,84]. The hallmark of the family is its replicative strategy which includes as essential steps reverse transcription of the virion RNA into linear double-stranded DNA by a viruscoded polymerase, "reverse transcriptase" and the subsequent integration of this DNA into the genome of the cell [40]. ...
Human milk samples contain microvesicles similar to the retroviruses. These microvesicles contain single-stranded RNA and possess reverse transcriptase activity. It has recently been reported that RNA content of the microvesicles could be transferred to other cells and be functional in their new location. These microvesicles may therefore transfer genetic signals from mother to neonate during breastfeeding. Moreover, transfer of wild type RNA from a healthy wet-nurse to the suckling neonate through the milk microvesicles and its subsequent reverse transcription and integration into the neonate genome could result in permanent correction of the clinical manifestations in genetic diseases.
... As packaging of the viral RNA is not selective for specific RNA copies (D'Souza and Summers, 2005), a heterozygous virus can be formed, by encapsidation of an RNA copy from each viral variant. Heterozygous virions can then infect other cells and recombination between the two co-packaged viral RNAs can occur during reverse transcription (Duesberg, 1968). (Figure 3). ...
A wild mouse embryo-derived nontransforming type C virus, WM-1504E, which showed in vivo neurotropic and lymphomagenic activities, was analyzed for biochemical and antigenic characterizations. The virions contained two major species of RNA, 70 S and 4 S. The 70 S RNA was heat dissociated into a heterogeneous species with peak distributions at 35 S, 18 S, and 4 S regions. The gel filtration pattern in guanidine-hydrochloride and neutralization by antibody against MuLV polymerase of the virion associated reverse transcriptase resembled those of MuLV enzyme. Virions contained six major protein groups as detected by agarose gel filtration in guanidine-hydrochloride. The two largest proteins were glycoproteins, and the next largest protein contained the group-specific antigenic determinants of murine type C oncornaviruses. The approximate molecular weights of the major proteins as determined by SDS-polyacrylamide gel electrophoresis were: 100,000 (and 11,000), 76,000, 29,000, 16,000, 13,500, and 11,000 daltons. The last group also contained a polypeptide of 10,000 daltons. These values correspond closely to those of MuLV (Moloney) proteins. It is concluded that WM-1504E virus represents a murine type C oncornavirus.
Die Hühner-Leukose-Sarkomatose-Viren stellen eine Gruppe morphologisch einheitlicher und serologisch verwandter Viren dar. Sie unterscheiden sich vor allem hinsichtlich ihrer Pathogenität in Vögeln und Säugetieren. In ihrer Struktur sind sie den Myxoviren ähnlich, aber von diesen morphologisch unterscheidbar. Wie diese enthalten sie Zellbestandteile, deren Quantität und Qualität vom Wirtszelltyp bestimmt wird. Als virusspezifische Bestandteile konnten eine einzelsträngige hochmolekulare RNS, eine gruppenspezifische Innenkomponente sowie ein typspezifisches Hüll-Antigen nachgewiesen werden. Mit Hilfe von gruppenspezifischen Antiseren kann man die Vermehrung der einzelnen Leukose-Virus-Typen in Zellkulturen verfolgen. Während Mechanismus und Lokalisation der Virus-RNS-Synthese in der Zelle noch unbekannt sind, können die Virusantigene im Cytoplasma nachgewiesen werden.
The effect of formamide on the dissociation of aggregate structure of high-molecular-weight RNA of avian myeloblastosis virus, an oncornavirus, was studied. It has been found that the pretreatment with increasing formamide concentration leads to the stepwise conversion of 60 – 70 S RNA molecule to 50 – 54 S and 30 – 40 S components; the 50 – 54 S intermediate is then further converted to 30 – 40 S subunits and smaller heterogenous RNAs. It is suggested that the subunits forming the aggregate RNA molecule of oncornaviruses are held together by not equally stable double stranded regions.
Oncornavirus-like particles observed and purified from HeLa cell culture supernates have a density in sucrose of 1.16-1.17 g/cm3. They contain DNA polymerase and ribonuclease activities with properties characteristic of reverse transcriptase and ribonuclease H of known oncornaviruses. The DNA polymerase and RNase H are antigenically similar to those of Mason-Pfizer monkey virus.
Particules du Type Oncornavirus dans les Cellules HeLa. III. Caractérisation Biochimique du Virus
Les particules du type oncornavirus observées dans des surnageants de cultures de cellules HeLa et purifiées à partir de ces surnageants ont une densité de 1.16-1.17g/cm3 dans le saccharose. Elles contiennent de I'ADN polymérase et de la ribonucléase ayant des propriétés caractéristiques de la transcriptase reverse et de la ribonucléase H des oncornavirus connus. L'ADN polymérase et la R Nase H sont antigéniquement analogues à celles du virus de singe Mason-Pfizer.
This paper describes some current work pertaining to transformation of cells by oncogenic viruses.
Part I includes: (1) the effect of a deoxyribonucleic acid (DNA) tumor virus (SV40) on the antigenic characteristics of transformed
cells; (2) in vitro and in vivo methods of detecting virus-specific surface antigens; (3) the role that the host cell may
play in the expression of virus-coded antigens; and (4) the presence of virus-induced antigens as a possible mechanism of
the apparent nononcogenicity of certain virus variants.
Part II discusses (1) the physicochemical properties of the nucleic acid of a ribonucleic acid (RNA) tumor virus-the Moloney
sarcoma-leukemia virus (MSV-MLV) complex —(2) a preliminary analysis of viral RNA replication in cells transformed by MSV-MLV,
and (3) application to human tumors.
All classes of vertebrates harbor tumor viruses that are capable of inducing either tumors or leukemias. After infection, their genomes become integral parts of the host cell's genetic material (DNA). Many biological functions such as the capacity to code for the synthesis of new proteins and, in particular, the oncogenic property (oncogen) have already been assigned to specific regions (on physical maps) of their DNA.
The harmful presence of retrovirus in CD4cells of the human immune system can result in the syndrome of human immunodeficiency
known as AIDS, a disease that has extended widely across the entire planet.This paper proposes to obtain characteristic RAMAN
spectra with specific peaks detected, eliminating the noise of high frequency (HF) and fluorescence of the signal obtained
with SERS and improved with ANFIS. With the spectra cleaned of this noise (HF and fluorescence) the characteristic RAMAN spectra
of each microorganism or retrovirus (HIV) in this case is defined. This method provides the specialists with important clinical
tools to express an efficient diagnosis of AIDS.
The 18- and 28-S ribosomal RNA components from chicken leukemic myeloblasts have been purified and separately analyzed for terminal nucleotides. Alkaline hydrolysates of 18-S rRNA contain, per mole RNA, about 1 mole of cytidine 3′(2′),5′-diphosphate and 1 mole of adenosine. Similar analysis of the 28-S component gives only guanosine 3′(2′),5′-diphosphate and uridine. Fractionation of nuclease hydrolysates of each RNA by a “diagonal” electrophoretic method (which exploits the absence of monoesterified phosphate on the 3′-terminal oligonucleotide) permits isolation of a terminal fragment from each. Analysis of these fragments indicates that the terminal nucleotide sequence of myeloblast 18-S rRNA is -GC[(AU)2C2U2]AOH and that of the 28-S rRNA is -PyAGGUOH. Analysis of the nucleosides released upon alkaline hydrolysis of avian myeloblastosis virus RNA show the 3′-terminus to be predominantly adenosine.
Sedimentation analyses in sucrose gradients of high molecular weight RNAs from several strains of avian tumor viruses revealed considerable variations. Analysis in gel electrophoresis likewise revealed differences in the rate of migration and experiments with various ionic conditions suggested that the observed differences are probably not due to conformational effects in the molecules but reflect variations in molecular size. The maximum difference obtained corresponds to a molecular weight of about 1.3 × 106 daltons. The observed differences were not correlated to (i) whether the agent was a sarcoma or leukosis virus or (ii) the virus subgroup, or (iii) the capacity of the virus to form tumors in mammals.It was shown, however, that mutagenization of a sarcoma virus to an infectious nonconverting virus was accompanied by a loss in molecular size of the RNA. The significance of these results is discussed.
Avian tumor viruses were induced in normal chicken cells treated with ionizing radiations or chemical carcinogens and mutagens. The induced leukosis viruses possess a buoyant density, DNA polymerase, polypeptides, and 70S RNA typical of avian tumor viruses. Induced leukosis viruses act as helper agents for the defective Bryan high titer strain of Rous sarcoma virus and with one exception belong to subgroup E as judged by host-range, interference, and neutralization patterns. Induction of leukosis viruses was successful in chicken cells lacking the “natural” group specific (gs) antigen of the avian tumor viruses as well as in cells carrying this antigen. This observation indicates that the viral genome is present in gs+ and in gs− cells. Therefore, the chromosomal locus which controls the presence of natural gs antigen in chicken cells does not represent the viral genome itself but regulates its expression in normal cells. These findings have implications for the origin of RNA tumor viruses and for theories of carcinogenesis.
Reticuloendotheliosis virus (REV) with several related viruses comprises a group of C-type, avian RNA tumor viruses which is distinct from the avian leukosis-sarcoma virus (ALSV) complex. The emphasis of the present investigation has been on the comparison of the properties of REV with those of a model mammalian RNA tumor virus, murine leukemia virus (MuLV), and with those of the ALSV.The structure and pathway of maturation of these viruses has been examined using electron microscopy. Conclusions derived from this work indicate that while the immature particles of REV can be morphologically distinguished from both MuLV and ALSV, the mature REV particle is very like that of MuLV and quite different from that of ALSV.The properties of the purified DNA-polymerase of these viruses were analyzed with the following significant findings: 1) Unfrozen, purified disrupted REV, but not purified REV-DNA-polymerase is enzymatically active using natural viral RNA as template-primer; 2) the DNA-polymerase copurifies with an RNase-H activity which probably resides on the same polypeptide; 3) the size of the DNA-polymerase-RNase-H complex is indistinguishable from that of the MuLV, a single polypeptide of 84,000 daltons; 4) the divalent cation preference of the REV-DNA polymerase, like that of MuLV, but unlike that of ALSV, is for Mn2+; and 5) serological cross-reaction between the DNA-polymerase of REV and ALSV could not be demonstrated.Apart from these structural and biochemical analogies, no direct relationship between REV and MuLV has been established. Infectivity of REV in two strains of mouse cells could not be demonstrated. Immunodiffusion tests for reaction of purified, disrupted REV with antisera specific for ALSV structural components and for the interspecies specific reaction characteristic of mammalian RNA tumor virus p 30 protein were uniformly negative. After consideration of all the available data, it seems that the REV must be considered a distinct group of avian RNA tumor viruses with significant structural similarities to mammalian viruses, but nonetheless differing antigenic determinants.
1. Nucleic acid was extracted from radioactively labeled visna virus particles and analyzed by equilibrium zonal centrifugation in glycerol density gradients.2. The major component recovered from virions has a sedimentation coefficient of 60–70 S; it is single-stranded RNA as shown by its complete sensitivity to ribonuclease and density after isopycnic gradient centrifugation in Cs2SO4. The virion also contains a slowly sedimenting 5-7-S nucleic acid species.3. Visna virions contain nucleic acids that resemble in size and composition those present in RNA oncogenic viruses.
A preferred site for HIV-1 recombination was identified in vivo and in vitro surrounding the beginning of the HIV-1 gag gene. This G-rich gag hotspot for recombination contains three evenly spaced G-runs that stalled reverse transcriptase. Disruption of the G-runs
suppressed both the associated pausing and strand transfer in vitro. Significantly, this same gag sequence was able to fold into a G-quartet monomer, dimer, and tetramer, depending on the cations employed. The pause band
at the G-run (nucleotide (nt) 405–409), which was predicted to be involved in forming a G-quartet monomer, diminished with
increased HIV-1 nucleocapsid (NC) protein. More NC induced stronger pauses at other G-runs (nt 363–367 and nt 382–384), a
region that forms a G-quartet dimer, adhering the two RNA templates. We hypothesized that NC induces the unfolding of the
monomeric G-quartet but stabilizes the dimeric interaction. We tested this by inserting a known G-quartet formation sequence,
5′-(UGGGGU)4-3′, into a relatively structure-free template from the HIV-1 pol gene. Strand transfer assays were performed with cations that either encourage (K+) or discourage (Li+) G-quartet formation with or without NC. Strikingly, a G-quartet monomer was observed without NC, whereas a G-quartet dimer
was observed with NC, both only in the presence of K+. Moreover, the transfer efficiency of the dimerized template (with K+ and NC) reached about 90%, approximately 2.5-fold of that of the non-dimerized template. Evidently, template dimerization
induced by NC creates a proximity effect, leading to the unique high peak of transfer at the gag recombination hotspot.
Nascent M12 RNA has been isolated in large quantities from Replicative Intermediate and denatured by a short heat treatment. After sucrose gradient centrifugation of the denatured Replicative Intermediate in a zonal rotor, the nascent RNA strands were divided into six fractions with increasing sedimentation values. Each fraction was further purified by sedimentation conventional sucrose gradients. In this manner, a set of M12 nascent RNA strands with fairly uniform lengths, ranging from about 15 nucleotides to almost 90% of the complete phage RNA genome, have been obtained. The molecules in these fractions, all containing the same 5′-end as complete phage RNA but an increasing portion of the phage genome, provide suitable tools for studies of the structure and function of phage RNA.
The physical and biochemical characteristics of progressive pneumonia virus were found to be remarkably similar to those of the ribonucleic acid (RNA) tumor viruses. Significant findings included the presence of a 60 to 70S RNA genome, RNA-dependent deoxyribonucleic acid polymerase activity, and common morphological properties. This information correlates with previously reported biological observations and supports the provisional inclusion of enveloped RNA-containing "slow viruses" within the RNA tumor virus group.
System requirements: World Wide Web browser and PDF reader. Mode of access: Available through the Internet. Title from document title page. Document formatted into pages; contains vi, 119 p. : ill. Thesis (Ph. D.)--West Virginia University, 1999. Vita. Includes abstract. Includes bibliographical references.
System requirements: World Wide Web browser and PDF reader. Mode of access: Available through the Internet. Title from document title page. Document formatted into pages; contains vi, 143 p. : ill. (some col.). Thesis (Ph. D.)--West Virginia University, 2006. Vita. Includes abstract. Includes bibliographical references.
We have examined the interaction of several psoralen derivatives with the RNA genome of Rous sarcoma virus (RSV). In the presence of long-wavelength uv light, 4,5′,8-trimethylpsoralen (trioxsalen) and its 4′-hydroxymethyl (HMT) and 4′-aminomethyl (AMT) derivatives all efficiently crosslinked the subunits of the RSV 70 S RNA complex into a nondissociable state. These psoralen derivatives also were able to penetrate virions and crosslink the RNA complex in situ. The ability of psoralen to crosslink the subunits of viral RNA in situ is consistent with the presumption that the RSV genome exists as a complex within the virion and suggests an approach for a detailed examination of regions of secondary structure. The addition of psoralen adducts to viral RNA correlated with a loss of virus infectivity and a decrease in the amount and length of viral DNA synthesized in vitro and in vivo. Our results indicate that the RSV genome contains a significant amount of secondary structure that is reactive with psoralen compounds and that the addition of HMT to viral RNA causes premature chain termination during viral DNA synthesis.
RD cells, line 2, derived from a human rhabdomyosarcoma were used for these studies3. The FeLV was derived from an interference end point dilution beyond the focus forming end point of the Gardner-Arnstein strain of feline sarcoma virus in beagle cells4. The virus stock, in thirteenth passage in beagle cells, contained approximately 103 (
Le cytoplasme de cellules de souris STU produisant en permanence le virus de Friend contient des particules virales à différents stades de leur développement. Il est possible de libérer ces particules en faisant éclater les cellules dans un tampon hypotonique. Après sédimentation à 90 000 g, la très grande majorité des particules virales est concentrée dans le culot, ainsi que le montrent la microscopie électronique et la caractérisation de l'activité transcriptase inverse. Les Poly (A) RNAs de ce culot contiennent principalement 2 espèces moléculaires 35 S et 70 S. L'hybridation de ce Poly (A) RNA à du c-DNA homologue du génome viral révèle également la présence de 2 RNAs 35 S et 70 S ainsi que d'autres espèces qui sont peut-être des agrégats. Pour éviter l'interférence des RNAs messagers viraux, nous avons utilisé des conditions expérimentales qui permettent la dissociation des polysomes et empêchent leur passage dans le culot de centrifugation à 90 000 g.
Encapsidating the viral genome into virions is an essential step in generating infectious viral particles. Most HIV-1 particles contain two copies of full-length viral RNA indicating genome encapsidation is an efficient and regulated process. Interactions between the HIV-1 structural protein Gag and cis-acting elements in the viral RNA mediate the packaging of viral RNA. The HIV-1 genome selects its copackaged RNA partner, or dimerizes, prior to encapsidation. Several aspects of virus biology and host-virus interactions important for the packaging of HIV-1 viral genomes are discussed in this review. © 2013 Springer International Publishing Switzerland. All rights are reserved.
Hydrogels are supramolecular assemblies with both solute transport properties like liquids and mechanical properties like elastomers. To date, every type of biomolecules, except ribonucleic acid (RNA), are capable of forming hydrogel. Here we report an RNA that forms hydrogel by self-assembly. This RNA is originally identified by systematic evolution of ligands by exponential enrichment (SELEX) to enhance the activity of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors as a potential RNA drug for treatment of cognitive disorders. The RNA hydrogel exhibits elastic modulus plateau in the order of 10² Pa and shows dynamic RNA chain interactions with relaxation behaviors similar to living worm-like micellar solutions. Small angle X-ray scattering and cryogenic electron microscopy characterization support the RNA network structures. By sequence mutation and rheological measurement, we reveal two key sequence motifs in the RNA responsible for intermolecular recognition and the formation of polymer network by self-assembly.
The foregoing discussion of the phylogeny of tRNAs clearly indicates that the genetic mechanism had existed in living things prior to the advent of the prokaryotes. Thus the latter group per se throws very little light upon the origins and early evolution of the nucleic acids and other prominent features used in inheritance. To gain an understanding of those matters the search must be continued elsewhere: An exploration into the genetic processes in viruses may at least suggest models of the early events, even if not firm evidence of particular occurrence.
The biological importance of virion polymerases to the infection process of viruses can be gauged from the fact that many groups of viruses possess virion nucleic acid polymerases of one form or another. A list of those animal RNA virus groups (and their members) shown to possess RNA-directed RNA polymerases (RNA transcriptases) is given in Table 1. Included are representatives of the arena-viruses, bunyaviruses (and bunyaviruslike viruses), orthomyxoviruses, paramyxoviruses, reoviruses (diplornaviruses), and rhabdoviruses. Oncornaviruses and similar virus types (e.g., visna) possess an RNA-and DNA-directed DNA polymerase, otherwise known as a reverse transcriptase (Table 2). Of the various DNA virus groups, the poxviruses and possibly the icosahedral cytoplasmic deoxyriboviruses possess a virion DNA-instructed RNA polymerase (Table 3). Virus isolated from patients with serum hepatitis appears to possess a DNA-directed DNA polymerase.
In the late 1960s, virologists made the surprising discovery that retroviruses exhibit a form of sexual reproduction. Humans are genetically “diploid”, which means that, except in germline cells, they maintain two sets of genetic information (in DNA, on paired chromosomes), and of course they reproduce sexually.
It is many years since the existence of a human oncogenic virus that induces malignant tumors in man was first suggested. Many oncogenic viruses have been found in various species of animals, and it is not hard to imagine that a virus exists, that is oncogenic in man. If the existence of oncogenic virus in man is confirmed, it will allow substantial advances in the diagnosis and treatment of cancer and will have direct applications in the prevention of human tumor.
Avian retrovirus research began with the first transmission of chicken leukemia in 1908 by Vilhelm Ellerman and Oluf Bang (1) who demonstrated that cell suspensions and cell-free filtrates from tissues of a chicken with myeloblastosis produced a similar disease in recipient birds. Over the next 12 years eight separate virus isolations were reported from Ellerman’s laboratory and confirming experiments were described in independent investigations from three other laboratories (2). The fact that it was many years before this pioneering work gained the recognition it deserved, grew from the misunderstanding, in those early years, of the true nature of leukemia. Nearly 20 years were to pass until, in the late twenties, transplantation studies in mammalian systems established that leukemic cells behaved like cancer cells (3). Indeed, because of their viral etiology, scepticism as to the relevance of avian leukemias persisted at least until 1967, when one text referred to avian and murine leukemias as “leukaemoid reactions to infection” (4). Thus, the later studies of Peyton Rous with solid chicken tumors were to be much more influential in the early years of cancer virus research.
The earliest description of cancer of the breast and probably of cancer in any form dates back 3,000 years to the Egyptians. A recent report on cancer gives indirect evidence that the etiological agent in mammary carcinoma is a RNA tumor virus.
The essential requirement for studying the molecular biology of cancer cells is a tool which can change normal cells into cancer cells. Fortunately, this tool is provided by nature itself; cancer is induced upon infection by certain viruses. There are DNA and RNA-containing viruses which can transform normal cells into malignant cells as Dr. Pontén pointed out.
Es ist bekannt, daßRNS-Tumorviren in einer Reihe von Tieren wie Hühnern [1], Mäusen [2], Katzen [3] usw. Leukämien und Lymphome hervorrufen. Der Beweis für die kausale Rolle dieser Viren ist bei Tieren verhältnismäßig einfach zu erbringen. Zellfreie Extrakte von erkrankten Tieren werden in gesunde Tiere injiziert und rufen dort die betreffende Krankheit hervor. Beim Menschen ist diese Art der Beweisführung aus verständlichen Gründen nicht möglich. Indirekte Evidenz muß zusammengetragen werden, um die Ätiologie menschlicher Malignome mit ihren Implikationen für Prophylaxe und Therapie zu klären.
Avian RNA tumor viruses are a group with certain characteristics that can be defined by both pathogenic and molecular criteria. Other designations which have been used for RNA tumor virus include leukovirus, oncornavirus (oncogenic RNA), Rous virus, retravirus (reverse transcriptase positive virus), rnadna virus (RNA → DNA virus), and type C Virus. Historically, the viruses have been isolated from birds with certain diseases and so have been named after these diseases. The following viruses are considered to belong to the avian RNA tumor virus group.
Die mit 32P markierte RNS 70S der AMV wurde durch Wärme, mit DMSO oder Formamid denaturiert und durch Zentrifugieren im Glycerolgradienten fraktioniert. Verschiedene Fraktionen wurden mittels einer Methode, die Aufschluss über multiple Kopien der DNS gibt, mit der Zell-DNS hybridisiert. Im Hinblick auf den Rest der viralen DNS sind die Fraktionen mit relativ niedrigem Sedimentationskoeffizienten zugleich an Poly A und an mit der DNS schnell hybridisierbaren Sequenzen angereichert. Nach der Behandlung der Hybride mit der RNase der Bauchspeicheldrüse oder T1 spaltet die RNase T2 etwa die Hälfte der viralen an die DNS gebundenen RNS. Die durch T2 freigesetzte Fraktion setzt sich hauptsächlich aus Poly A zusammen. Diese Ergebnisse bestätigen und verbessern die alteren Untersuchungen über die teilweise Gemeinschaft zwischen der RNA der onkogenen Viren und der DNS oder mRNS der Zellen. Sie werden im Licht der neueren Arbeiten über die an mRNS gebundenen Poly A- Ketten und die Entdeckung von Kopien der komplementären DNS der viralen RNS in den durch die onkogenen Viren veränderten Zellen erörtert.
RNA tumor viruses are useful tools for the study of oncogenesis because they rapidly induce tumors in animals and efficiently transform cells in culture. These viruses are distinguished by certain morphological features (Bernhard, 1960; Sarker et al., 1971a), an exceptionally large single-stranded RNA genome (about 30,000 nucleotides, Duesberg, 1970) and an RNA-directed DNA polymerase which transcribes the viral genome into single- and double-stranded DNA (Baltimore, 1970; Temin and Mizutani, 1970; Temin and Baltimore, 1972). This transcription, the mechanism by which it occurs, the fate of its products in the infected cell, and the role of the products in both the viral life cycle and virus-induced transformation of the host cell are the principal subjects of our discussion.
Along with other carcinogens of physical or chemical origin, viruses are known to be associated with and to cause tumors in a variety of experimental animals. RNA tumor viruses in particular are widespread in many species of animals, and frequently cause sarcomas or leukemia. The isolation of fowl sarcoma-leukemia virus in the early 1910s by a number of investigators marked the first successful identification of such tumor viruses (Ellermann and Bang, 1909; Rous, 1911; Fujinami and Inamoto, 1914). The cell-virus systems used for the studies of basic aspects of the pathogenesis of avian tumor viruses illustrate the progress in methodology for studying animal viruses in general. Early work in animal hosts was gradually replaced by a system using the chorioallantoic membrane of eggs (Keogh, 1938), then by tissue culture cells (Manaker and Groupé, 1956). The establishment of an assay system for Rous sarcoma virus (RSV) in tissue culture cells (Temin and Rubin, 1958) eventually led to an era of quantitative studies on the mechanism of cellular alteration by viruses.
Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Traditional chemical and enzymatic reagents and backbonebased cleavage are useful for mapping RNA secondary structure, and on going advances in nucleotide resolution RNA structure probing have made possible increasingly rigorous and quantitative analysis. Although chemical and enzymatic probes are recently coupled to high throughput sequencing, these techniques still suffer from some disadvantages. This project aims the optimization of two existing techniques and is divided into two major topics. The first part aims the optimization of "hSHAPE chemistry". A practical method is the synthesis of energy transfer dyes from the "BigDyes" family that are useful to study small amount of RNA and are compatible with DNA sequencing. We have done the synthesis of 5 modified dyes and one BigDye. The characterization of this BigDye shows that it exists excitoncoupling mechanism due to the strong interaction between the two transition dipoles. In addition, the solvent influences strongly the photophysical properties of this BigDye. The purpose of the second part is to develop and synthesize new class of isatoic anhydride derivatives useful to map the entire genome of HIV-1 in virio. We have done the synthesis of 4 N-substituted molecules with a propyn-2-yl function and 8 reference molecules. In addition, preliminary results on HIV-1 1-311 RNA showed that these compounds are able to modify RNA and that it is possible to fix a biotin-azide by click chemistry.
The paramyxovirus group is a large one which includes the parainfluenza viruses types 1–5, Newcastle disease, and mumps viruses. Measles, canine distemper, and rinderpest viruses form a distinct subgroup on the basis of antigenicity, hemagglutinating characteristics, and lack of evidence for a virion-associated neuraminidase or neuraminic acid-containing cellular receptors. However, it is now generally accepted that these viruses should also be included in the paramyxovirus group because of their similar structural properties. Other more recently isolated viruses which have been classified as paramyxoviruses on the basis of morphological and biological properties are Yucaipa (Dinter et al., 1964) and Nariva (Walder, 1971) viruses. Table 1 lists paramyxoviruses and their primary hosts.
The isolation of RNA requires an effective means of cell disruption, a procedure for separating the nucleic acid from the protein or lipoprotein with which it is intimately associated, and a method for purification. To avoid degradation or denaturation, the RNA must be protected throughout from liberated nucleases, strong mechanical forces, high temperatures and extremes of pH and ionic strength. A number of methods which have been reported appear to meet these requirements when applied to one or a number of plants, animals or micro-organisms. However, none of them are universally applicable, due to a number of tissue and species specific differences. For example, variations in cell or organelle structure dictate different methods of disruption. Differences between tissues in the pH optima and inhibitor sensitivity of their nucleases necessitate procedures adapted to each material which minimize enzymic breakdown. Other variables of which a satisfactory method must take account include the resistance to denaturation of the RNA to be recovered and the nature of the principal contaminants. As a guide to the isolation of RNA from tissues for which no satisfactory procedure has been reported, a number of methods of cell disruption, nucleoprotein dissociation and RNA purification are described and evaluated.
Viruses have been shown to share unique properties, which may help in finding approaches for elucidation of the patterns of their gene expression and regulation. This chapter discusses these approaches for five groups of large RNA viruses of animals, such as reoviruses, rhabdoviruses, paramyxoviruses, myxoviruses, and oncornaviruses. These groups differ significantly from each other in structure and mechanisms of replication. Reoviruses represent a unique group of viruses with the segmented double-stranded RNA genome. Unlike the other four groups, they have an icosahedral type of subunit arrangement and lack a lipid-containing envelope. The genome of rhabdoviruses and paramyxoviruses is a continuous single-stranded RNA, whereas the genome of myxoviruses and oncornaviruses consists of several separate segments. Oncornaviruses differ from the four other groups by their unique mode of reproduction, involving the synthesis of DNA. The aim of this chapter is to review present knowledge concerning involvement of viral cores and nucleocapsids in transcription in and in vivo and to discuss the possibility that nucleocapsids represent functionally active units in this process. These sections are preceded by a section where structure, composition, and some properties of cores and nucleocapsids are briefly summarized, the data being used for further discussion. Finally, this chapter includes a section concerning some aspects of template-enzyme interactions.
The genetic analysis of RNA tumor viruses has two main objectives: (1) to provide an understanding of virus replication and (2) to explain virus-induced transformation of the host cell. Virus replication results from a complex interaction of viral and cellular genomes. Viral genetics, however, considers only the virus side of this interaction; the numerous and specific cellular functions which are required for the synthesis of infectious virus will have to be defined by a genetic analysis of the host cell. In virus-induced transformation, viral genetic information presumably interferes with the genetic regulatory apparatus of the host cell, and here again it is important to realize that focusing on the viral information will reveal only part of a very complex interaction between two organisms. Despite these obvious limitations, the viral genome is at the moment that partner in this interaction which is more amenable to experimental study and offers realistic opportunities for an increase of our insight into virus replication and cellular transformation.
Nuclease S1, specifically splitting only single-stranded polynucleotides has been used to detect the double-stranded regions of high-molecular-weight AMV-RNA. Nuclease S1-resistant material comprising approx. 8% of 60S AMV-RNA molecule was isolated, purified and found to be completely nuclease S1-resistant when native and completely nuclease S1-sensitive upon heat denaturation. The symmetric nucleotide composition with equal G-C and equal A-U contents is also consistent with double-stranded nature of this material. Poly A does not participate significantly, if at all, in nuclease S1-resistant structures. It is suggested that those base paired regions might participate in linking the RNA subunits together to form an aggregate 60S RNA molecule of oncornaviruses.
The co-packaged RNA genomes of human immunodeficiency virus-1 recombine at a high rate. Recombination can mix mutations to
generate viruses that escape immune response. A cell-culture-based system was designed previously to map recombination events
in a 459-bp region spanning the primer binding site through a portion of the gag protein coding region. Strikingly, a strong preferential site for recombination in vivo was identified within a 112-nucleotide-long region near the beginning of gag. Strand transfer assays in vitro revealed that three pause bands in the gag hot spot each corresponded to a run of guanosine (G) residues. Pausing of reverse transcriptase is known to promote recombination
by strand transfer both in vivo and in vitro. To assess the significance of the G runs, we altered them by base substitutions. Disruption of the G runs eliminated both
the associated pausing and strand transfer. Some G-rich sequences can develop G-quartet structures, which were first proposed
to form in telomeric DNA. G-quartet structure formation is highly dependent on the presence of specific cations. Incubation
in cations discouraging G-quartets altered gel mobility of the gag template consistent with breakdown of G-quartet structure. The same cations faded G-run pauses but did not affect pauses
caused by hairpins, indicating that quartet structure causes pausing. Moreover, gel analysis with cations favoring G-quartet
structure indicated no structure in mutated templates. Overall, results point to reverse transcriptase pausing at G runs that
can form quartets as a unique feature of the gag recombination hot spot.
The denaturation of single-stranded and double-stranded RNA's in solutions with varying proportions of dimethyl sulfoxide has been followed by changes in absorbancy, optical rotation, and—with a double-stranded form of bacteriophage of MS2 RNA— infectivity for bacterial spheroplasts. By these criteria the RNA's studied, including the synthetic polynucleotide rG:rC, are completely denatured at room temperature in high concentrations of this solvent. In lower concentrations, the Tm of the RNA preparation is decreased only slightly as the dimethyl sulfoxide concentration is raised until a critical concentration is reached. The Tm falls sharply with small further increases in dimethyl sulfoxide concentration. Sedimentation studies can be conducted directly in these media. The determination of sedimentation velocity in 99% dimethyl sulfoxide containing 0.001M EDTA provides a reliable estimate of RNA molecular weights.
Ribosomal ribonucleic acid preparations of exceptional stability were obtained from the ribosomes of Escherichia coli by a method of isolation employing phenol, sodium dodecyl sulfate, and a purified hectorite, Macaloid. The sedimentation and viscosity properties of the total ribosomal RNA and of the separated components were extensively investigated and molecular weight determinations were made by sedimentation viscosity, sedimentation equilibrium, and viscosity kinetics at elevated temperatures. Molecular weights of 1.07 × 106 and 0.55 × 106 g/mole were found for the 23 and 16 S components, respectively. The influence of RNA aggregation upon hydrodynamic parameters was evaluated and several methods (organic solvents, reaction with formaldehyde, low ionic strength, and heat) are suggested for the detection of aggregates within RNA preparations. Chromatography of undegraded ribosomal RNA upon DEAE-cellulose was found to be complicated by the formation at equilibrium of a nonelutable complex between the ion exchanger and the polynucleotide. Base composition analyses were performed upon the separated ribosomal RNA components, and slight but significant differences were found between the 23 and 16 S molecules. It is concluded that the ribosomal RNA of E. coli is composed of two classes of polyribonucleotide chains, each class being covalently continuous and thus not containing polynucleotide subunits.
A method for separating high molecular weight RNA or DNA by electrophoresis on polyacrylamide gels has been described.Ten viral nucleic acid species and four Escherichia coli RNA species have been used to calibrate the system and demonstrate a general relationship between the logarithm of the molecular weight and the relative electrophoretic mobility.The elution of viral RNA from gel slices and the demonstration of infectivity after electrophoresis are described.
Turnip yellow mosaic virus (TYMV) has been treated at alkaline pH (10.5–11.0) and high ionic strength (1.0 M KCl) at 30° for 8 minutes. According to Kaper and Halperin (1965) such a treatment causes in situ breakage of the viral RNA chain, yielding fragments of uniform size (about 5 S). In the present paper it is demonstrated that in situ fragmentation is accompanied by in situ aggregation of the RNA fragments. The aggregate can be released as such from the capsid with phenol and sediments more rapidly and more uniformly than TYMV-RNA. It is assumed that each aggregate molecule is derived from one virus particle and has adopted a structure which is more compact than that of TYMV-RNA. Deaggregation, which is essentially irreversible outside of the capsid, can be accomplished by (a) heating at 55° for 2 minutes; (b) treatment with dime thylsulfoxide; (c) the successive removal of divalent and monovalent cations. Below limiting temperatures aggregates of intermediate sizes persist when the heating is prolonged. Possible models for the structure of the aggregate are discussed.
Influenza virus PR8 has been purified from the allantoic fluid of infected chick embryos and from tissue culture medium. Preliminary characterization indicates that the virus contains at least two major proteins and several RNA components with sedimentation coefficients (S20,w) ranging from about 9 to 18 s.Synthesis of virus-specific RNA in influenza virus-infected chick embryo cells has been demonstrated under conditions where cell RNA synthesis is suppressed by actinomycin D. In addition to the single-stranded viral RNA components, a smaller fraction of virus-specific double-stranded RNA is labeled with [3H]-uridine in the presence of actinomycin D. This RNA has been purified after incubation of the RNA from infected cells with RNase. It has an approximate sedimentation coefficient of 8 to 10 s, shows a sharp thermal transition and has a lower buoyant density in cesium sulfate than single-stranded influenza virus RNA. Preliminary evidence indicates that the double-stranded RNA may be an intermediate in viral RNA synthesis.
Infectious RNA from turnip yellow mosaic virus (TYMV) has been prepared by phenol extraction. The molecular weight determined by sedimentation and viscosity measurements is 2·3 ± 0·15 × 106, corresponding with the maximum molecular weight of RNA per particle. The heating of TYMV RNA to 60°C in 0·01 M-salt resulted in a rapid drop in molecular weight for a variable proportion of the RNA. The proportion of thermally stable RNA was found to be positively correlated with the infectivity of the virus from which the RNA was prepared. Virus inactivated by incubation in sap at 37°C prior to purification yielded RNA which contained no thermally stable molecules, and which was noninfectious.
Zone centrifugation of heated, infectious RNA on a sucrose gradient indicated that nearly all the infectivity is present in the small fraction of thermally stable molecules, corresponding with M = 2·3 × 106. These data are consistent with two alternative hypotheses:
(a)infectious RNA consists of a single polynucleotide chain. Inactivation and thermal lability are caused by chain scission by degradative agents;
(b)infectious RNA consists of several polynucleotide chains crosslinked by a factor conferring thermal stability to the complex. Inactivation and thermal lability are caused by loss of this factor.
The electron microscope was used to make particle counts of the viruses of Newcastle disease of fowls, fowl plague, mumps and influenza C. Two counting techniques previously described were also used, and correlations with simultaneous measurements of haemagglutination and infectivity were made.
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