The low molecular weight RNAs of Rous sarcoma virus

Using a novel chemical tritium derivative method, we have determined the base composition of 4S RNA isolated from an RNA tumor virus, the avian myeloblastosis virus, and from normal and neoplastic host cells. Extensive differences were detected, particularly with respect to the amount of methylated bases in the viral RNA. The (7), with respect to the four major bases: adenine, guanine, cytosine, and uracil. No quantitative data on modified ("minor") bases have, however, been reported thus far for 4S RNA from any RNA tumor virus. 4S RNA from AMV does not have the same amino acid-accepting activity as host-cell 4S RNA (2, 3, 6), and lysine tRNA elution profiles have been shown to differ (9).We report in this communication major and minor base constitution data for normal chicken liver, myeloblast, and AMV 4S RNA. Base composition was analyzed by a novel isotope derivative method, which involves the stoichiometric incorporation of tritium label into periodate-oxidized enzymatic digests of RNA, chromatographic resolution of such labeled digests into individual radioactive nucleoside derivatives, and evaluation by liquid-scintillation counting (10)(11)(12)(13)(14). Due to its sensitivity, this method is capable of assaying minute amounts of RNA (<1 Mg) without requiring in vivo labeling of the RNA with isotopes. MATERIALS AND METHODSVirus Growth and Purification. Avian myeloblastosis virus, Beard's BAI strain A, was propagated by intraperitoneal injection of 1-day-old White Leghorn chickens (15) (Spafas, Inc., Norwich, Conn.). The morphology of peripheral blood was observed by Wright-stained smears. Plasma was obtained by cardiac puncture (heparin was used as an anticoagulant) from chickens with advanced leukemia. The virus was purified by centrifugation of plasma that contained 101I1012 viral particles per ml at 7500 rpm for 10 min in a Sorvall RC-2B refrigerated 40C centrifuge. The supernatant plasma was layered onto the top of 100% glycerol pads and centrifuged at 50C for 90 min at 29,000 rpm in a Spinco no. 30 rotor (16). The virion was collected from the top of the glycerol pad and diluted with 0.1 M NaCl-1 mM Tris (pH 7.4)-i mM EDTA. The virus was purified further after two cycles of differential centrifugation (17).Isolation of Viral RNA. Viral RNA was isolated from purified preparations of AMV by either of two methods. In the first, purified virus from 300 ml of plasma was extracted according to the procedure described by Travnicek (3). The portion of RNA that was soluble in 2 M NaCl was designated as crude viral tRNA (3), which was further purified by Sephadex column chromatography, as described below. A final yield of 120 1g of purified 4S RNA was obtained.The second method for extraction of the viral RNA used a buffering system described by Duesberg (18). Purified viral pellets were obtained from 650 ml of plasma. The second method appears to be preferable because of better preservation of m7G (see Results section). 320 ,ug of purified 4S RNA were obtained after purification by column chromatogr...

Section: Resultsmentioning

confidence: 94%

mentioning

confidence: 98%

Base Composition Differences between Avian Myeloblastosis Virus Transfer RNA and Transfer RNA Isolated from Host Cells

Randerath

Rosenthal

Zamecnik

1971

“…It-also was established that the 7S RNA isolated from Rous sarcoma virus does not anneal with the viral 65S genome RNA (4). The 7S RNA, therefore, appeared to be derived from host rather than viral genetic information.…”

mentioning

confidence: 95%

The 7S RNA Common to Oncornaviruses and Normal Cells is Associated with Polyribosomes

Walker¹,

Pace²,

Erikson

et al. 1974

The 7S RNA species first demonstrated in avian and murine oncornaviruses and later in normal, uninfected cells is' found associated in part with cellular polyribosomes. A molar ratio of 7S RNA to 5S ribosomal RNA of 0.05 indicates that there is approximately one mole of 7S RNA per mole'of messenger RNA. Dissociation of polyribosomes with dimethylsulfoxide results in a marked decrease in the sedimentation rate of the 7 S RNA.The dimethylsiilfoxide-induced dissociation of polyribosomes and the concomitant movement of the 7S RNA from the polyribosome region into lighter regions of a sucrose gradient are both inhibited by cycloheximide, indicating that the 7S RNA is indeed associated with polyribosomes and not with a ribonucleoprotein particle sedimenting with polyribosomes.The avian oncornaviruses contain a variety of RNA components, including the 65S. genome complex (1) and discrete populations of low-molecular-weight species designated 4 S (2), 5 S (3), and 7 S (4). The virion 4S RNA is structurally (3, 5, 6) and functionally (2, 7) similar to that of the host, and the 5S RNA apparently is identical in hucleotide sequence to the host 5S ribosomal RNA (3). The function of these small RNA species remains enigmatic.The viral 7S RNA, first described from oncornaviruses by

“…The low-molecular-weight population includes 7S, 5S, and 4S RNA and is generally referred to as RSV-free (not associated with 70S RNA) RNA (1)(2)(3)(4)(5)(6). The free 4S RNA exhibits structural features of transfer RNA (tRNA) and can be acylated with amino acids (3,(5)(6)(7)(8)(9)(10)(11).…”

mentioning

confidence: 99%

RNA-directed DNA synthesis by the DNA polymerase of Rous sarcoma virus: structural and functional identification of 4S primer RNA in uninfected cells.

Faras¹,

Dibble²

1975

The RNA-directed DNA Virions of Rous sarcoma virus (RSV) contain two major size classes of RNA: 70S RNA presumed to be the viral genome, and a low-molecular-weight population consisting of a number of discrete RNA species. The low-molecular-weight population includes 7S, 5S, and 4S RNA and is generally referred to as RSV-free (not associated with 70S RNA) RNA (1-6). The free 4S RNA exhibits structural features of transfer RNA (tRNA) and can be acylated with amino acids (3, 5-11). RSV 70S RNA is segmented in nature, containing three to four high-molecular-weight 30-35S RNA subunits (1) and several low-molecular-weight species. This latter group includes 7S, 5S, and 4S RNA and is referred to as 70S-associated (70S-a) RNA (5,(12)(13)(14).Transcription in vitro of the 70S RNA genome of avian RNA tumor viruses by the RNA-directed DNA polymerase is initiated by covalent attachment of DNA to the 3' terminus of an RNA primer molecule (15-17). We have recently identified the RNA primer molecule as a low-molecularweight RNA species, 75 nucleotides in length, exhibiting structural features analogous to those of tRNA (18)(19)(20). This molecule appears to be the principal primer utilized by the RSV DNA polymerase, since its removal from 70S RNA results in the loss of 80-90% of the template activity of the 70S RNA complex (14, 17). In the absence of this RNA primer molecule the template activity of RSV RNA is no better than that of other natural RNA molecules (14, 17), suggesting that the preference exhibited by the RNA-directed DNA polymerase for oncornavirus 70S RNA as template is a special feature of the RNA and not a property of the enzyme. Although no virus-specific functions have previously been assigned to any of the other low molecular weight RNA species associated with virions of RNA tumor viruses, many of the virion-associated tRNA species appear to be cellular in origin and selectively incorporated into the virus particle during budding of the virus from the cell membrane (3, 7-11). It was of considerable interest, therefore, to determine whether the RNA primer molecule was a normal constituent of uninfected cells, or a virus-specified or virus-induced molecule.In