Virus-Specific Ribonucleic Acid in Cells Producing Rous Sarcoma Virus: Detection and Characterization

Leong, J. A. C.; Garapin, Axel-Claude; Jackson, Nola L.; Fanshier, Lois; Levinson, Warren; Bishop, J. Michael

doi:10.1128/jvi.9.6.891-902.1972

Cited by 298 publications

(115 citation statements)

References 35 publications

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“…Reactions were incubated at 68" under mineral oil in plas-tic tubes. Aliquots were removed and the percentage of single-stranded cDNA was determined using Sl nuclease (Calbiochem), which specifically digests singlestranded DNA, using the conditions of Leong et al (1972).…”

Section: Methodsmentioning

confidence: 99%

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Intracellular murine hepatitis virus-specific RNAs contain common sequences

et al. 1981

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A major polyadenylated viral RNA of approximately 0.8 X lo6 daltons was isolated from murine hepatitis virus (A59)-infected cells by preparative polyacrylamide gel electrophoresis in formamide. This RNA was shown to encode the viral nucleocapsid protein by direct in vitro translation in a cell-free, reticulocyte-derived system. Single stranded q-labeled complementary DNA was prepared from this RNA and was demonstrated to be virus specific. Using this complementary DNA in a Northern blotting procedure, we were able to identify six major virus-specific intracellular RNA species with estimated molecular weights of 0.8, 1.1, 1.4, 1.6, 3, and 4 X l@ daltons. All of these RNA species were polyadenylated. Our results support the idea that coronavirus-infected cells contain multiple intracellular polyadenylated RNAs which share common sequences. et al., 1959) of mouse fibroblasts.

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Section: Methodsmentioning

confidence: 99%

“…The DNA was then annealed with r2P]cDNA as described by Morris et al (1977) except cell DNA was 6.1 mg/ml in the annealing mix instead of 3.5 mg/ml. Single-stranded DNA was assayed with Sl nuclease as previously described (Leong et al, 1972).…”

Section: Methodsmentioning

confidence: 99%

Intracellular murine hepatitis virus-specific RNAs contain common sequences

et al. 1981

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“…Infected and uninfected cell RNA were isolated as described . For the analysis of hybridization kinetics (Varmus et u& 1973;, the percentage of singlestranded cDNA remaining in aliquots of the annealing mixture was determined with S-l nuclease (Calbiochem) according to the procedure of Leong et al (1972). RNA was denatured with glyoxal, subjected to electrophoresis in 1.5% agarose gels, transferred to diazobenzyloxymethyl (DBM)-paper, and hybridized with [%P]-cDNA as previously described (Coulter-Mackie et a& 1980;.…”

Section: Methodsmentioning

confidence: 99%

RNA and polypeptide homology among murine coronaviruses

et al. 1981

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Using a =P complementary DNA (cDNA) prepared against the A59 nucleocapsid protein messenger RNA, we have investigated the extent of homology between A59 and four other strains of murine hepatitis virus (MHV). Analysis by hybridization kinetics of the annealing between A59 [S2P]cDNA and infected cell RNA from the other four MHV strains demonstrated 7040% homology. By gel transfer analysis, the A59 [S2P]eDNA was able to detect subgenomic-size virus-specific RNAs in cells infected with all of the five MHV strains. A similar pattern of seven viral RNAs was detected in cells infected with A59, MHV-1, MHV-3, and JHM. In contrast, cells infected with MHV-S contained seven virusspecific RNAs, of which only the two smallest species comigrated with RNAs from the other four strains. The results suggest that as previously shown with A59 (S. Cheley, R. Anderson, M. J. Cupples, E. C. M. Lee Chan, and V. L. Morris (1981) Virdogy, 112,596-604), all MHV strains tested encode a nested set of subgenomic RNAs. Analysis of [?l]methionine-labeled viral proteins by SDS-polyacrylamidegel electrophoresis revealed that each strain of MHV specified four major viral polypeptides with apparent molecular weights very similar to those previously reported for the E2, N, El, and PEl polypeptides of A59. The strong degree of interstrain homology among the five MHV strains investigated was confirmed by comparative chymotryptic peptide mapping of the viral N proteins. A majority of the chymotryptic peptides from each of the [35S$nethioninelabeled N proteins was found to coelute by high-performance liquid chromatography. Moreover, this technique of peptide mapping indicated a particularly strong relatedness between MHV-1 and MHV-S and among MHV3, JHM, and A59.

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“…DNA product from KiMuLV(Ki-MSV) virus preparations grown in normal rat kidney (NRK) cells (17) and hybridization methods using S-1 nuclease are as described (25,26). Over 70% of the [3H]thymidine DNA product hybridized to 70S RNA from purified MuLV virions (26).…”

Section: Nucleic Acid Llybridizations--the Procedures Employed For Tmentioning

confidence: 99%

Radioimmunoassay of Mammalian Type C Viral Proteins

Parks

Livingston

Todaro

et al. 1973

The Journal of Experimental Medicine

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Induction of infectious type C viruses by physical and chemical means from avian and routine cells (1-4) strongly supports the view that most, if not all, mouse and chicken cells contain at least one normally repressed copy of a complete type C viral genome (5). The state of this endogenous viral information is presently unclear. The recent development of a radioimmunoassay for the major internal viral protein (6-8), the group-specific (gs) ~ antigen of murine type C viruses (9-] 2), provides a method for the detection of small quantities of this protein in routine cells below the level of sensitivity of complement fixation or gel diffusion assays (9, ] 1, 13).Using this technique, we have been able to measure an immunological reactivity that is similar to the virion gs protein in both virus-producing and virusfree mouse cell lines and also in natural nontumored murine tissues. The findings reported here provide additional evidence for the widespread prevalence of type C viral genes in a variety of mouse cells and strongly suggest a low level of synthesis of at least one virion protein both in apparently virus-negative tissues in the animal and in virus-free cultured cells. Materials and MethodsCdls.--The cell lines and strains listed in Table I were employed for the present studies. Cells were grown and maintained with Dulbecco's modification of Eagle's medium supplemented with either 10% calf serum or 10% fetal calf serum (Colorado Serum Co., Denver, Colo.).Virus.--The Rauscher strain of rnuriue leukemia virus (MuLV) was obtained from Electro-Nucleonics, Bethesda, Md., as twice sucrose density gradient purified material concentrated 1,000-2,000-fold relative to the starting material.Antigens and Antisera.--The preparation of murine gs antigen [MVP3(gs)], the major polypeptide (30,000 daltons), from disrupted MuLV by Sephadex G-100 gel chromatography and electrophoresis in ampholytes has been described (5), as has the preparation and specificity testing of rabbit anti-MVP3(gs) (6). Cell extracts were prepared by several methods designed to solubilize maximally the gs reactivity. Generally, cell monolayers were washed one to two times with phosphate-buffered saline, pH 7.2, and after removal with a rubber policeman, pre-1 Abbr~iations used in this paper: CF, complement fixation; gs, group specific; MuLV, murine leukemia virus; MVP3(gs), murine group-specific antigen; NRK, normal rat kidney.

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Virus-Specific Ribonucleic Acid in Cells Producing Rous Sarcoma Virus: Detection and Characterization

Cited by 298 publications

References 35 publications

Intracellular murine hepatitis virus-specific RNAs contain common sequences

Intracellular murine hepatitis virus-specific RNAs contain common sequences

RNA and polypeptide homology among murine coronaviruses

Radioimmunoassay of Mammalian Type C Viral Proteins

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