Partially separated double-stranded RNA from purified Rous sarcoma virus, Schmidt.Ruppin strain, was observed by electron microscopy utilizing 8.M urea and protein monolayer technique. Furthermore, viruses in pair were frequently and viruses with two nuc1eoids were occasionally
observed in ultrathin. sectioned specimens of chick cells transformed by RSV.
From these results taking other reports in consideration, a possible mechanism of RNA replication in Rous sarcoma virus is proposed.</p

Oda, Takuzo

Ogura, Hajime

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A possible mechanism of RNA replication in Rous sarcoma virus

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Acta Medica OkayamaVolume 25, Issue 5 1971 Article 6OCTOBER 1971A possible mechanism of RNA replication inRous sarcoma virusHajime Ogura∗ Takuzo Oda†∗Okayama University,†Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.A possible mechanism of RNA replication inRous sarcoma virus∗Hajime Ogura and Takuzo OdaAbstractPartially separated double-stranded RNA from purified Rous sarcoma virus, Schmidt.Ruppinstrain, was observed by electron microscopy utilizing 8.M urea and protein monolayer technique.Furthermore, viruses in pair were frequently and viruses with two nuc1eoids were occasionallyobserved in ultrathin. sectioned specimens of chick cells transformed by RSV. From these resultstaking other reports in consideration, a possible mechanism of RNA replication in Rous sarcomavirus is proposed.∗PMID: 4344875 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 25, 567-571 (971)A POSSIBLE MECHANISM OF RNA REPLICATIONIN ROUS SARCOMA VIRUSHajime OGURA and Takuzo aDADepartment of Biochemistry, Cancer Institute, Okayama University Medical School,Okayama, Japan (Director: Prof. T.Oda)Received for publication, September 2, 1971RNA dependent RNA polymerase (EC 2. 7. 7. 6) has been detected inpurified virions of vesicular stomatitis virus (1), influenza virus (2, 3) andNewcastle disease virus (4), which have single stranded RNA in theirvirions. The same enzyme was detected in E. coli infected with the RNAbacteriophage MS 2 (5) and Q,j' (6). In these viruses, replication ofthe nucleic acid is mediated by virus.induced RNA dependent RNApolymerase.Recently RNA dependent DNA polymerase has been proved in vitroin the virions of RNA type oncogenic viruses (7, 8), which have also singlestranded RNA. It strongly supported the idea of participation of DNA(9) in transformation and replication of these viruses.The activity of RNA dependent RNA polymerase from myeloblasts(10, 11) infected with avian myeloblastosis virus and from tumors inducedby Friend virus (12) was reported, while the enzyme activity in chickembryo cells infected with Rous associated virus was not detected (13).The presence of the enzyme in RSV.infected cells was also suggested (14).At the present time, very little is known concerning replicationmechanisms of oncogenic viral RNA in permissive cells.In our electron microscopic studies of RSV and its RNA, interestingfigures were observed. On the basis of these findings, a possible mechanismof RSV.RNA replication is proposed.MATERIALS AND METHODSThe source of RSV, Schmidt-Ruppin strain, and purification procedureswere as described before (15). The purified viruses through sucrose densitygradient were treated with 0.05 % deoxycholic acid and 1% sodium dodesylsulfate for 10 min at 3rC to release viral RNA from virions (16). The releasedviral RNA was precipitated with 2 vol. of cold ethanol. Discarding the super-natant after centrifugation, the precipitate was dissolved in a small amount ofsaline sodium citrate (pH 7.4). The RNA solution in saline sodium citrate was5671Ogura and Oda: A possible mechanism of RNA replication in Rous sarcoma virusProduced by The Berkeley Electronic Press, 1971568 H.OeURA and T.ODAdiluted with 8 M urea (17), and protein monolayer technique (18) was appliedfor electron microscopic preparation.Transferred cultures of chick cells transformed by RSV were fixed withglutaraldehyde and osmium tetroxide. The fixed samples were ultrathin section.ed, and were stained with uranyl acetate and lead citrate. The specimens wereexamined in an electron microscope, Hitachi HU.IIC.RESULTS AND DISCUSSIONAmong linear structures of many RSV.RNA isolated from purifiedRSV through sucrose density gradient (15), partially separated doublestranded RNA of 3.86 ,ft in length (Fig. 1) was 0 b3erved. At the separatedparts, both of the strands were the same in length. RSV with two nucleoids(Fig. 2) was rarely observed in extracellular space in an ultrathin. sectionedspecimen of cultured chick cells transformed by RSV. The size of theRSVs with two nucleoids were 81 ~ 100 mr~ in diameter and the shape ofthe two nucleoids were elongated showing an ovoid shape. Furthermore,RSVs appeared in pair were frequently observed in extracellular spaces(Fig. 3).It has been reported that the cell nuclei transformed by murinesarcoma·leukemia virus contain RNA complementary to viral RNA andsome nuclear RNA are double stranded (19). In pair arrangement of virusparticles in the specimens of Rous sarcoma cells (20) and avian leukosisvirus.infected cells (21) were reported before. Virus particles with twonucleoids were also reported in the specimens of avian leukosis virus-infected cells (21) and passenger C-type viruses with two nucleoids in"mirror image" were sometimes ob3erved in adenovirus-transformedhamster cells (22). In those reports, a possibility of virus replication byfission was suggested. The hypothesis on division of virus particle isinteresting but lacks experimental evidence yet. The virus particles withtwo nucleoids might, however, be explained better as malformation ofvirus particles during the formation or maturation of virions. Partiallyseparated double-stranded RNA from purified virus may be interpretedas RNA from malformed RSV which is formed in the process of double.stranded RNA formation by RNA dependent RNA polymerase and isFig. I Electron microscopic figure of double stranded RSV-RNA isolated from purifiedRSV (Schmidt-Ruppin strain). Arrows indicate the parts of a partially separated RNAstrand. Mar:<er presents 1,11.Fig. 2 RSV with two nucleoids appeared in an e. tracellular space in ultrathin-sectionedspecimen of cultured chic:, cells transformed by RSV. n: nucleoidsFig. 3 In pair appearance of RSVs in extracellular spaces. p: RSVs in pair2Acta Medica Okayama, Vol. 25 [1971], Iss. 5, Art. 6http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss5/6Mechanism of RNA Replication in Rous Sarcoma Virus 5693Ogura and Oda: A possible mechanism of RNA replication in Rous sarcoma virusProduced by The Berkeley Electronic Press, 1971570 H. QGURA and T. QDAcovered with coat protein before the complete separation of double-stranded RNA. It is not yet clear whether one of RSVs appearing inpair contains complementary minus strand RSV-RNA or not. From theseobservations, we propose a possible model of RSV.RNA replication asshown in Fig. 4. In order to clarify these hypothesis, experiments arenow in progress.~RSV~RSV ·RNA{.=Y- -~~~--RNA( +) .- RSV - RNA (:-)________ l' ~" ... - tds RSV-ONA .;.,.. -. RSV-ONA(-)t ". ' ,j,RSV'RNA(-), ds R5V-RNA------- -'" --------Provirus in ChromosomeFig. 4 A possible model of RSV-RNA cycle.(+ ): plus strand of viral RNA, (-): complementary minus strand of viral RNA,ds: double strandedSUMMARYPartially separated double-stranded RNA from purified Rous sarcomavirus, Schmidt.Ruppin strain, was observed by electron microscopy utiliz.ing 8.M urea and protein monolayer technique. Furthermore, viruses inpair were frequently and viruses with two nuc1eoids were occasionallyobserved in ultrathin. sectioned specimens of chick cells transformed byRSV.From these results taking other reports in consideration, a possiblemechanism of RNA replication in Rous sarcoma virus is proposed.ACKNOWLEDGEMENTThis work was supported by a Grant-in-Aid for Scientific Reserch from the Ministryof Education of Japan.REFERENCES1. BALTIMORE, D., HUANG, A. S. and STAMPFER, M.: Ribonucleic acid synthesis of vesicularstomatitis virus 11. An RNA polymerase in the virion. Proc. N atl. Acad. Sci. 66, 572,19702. CHOW, N. and SIMPSON, R. W.: RNA-dependent RNA polymerase activity associatedwith virions and subviral particles of myxovirus. Proc. Natl. Acad. Sci. 68, 752, 19713. DOYLE, M. and BLATTI, S.: RNA-dependent RNA polymerase activity in influenzavirions. Proc. Natl. Acad. Sci. 68, 1369, 19714. HUANG, A. S., BALTIMORE, D. and BRATT, M. A.: Ribonucleic acid and polymerase in4Acta Medica Okayama, Vol. 25 [1971], Iss. 5, Art. 6http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss5/6Mechanism of RNA Replication in Rous Sarcoma Virus 571virions of newcastle disease virus: Comparison with the vesicular stomatitis virus poly-merase. j. Virol. 7, 399, 19715. HARUNA, I., Nozu, K., OHTAKA, Y. and S?IEGELMAN, S.: An RNA replicase induced byand selective for a viral RNA: Isolation and properties. Proc. Natl. Acad. Sci. 50,905,19636. HARUNA, I. and SPIEGELMAN, S.: Specific template reqirements of RNA replicases. Proc.Natl. Acad. Sci. 54, 579, 19657. BALTIMORE, D.: Viral RNA-dependent DNA polymerase. Nature 266, 1209, 19708. TEMIN, H. M. and MIZUTANJ, S.: RNA-dependent DNA polymerase in virion of Roussacoma virus. Nature 266, 1211, 19709. TEMIN, H. M.: Nature of the provirus of Rous sarcoma. Nat!. Cancer Ins!. Monogr. 17,557, 196410. WATSON,K.Fo, HARUNA,I. and BEAUDREAU,G.S.: Purification of an RNA-dependentRNA polymerase from leu:iemic cells. Proc. Amer. Assoc. Cancer Res. 8, 71, 196711. WATSON, ¥. F. and BEAUDREAU,G. S.: Isolation of an RNA-dependent RNA polymerasefrom Friend murine leu':emia cells. Biochim. Biophys. Res. Commun. 37, 925, 196912. HARUNA, I., OHNO, To, WATANABE, I. and IKAwA, Y.: Isolation of an RNA-dependentRNA polymerase from Friend murine leu\emia cells. Proc. jap. Acad. 46, 1016, 197013. WILSON, R. G. and BADER,j. P.: Viral ribonucleic acid pol) merase: Chick embryo cellsinfected with vesicular stomatitis virus or R ous associated virus. Biochim. Biophys. Acta103, 549, 196514. YAMAMOTO, T.: Viral genome in SR-RSV induced mouse ascites sarcoma (SR-C3H/He).Igaku no Ayumi 64, 30, 1963 (in japanese)15. OOURA, H. and ODA, T.: Molecular length of Rous sarcoma Virus (SR-RSV) RNA.Proc. jap. Cancer Assoc. p.61, 1971 (in japanese)16. CARTWRIGHT,B., SI4ALE,C.j. and BROWN,F.: Structure and biochemical relations invesicular stomatitis virus. The biology of large RNA viruses. BARRY, F. I\ and MAHY,B. W.j. eds. Academic Press, Inc., New Yor:i, p.115, 19iO17. GRANBOULAN, N. and SCHERRER, K.: Visualisation in the electron microscope and sizeof RNA from animal cells. European j. Biochem. 9, 1, 196918. KLEINSCHMIDT,A. K., LANG,D., jACHERTS,D. and ZAHN,R. K.: I'arstellung und Lan-genmessungen des gesamten I'eo q ribonucleinsaure-inhaltes von T2-Pa1{teriophagen.Biochim. Biophvs. Acta 11, 857, 196219o BISWAL, N. and BENYESH-'dELNIK, M.: Characterization of the complementary nuclearRNA of murine sarcoma-leu1,:emia virus. Virolog)l 42, 1064, 197020. GAYLORD, W. H.: Virus-li':e particles associated with the Rous sarcoma as seen in sec-tions of the tumor. Cancer Res. 15, 80, 195521. KARRER, H. F. and Cox,j.: Virus particles in normal chic", embrJ"os. j. UltrastructureRes. 4, 360, 196022. OHMORI, M.: Fersonal communication.5Ogura and Oda: A possible mechanism of RNA replication in Rous sarcoma virusProduced by The Berkeley Electronic Press, 1971

A possible mechanism of RNA replication in Rous sarcoma virus

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