“5517.{ was. “Mpg.“ .- xjflfl? M .mn—nrufiu “nanny.“ cit—5.. ratsfln Skukmu .. «N . .mammmmg .3... Mia... 1» , .2. . .s \. .3. T. .. IS.» u. . T”. , I... I3! 12.. . , Baku—2%“? .3. v4... n. W... 14:. Minna an“; .. i... . .9... .Q ,.hu.....nuuu...snfi.n% . a. #11 . sum. . 1 .XhXfia ngfl“..\\1.\\. RN .Ivd... V1.\ 133‘. . mm. . a... m... . u. Ewhw? $1.”... m? H... a , fianfimmk “83% vs! 51.5.}... k}... ”Infihfiih. . .wufldfiali. g k s fikrgvfiwx. Vii 1 u 3 , ' O. 3 . . V . . . . . .- . , 1.... i. v. n , . . 5% Ema. i§¥vf w$vfl . am. m . a. . . . . . .E ..%.némn.in. a... . x...“ n. I]. .. k6? RfiWuAEfifl iwflwihfiqnmfiaga W. «Y . .3: 1...! .. ._ a... . nazfimxmwfin fl... aka n... . . . , 5K ... ,am.n..%....... .. Idfiunmm. Mat .. . -. n 24... ... .1. NR2... $.31»: WE$§§4§1§§§ 1 . . ”N“: in g. .. LE v . . . $113111 is. a 3. 813.. .. 5 .V1 i {31.1.1 .. Nu ..nu.bw.o¢4zn.hnhn.mmfl8..n¢. .. . Km... uni... 3.38m"... 12 u. fiffi... ..2.r§wn.mmfi.- , s 1-..... 1. - .0 Wmmfi; . , n I. bl . S. :13 v .. . .«I.n.. . affidwn. M. .. u. .. fix%firykflfil .3 fin... .M. . .1: nfifi... . 3.“. a... z . q . )1 IN ' ‘1': ' .u' H .1! . %¥n«mfi&$.fld§h¥. . 5H 5.5.) x. . n. . 1 1...... . . . a...“ ugh... 1 . .. n: . arfimfim. .mmwudfifimmhq mm. .3...wm.. .1. . .3. _ ,. nu n.-. £3... 3...... {.2 I: .. fi. n... .2?.........:. .m... “fa... flawfisz. 5., 8...... as. us... hm? , .u.. n sainVLHWflWW $.ch%huww.§§fi§. .51 .6. W. . .. I . . ~ i‘u . no , . a . . . 3>:.....I.......u.. {tag .4 an R... . . 3.... e 5.. .\ n . r .. n. s 5.... 5.? gran- - ,......... a . . .- .m. .3. sum 1.. . T. . . 3.13.23... .n?... 31%. .3... . . . a. 534%“... n- .. . g In.” .. .31.dufl»§lwuw$. 1.. «3.... ammuk .. \ANfla. . .6 z z... .. an. .. .JM. .3 113,3“... 3... : , ,, .15... 312... x . in": .32K . uhflzintr 1. 8.3% «.332. :6 k... 1%. ¢§.ummrn.thhfiui . g... .Iflmmmfingkiuzifihlflmxuv a}... u.” afiiflmfinzar 9.1g. , (.1...va 2. 51442.1... . y .2... .9... . (.0 n. s LL..- Ed, 1..-. 3L. 5..., 2. 2...... .30. aw lg“... 95nfl%k12 , a u. I:3.¢.A$%QZ§IJ s£\\.$\....5k. 1. . . )1. 3.3 .‘utilstn KG... x I» in... k. «mar xi uh»... mm 5303?. .{I a 3.1.3». . . ’02.! ’77. .. . Iv . .1... 2L)“. {.19. a. . $3 , i. Jaw. . l “‘1‘ )V.:v..}1\lnm( .3... 113$). .43).! 4.6.5.3.... .thLWhlmW.{$.z...7 .13....” , . .. .2 . 5.9%.. 3,. .. 5 .. - ,. Inigflwinbfi . gig; I. .3433: n. nu. . . Bo??b...h4u 5.5.833. .53....5... . . .3».. ha. . .341 .n , a. Idyf.§>.n.iau.fln.n?}m¢ha. .3 t... .. u .c\; 3 . . \Vufié‘ r Emmy. $5 gm angefibcnwnumx-anlhf I . 715V . 5?}. g #5 198%?1331 - _ . k n y u... 1:301“- .2... 1, 52.5.: . 1?...V 2...?) was. 1‘? \\~ . HI}: $§§i~1 waving 1.. . x .. >33... %. knit.n)\\1 u 2 int}. 5311...... 5...». 1 «V 1 a .s s. .1. 3“... , .I. Eg§an;w..zh.¢n9. 1.16.1.3“... n.‘ . J...‘ {Iris .5 . . “kg. 11%. taut)»: urn... , greg. .333: :5? .. 3.46.33. .9431 Jon... .. $.25... A. 51...... 59.453. 3.00.433. 9.... x . nx. .xlrxafiuvhr Zhfisnnsll. g. .. ah. v1.14... .21. 2?... «.41.... ”“54... s .11.)... I £59... 1.2:... flung... b¢1%.§§8¥ .. 3.3.. . . , .anfifiwnfi fig... 1. VII} 53.73i \n 1nflia ngm...~.....5.w.~u.fl..... {3.73.3 ,. um... . .. . . )1, . a. 3:; 12.... ,2 I nsin 1 a , 314...}... n. 2?... 5 on... ibis... 5153‘. 31...... . n 13,313.91... «5 In. 15.2.}. ) . 13...; .v .. . in... 5).. ~21rnnwmmwnfiflnvs. Wasncwhflf «1.): . . ). lir‘L .2. .1 5.003. ‘1‘... . In”? 5 .. .. , .1 . x a 12. .. L. 1.. 2.. . 1561., Viki". {33.3.} n. E... 2.33... «H. 25:132.. , . , 3% 1? l. L n. .30 1 , $3.3. h. \ Nisnfienfis... Jaw. ..).13.3$1)h.$~513215 31%. 3).. . . wmuwu..fi . I with... . .51 1.. ..«fi.nl$nk.1n\ir 1, 1\J.. niuzvzfizkgqfiii . 1.. n 4h§§§ .. . I 71». ..3...l~.l.Vn..».. .1s .35»... .V\ . 3. 1‘7... « . 8.5.1} : , .. .Ixslt,.!»3q\n.aa1{anufirs n18... nfiuflflmz n.2.i...nx...h.y.§¢ I... .nn.qPJn.«.Tu.#.h...va .\ a s... ,n . .. i... gfifiufiflfikflhfiq .3. .5 $21.... awn"ssn$asa flnfimgflfluwv 34.9...Pnhuwwwfi. .5 “4.135 5.115.. 55%:mmvw 2......”5; . . in... I . ,rnff...}.mwuw_ . 1.... .311... a. n3 . 1.1%. . 4.. .3m. . .5 Ln... ban... 3 .1. ,) g» 3 .4518.“ 1 ($3.45.“ .ngfli Tau}. 7) . 5 {SUV} 1.“ . .....n....m. 3.. 3413?... ......,mz....§...3.z .........._,...mf¢. ”3mg? 5. Java , . ....I .\.. 1...; , \mflflfiv . . 1.1.2 .(2...\ \bsi$..dwr1s%}.nun.hn§5 ix. arhNJ‘}; \ qufi. 5 .253... ..xk~$n.....0fl.\ ‘2... 31 3.4.93?»“ , . Lt. V I}: 1.1”: $.59... .. uh x . , n... \ . ‘ 1... «at... 1 . e.¥s,.§u%w1£wrin .. . . . \nvr 3.3.3.5 . \(Sllx Luv. .01} .1‘\$fi)2.3h.LLo-fi)$fl§fif§tfi n. 93?. . . §?.\ 51:4“. f 1131 01:.” $125315“? , «. Munitfigihrvgj (1‘ 3. r\ i 3..» . ‘Y g1 . v wk: 'l .1 .2.th .4 ! 1 .1 3.. \l . . n H n 7 \n I 3h . .firsalfihh 51%; .33 a T 31... nkéffifin .wnfi. .figwfinwznls b....n..u.3.. in“? n. . A n. J . , .n . .3. < gays. flaw“... .knum....§§ .3... 3.8». .nnwfibgfl . nhmnmu . . . I .1. . ‘Innx. 3.1.... ., 1. (Ir. 1. it .vnitn... . . I «2. In“. . jun .3 . .13).}... n ‘31:... ,. Inshfl.’ $3ij t r :n‘sflfl 31. In Aw I. 5% .01.. x . n. navyfivmif .nxgmw. .%$§7¢hnm§f$a}niwfi¥3:n u . . n n 393...... 3...... 2.1.9.. .9351... hi. 3.... 33...] hum. .1. .. I 11.1.. at» .! \Txizagfi . waif-nu... . 11:53... .1 11W! 31 .. 1?. . . in vhgqfi.‘ Hi3“. 3.81.521. . a} . . . 53.11.. 3.) .5. it 51. 135314, 1.3.2.5.. mi 37‘. infiéq :1! I n. , fig... .n?.z.fin$¢§maiu.ru.uwsziz n. . 1 e, 3‘. .tfinfi‘l..‘.\>\' 31V . .. . 5. \$.l in AC5. 7.1“.) .\. . ‘ a, '3 15.x .V. “I...” M 5&3 3% . n. .5... . .7... ’41.)!!! . 2.. ha“... . 91.! .V... x. a n. .\. . ...,\u.hn.1.\.flc. W...) ,3! .,.. . flu.“\i\\vl.w..nrdmmh.u( .1 3.11%....“ . \ ... 1 91 :. . . . u y. 1... 1 , 3... . Liganfinbrnfluw? . an. bf... 352... 3.5.4.... 3.9."... .. ,. .. ”nun... . (.35.... . §§I_hwmm§7\: 9r ., barn)! «.1151: . airflavnllf.i:.. , 15%.... 1.... In .. n. i}... .15 .. . . a. . .1}§\\nflk.l u. .11. 72134.5 . 1.5.1713“... . g . . 1 .Ififi . . 1.9 . .. . .57 ~ ) n x \..1. ’ffi . . .. 139M. 1. i}. .11... 9.818.}... .14 . \. ;5§!\ . 139:5}. A.) t. ,, . “of... n . .l I ’1 , . - §§n§§§.33§ . 1.3!... , I}. .4.§§th n .2. :1 .1 .n .. 1.. )1 1.3 4659.! 33.131. .n.t"\i1.1\\\§i . i. o .4! . g? 3.33“... .. . . $27. _ . .3»... ghfi. a..- n film! .1. (1% - .- 2.... 1mm! \ .n... 3. .. , . Jig haw. 1“ \4 I I. n... v 5.... 1.. In..- t :fivnfius .. .wnvfifiwstnzg . V 2.)}: g _ . . . n u . n . 5'7. .. at} . .~.. . It: 0 u i at! LA. .1 .. {If-Int! . Mmfim. LES... .. . . . mflm Hf fig? .. if... . . .. a... . Etta}? 42. r .\ A. ntmcnih: .. .l.’ VII...“ . , . 25.13,)... . . L . (“fawnfin {‘.Wflv“ .. , hhnkflw; v A ‘ K5. .5... . - .oA. "won—.0375rtsk . ,fihuatzé . K . . .15 3?. I. {1.5 if... 4&1! . . .. g. infiuufii An. vflnfiufitrl ozfi .rn vanléknndohhuw . h 1) in... . urn} . . L t). I. at it“?! in. \5 no“. in»: .7... 5 n: nth»! v. ,tctiiuk... £3.71 . yup... .. [£1.1nt nu... ”Winn...“ . .3 A . .fir.l§hn.uflfll?lnt.h:. “n1... .....nr«§..£4..t....a$.r..y it. .. ..... t. A.» Z‘nn. nIfifimuonX... .ra . . I. 5.1%: l silt . J/Nn. startlns. . . tn. . .. .§~K..n.\§...~.. 3.5.1 gr... p.» .. . ll u In Al t‘bru. .fi '-!0 o I . . ..I.n.....,...r..-nn.y..uu.tt , . .. . 1:31.... . L . .. . - . . . . ..-- y. in... . Lafitte... I. x- 21.....- . . . II... 3.3...» .«tiln! $1....(Lv3in‘ n10.1L n52.3l i!.(.«§n. 1.. . . . . . SILS!YV.~.‘I;£(L an. 2.1.11.5...‘r... . .VALLOK I? 66. t........n.nnxu.un...un.- «nu: $1.... 2. 8.4.1.5.; . , .....c...: an--. n 1;." . . _ I13 .. . . .f F. » 5.1.» “I... “If?! n. y .. . . ... . n . .. . . . 5. Kit. n. . . , . _.-...n..n. yaw .... . .t... a... 5.. . . . ) fl ‘lv’nlvntgf n . . (iii... 2...? x. 331...... . iflt--.{tt§:obnzt3gii . . & Iii--.I....§..!cl.).n.- . . . . \Ihr.‘... .o."||$:?. .. . . . . THESIS LIBRARY Michigan State University This is to certify that the dissertation entitled , :D\U(9,(/,U€Wv3t \Dfi/ CUZQL‘JZQW Una M4; WK M09“ [aft/LWZA/ MM .MOLLLQUL Ail/CFKWAWC €41 I 1&th presented by \fuew ~ KA‘ 7- _FLLV\3 has been accepted towards fulfillment of the requirements for M— degree in Final“ m l SW 1' 4 *2, / ,1 R q ' )9 vf . V Major professd’ Date %‘4 /8L Msu;.n.ur .- 1 ‘ F1 m . , A . 042771 RETURNING MATERIALS. MSU P1ace in book drop to “BRAMES remove this checkout fro “ y FFFFFFFF d. FI__N__ESW “1m ; hm W INVOLVEMENT OF CELLULAR ONCOGENES IN AVIAN LEUKOSIS VIRUS INDUCED NEOPLASTIC DISEASES: LYMPHOID LEUKOSIS AND ERYTHROLEUKEMIA By Yuen-Kai T. Fung A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1981 /. A’l‘ ‘ 1’ I. ,A 1" - t 4 l 9‘ -' . (g I " ' I, I/ 1 ABSTRACT INVOLVEMENT OF CELLULAR ONCOGENES IN AVIAN LEUKOSIS VIRUS INDUCED NEOPLASTIC DISEASES: LYMPHOID LEUKOSIS AND ERYTHROLEUKIMIA By Yuen-Kai T. Fung Avian leukosis viruses (ALVS) are a group of chronic RNA tumor “viruses that do not encode an oncogene in their viral genome, yet can induce a variety of neoplasia in chickens after a long latent period. It has been proposed that ALV induces neoplasia in chicken by integrating upstream from cellular oncogenic sequences, thereby enhancing their expression with the promoter sequence in the 'C' region. In this thesis it is shown that in ALV induced lymphoid leukosis, the ALV provirus has integrated next to the cellular sequence, c-myc, homologous to the oncogene of MS 29 (avian myelocytomatosis virus). Similarly, in ALV induced erythroleukemia, many of the clonal tumor erythroblasts were found to have tumor specific fragments comprised of ALV and c-erb, the cellular counterpart of the oncogene of avian erythroblastosis virus (AEV). It has been preposed that the erb sequence in AEV is a hybrid of two gene 1001, A and B, transduced from the cell genome. Previous studies using a temperature sensitive mutant and a non-conditional mutant of ARV have implied a role of the A gene in the transformation of erythroblasts. In the present study, most tumor Specific fragments were found to be a hybrid of ALV and the c-erb sequence corresponding to the B Yuen-Kai T. Fung In one case, both A and B were shown to be in the same tumor specific fragment associated with some ALV sequences. The erb gene expression was found to be elevated in some tumors to a level compatible with leukemic tissue from AEV infected birds. On the other hand, other tumors do not Show enhancement of erb gene expression although they are pathologically' similar. The implication of these findings on the transformation mechanism is discussed. ACKNOWLEDGEMENTS I wish to thank Mr. Leonard Provencher, Miss Deborah Eagen, Dr. .Aly Fadly and Dr. Lyman Crittenden of the 0.8. Department of Agriculture Science:& Education Administration, Agriculture Research, Regional Poul- try Research laboratory, East Lansing, Michigan, for their valuable help and education throughout the course of my study. I also wish to thank Dr. C. Sweeley for his understanding and tolerance in setting me on the path of scientific research during my early years. The helpful and stimulating discussion and valuable advice of Professor Jerry Dodgson, Edward Fritsch and John Wang are deeply appre- ciated. I wish to also thank Dr. Sweeley for the stipend support during the first three years and last three months of'my stay here, and Dr. Kung for the stipend support in between. Dr. Kung has truly been an inspiration to me. He has forged the right laboratory environment in which a student can be as creative as he/she desires with frequent rewarding results. During my stay in his laboratory, I have enjoyed unlimited freedom in setting research goals, research planning and execution. II also thank him fer putting at my ii disposal all laboratory equipment and chemicals. It has been quite enjoyable working with my lab colleagues, espe- cially Mr. Robert Swift for his late night discussions and companionship in the laboratory. TABLE OF CONTENTS Page LIST OF FIGURES . . . . . . . . . . . . . . . . v LITERATURE REVIEW . . . . . . . . . . . . . . . . 1 Introduction . . . . . . . . . . . . . . . . 1 I. The Architecture of the Virion Viral RNA 2 Viral Genes . 6 II. Replication of Retroviruses . . . . . . . . . 7 III. Pathology of Retroviruses . Acute Viruses . . . . . . . . . . . . . . 8 Chronic Viruses . . . . . . . . . . . . . 25 REFERENCES . . . . . . . . . . . . . . . . . . 30 ARTICLE - On the Mechanism of Retrovirus-Induced Avian Erythro- leukemia: Alteration of Cellular Erb Gene Structure and Expression. Yuen-Kai T. Fung and Hsing-Jien Kung, (1981) (to be submitted to Cell). APPENDIX - 0n the Mechanism of Retrovirus-Induced Avian Lymphoid Leukosis: Deletion and Integration of the Proviruses. (1981) Yuen-Kai T. Fung, Aly M. Fadly, Lyman B. Crittenden and Hsing-Jien Kung, Proc. Natl. Acad. Sci. USA lg, 3418-3u22. iv Figures LIST OF FIGURES LITERATURE REVIEW 1 2 ARTICLE 1 2 Page Architecture of the retrovirus Virion 3 Schematic representation of the replication of 4—5 retroviruses, using the prototype ASV for illustration Schematic of retroviruses 10-11 Expression of the genome of AEV, adapted from published data (69,70) 17 Kinetics of development of chicken erythroleukemia 25 Blood smear slides of AEV and RAV—l infected birds 27 Analysis of DNA from tumor and normal tissues for 29 the presence or absence of AEV proviruses Analysis of EcoRI and BamHI digestion patterns of 31 genomic DNA samples from infected and uninfected chickens Analysis of genomic DNA with DNA probes specific 35 for ALV and erb Kinetic of development of disease in chicken 4 38 Dot blot analysis of erb gene expression in avian 40 erythroleukemia Restriction maps of tumor specific fragments from 42 chicken 4 LITERATURE REVIEW Introduction The retroviruses are a group of RNA-containing animal viruses that have been found in virtually all species of vertebrates. Many different retroviruses exist, and, as our knowledge about them has increased over the yearssince their early identification as disease causing agents, an extensive system of taxonomy has been developed for their classifica- tion. Retroviruses differ from each other in the size and number of Virion proteins, morphology, host range, genomes, and pathogenicity. Among all retroviruses, the avian RNA tumor viruses are the best characterized with respect to their biochemical and genetic make-up as well as possible mechanisms of replication and transformation. The following review is thus limited mostly to the avian tumor viruses. This includes a brief review of the architecture of these viruses, their mode of replication, and, in greater detail, their pathogenicity. Readers interested in biochemical archeology are referred to the excellent review by Gross (1). I. The architecture of the virion Figure 1 shows the architecture of a typical virion. The outer envelope of the virion is a lipid bilayer derived from the plasma membrane of the host cell during the virion budding process. Protruding from the exterior of the viral envelope are glycoprotein(s) encoded in the viral genome (env). These glycoproteins are involved in the recogni- tion of host receptors, essential to the virus's ability to penetrate cells. Moreover, these glycoproteins may elicit an immune response in the host animal. Enclosed inside the envelope is a protein core, the center of which is the ribonucleoprotein consisting of the diploid RNA genome, RNA binding protein and reverse transcriptase (pol). Viral RNA Two single stranded RNA molecules of ca 5-10kb, each of which contains the entire genetic information of the virus, are held together near their 5' ends by a base-paired structure. A haploid subunit of the retrovirus genome is illustrated in figure 2. The RNA genome exhibits many features of eukaryotic mRNA. Thus the genome is bound at the 5' end by a cap structure 5'-m7Gppme and has a poly adenylic acid (Poly A) tail at the 3' end with a low level of internal methylation. In ASV, a molecule of tRNATrp serves as the primer for the initiation of DNA synthesis by binding at a site ((-)PB) 101 nucleotides from the 5' end of the genome. ENVELOPE (H087) GLyCo PROTEIN (6W) REVERSE TRANSCEIPTASE (m) RNA AENOME. INTERNAL PROTEIN C 6&6!) F—lOOnm ————>| Figure 1. Architecture of the retrovirus virion . no newton TV SEE was. co 53.3 23 L8 3E M523 qéazfi 23 3 .BR SENSE 22% SEE .mnzuv .A::NV umoaon Hm2HEqu ocwuooaosc IFN on [or m we emefimoe wH m .cOHmepmsaafl tom >m< on>p0ponn on» MCHm: .wom:ew>oeuon ho soameAHQwL on» no GOHDMpcmmmLamL oaumsosow .N ogswflm N ogswwm :ng » 6.233: . » qum as; €u+nhzom§wr . Afiacuo 2.35 3:9. b . h. .. Aw E. 3 So 3 ,3 .fflleHfllg TRMWAIH 120:3» WW+CH I 2 Q Q ¢o.+4nw>c_se._u % . m c. ._ Aswfii W 12 a. «H a «H eou+LTumch 9.63m .— 9. 3 93 .wdw ([1. m3 H_m5_m 42w don? \ 08:9 r. E A mu .0 < mom .E 3 .126 3:0. Viral Genes Three viral genes are essential for the replication. Gag codes for the structural protein of the viral core. Pol codes for RNA-dependent DNA polymerase which copies the RNA genome into DNA. Env codes for the viral envelope glycoproteins. A fourth viral gene, src or one, is not essential for the survival of the virus and is found only in viruses with the ability to rapidly transform cells to the oncogenic state. In addition to these coding sequences, other portions of the genome serve important functions in the life cycle of the virus. The terminal _ repeats provide "sticky ends" for circularization of the viral RNA genome whereby reverse transcription can proceed through the 5' terminus and reinitiate at the 3' end of the RNA (7). The 5' and 3' sequences that are duplicated during replication are termed U5 and U3, respectively. The U5 and U3 together (and R) form the large terminal repeat (LTR) of the viral DNA. The LTR sequence may serve regulatory functions in the synthesis and processing of viral RNA (8). Recent DNA sequencing data on the LTR and related neighboring sequences (9-11) has yielded the following information about the U3 region of the genome. (1) Judging by the presence of stop codons in all three reading frames, this region probably does not code for a protein. (2) Two possible promotors for transcription by RNA polymerase II exist, one resembles the promotors for -globin and SVNO; the other is a stretch rich in A+T. (3) There is a poly A addition signal in this part of the genome. (4) A structural feature of the U3 5' end, may account for the high frequency of deletion of the non-essential oncogene. (5) The presence of a direct repeat in the U3 and U5 region may help circularization of the linear viral DNA into covalently closed circles. II. Replication of Retroviruses Figure 2 shows a schematic representation of the replication of retroviruses (12), using the ASV as a prototype. The replication of the retrovirus begins with the infection of the virus into the host cell. The viral RNA is transcribed into DNA by the reverse transcriptase associated with the ASV genome. Synthesis starts from a site 101 nucleotides from the 5' end with an existing cellular tRNA as a primer. The short direct repeat (solid square labelled "R") at both ends of the genome is used to facilitate the necessary transfer of reverse transcrip— tase from the 5' end of the genome to the 3' end. Using this first (minus) strand of viral DNA as a template, the second strand (plus) of viral DNA is synthesized (13). The resulting product is a linear duplex DNA molecule containing long terminal repeats at both ends (figure 3). DNA sequencing data indicates that the LTR's conclude with short and often imperfect inverted repeats. Notice that the viral DNA bears strong structural resemblance to transposable ele- ments, for example Tn9. In both elements, large direct repeats embrace gene coding domains. The direct repeats themselves, in turn, end with short inverted repeats. The linear DNA can then migrate to the nucleus where some of it is converted to a closed circular species (1U) possibly by several different mechanisms (15-17), generating circles with one or both copies of the LTR. The viral DNA integrates, apparently randomly, into the host genome. The proviral (integrated) DNA is colinear with the linear viral DNA (18,19). However, a few (generally two) nucleotide pairs present at the ends of the linear viral DNA appear to be missing in the proviral DNA. Reminiscent of the integration site of transposable elements, direct repeats of a few cellular base pairs, which are present only once in unoccupied integration sites, are found at the ends of the integrated proviral DNA. This proviral DNA can then serve as a template for transcription. Genomic RNA is produced for packaging into new virions. Subgenomic RNAS are produced with the 5' leader sequence spliced onto different parts of the RNA genome. Proteins produced are then available for packaging. Readers interested in the processing of viral proteins are referred to the recent review by J.M. Bishop (20). III. Pathology of Retroviruses RNA tumor viruses can be classified into the acute and the chronic viruses according to their pathology. Acute Viruses. The acute viruses can induce neoplastic diseases in vivo rapidly and efficientLy. Moreover they can usually induce cell transformation in tissue culture. This ability of acute viruses to transform efficiently is due to the presence of Specific oncogenes in the viral genome. The genome of’Rous sarcoma virus for example, includes the usual viral genes (gag, pol, env) essential for the replication of the virus. In addition, as was shown in figure 2, the virus carries a 1.5 kilobase "sarcoma gene", src, responsible for transformation. It has been shown that subgenomic fragments of RSV containing src are capable of transforming NIH 3T3 cells in culture (21), indicating that src by itself is capable of inducing transformation. Since RSV carries the entire set of viral genes needed for replication, it is replication-competent (see figure 3b). However, many acute viruses are replication-defective. In these replication-defective viruses, an essential portion of the RNA genome is replaced by the oncogene. Figure 3A shows a schematic representation of the genome of a typical acute defective virus. The extent and precise site of deletions of viral genes in the genome of defective acute viruses vary among different viruses. Many different viruses carrying specific oncogenes have been identified. For example, the sarcoma gene found in Fujinami sarcoma virus (FSV) (22,23) bears no sequence homology with the src gene of ASV or any other known avian oncogenes (22). The viral genome of FSV is a 4-5 kb RNA (the smallest known RNA tumor viral genome) containing a 5' gag gene-related sequence of 1 kb, an internal specific 3 kb oncogene, and a 3'-terminal sequence of about 0.5 kb related to the C region of avian tumor viruses. Like other replication-defective acute Viruses, FSV requires a helper virus to replicate, in this case FAV (Fujinami associated virus). Another recently isolated strain of avian sarcoma virus Y73 (24) has a genomic architecture similar to that of FSV. Y73 encodes a 90 k protein which, like pp60 src has kinase activity for tyrosine residue. FSV also encodes a 1u0k protein with tyrosine protein kinase associated activity (29,30). Again, Y73 is replication-defective and requires a helper virus for its propagation. In contrast ASVS can replicate on their own. Nevertheless, both FSV and Y73 induce sarcomas in chickens and transform chicken fibroblasts in culture as efficiently as ASV. This overlapping of the oncogenic spectrum of different sarcomagenic RNA subgroups is not unique 10 .Amscfi> msocowoonov ou> casewoococo: .Amspfl> mfimoxsoa cmfl>mv >q< .m.o mscfl> oflcocco .Amsew> maoonmm cmw>mv >m< .m.o mscfl> opsom .Am3LH> wflmoumenognuznw cma>mv >m< .w.m mscw> muzom pcouoqsooIGOHmeHHQom . ucmuoqsooucOHmeHHQmm unoquEOOIGOHumowfiqom o>apoomoouqoaamoflflamm 'o aic5c: .mmm:tfl>onuon mo OHDmsonow .m mcswflm m season J 11 —- J- Ah 12 to the avian system. Parallel examples can be found in the replication- defective retroviruses in mammals such as the Harvey-Kirsten sarcoma viruses (25,26). It now appears that the Harvey and Kirsten strains of sarcoma virus encode enzymatically and serologically related src pro- teins. The sarcoma genes in each virus, however, show only a small region of homology (32). Another group of replication-defective viruses that can induce neoplasia rapidly in vivo and transform appropriate target cells in vitro are the acute leukemia viruses. These include the Friend (27) and Abelson (28) viruses of mice and three groups of leukemia virus (AEV, MC 29, and AMV) in chicken. An excellent review on the molecular biology of Friend virus has appeared recently (31). The Friend virus complex consists of two components, a replication-defective rapidly-transforming virus (the Spleen focus-forming virus, SFFV) and a replication-competent type-C helper virus which helps the transmission of SFFV (37). SFFV can rapidly transform erythroid precursor cells in spleen of adult mice. The helper virus on the other hand can cause a lymphoid leukemia after a latent interval of up to 6 months (33-36). However, formal proof that SFFV can transform erythroid target cells in the absence of co-infection with a replication competent murine leukemia virus (MuLV) does not exist. Circumstantial evidence has been provided by the studies of'Hankins et a1 (38) who show that in zitrg infection of bone marrow cells with Friend virus complex resulted in a marked increase in the number of erythroid burst-forming units five days after initiation of the culture, whereas these were not observed with F-MuLV infection alone. Moreover, SFFV rescued from nonproducer cells with thymic leukemia-inducing Moloney 13 MuLV causes erythroleukemia and not thymic leukemia following inocula- tion into adult mice. Thus SFFV may indeed be capable of inducing erythroleukemia by itself. While F-MuLV most often induces lymphoid leukemia, studies done in Scolnick's laboratory have shown that several isolates of’F-MuLV have the capacity to induce a rapid Spleenic leukemia in newborn mice after a latent interval of only A to 6 weeks (39). In contrast to SFFV these clonal isolates of‘F-MuLV are not rapidly leukemo- genic for adult mice» McCarry et a1. (40) have isolated an F-MuLV strain that induces myeloid leukemia. It seems likely that the genome of'F—MuLV may have been modified by passage in rats or mice, generating the different strains of viruses observed. One such example is the F-MCF virus (Friend-Murine Mink cell focus-inducing virus) an env gene recom- binant of ecotropic F-MuLV and endogenous xenotropic virus isolated from the leukemic spleens of Swiss mice inoculated with cloned F-MuLV (A1). The genome of F-MuLV is typical of other type C viruses (figure 3c). Three-fourths of the genome of SFFV are identical to that of F-MuLV, the remaining one-fourth is derived from the env gene of murine xenotropic viruses. Interestingly, this SFFV specific env gene sequence shares extensive homology with the env gene of the MCF‘mentioned above. In fact with the exception that F-MCF is replication competent while SFFV is replication defective, F-MCF and SFFV are quite similar. This has led to the proposal that both F-MCF and SFFV are recombinants of F-MuLV and the env gene of murine xenotropic virus and that further deletion of F-MCF gives rise to SFFV. However it should be noted that MCF viruses derived in other murine leukemia virus systems are known to cause lymphoid and not erythroid leukemia (N2). The transformation of erythroid cells by SFFV may therefore reside in a portion of the SFFV genome other than or 14 in addition to the env gene common to both SFFV and MCF. As yet there is no formal genetic proof that SFFV encodes for a gene product responsible for leukemic transformation of erythroid cells. The Abelson murine leukemia virus, A-MuLV, on the other hand has been shown to encode for a specific oncogene responsible for transforma- tion of the target cells (N3,N7). ApMuLV is a replication-defective virus derived during passage of RFMuLV in mice (28). It can rapidly induce leukemia in 1319 as well as transform bone marrow cells in X3229 (28,N4,45). .A number of APMuLV strains have been isolated (A6); each encodes a protein corresponding to a fusion between the gag gene product of MuLV and a polypeptide encoded by a 3.6 kb A-MuLV specific gene sequence derived from. normal. mouse genome (NB-50). Moreover, this oncogenic sequence was found to be present in rat, hamster, human and chicken cells (48). The A-MuLV specifically transforms B-type lympho- blasts and fibroblasts of mice. It should be emphasized that the genome of A-MuLV resembles more closely the prototype structure shown in figure 3 than does SFFV. SFFV might simply be a recombinant of ecotrOpic and xenotropic MuLV sequences without actually carrying a cellular oncogene like other acute RNA tumor viruses do. In the avian system, more than ten strains of defective leukemia viruses have been discovered. Recent studies have provided the biochemi- cal (51,52) and genetic (53,54) basis for classification of these viruses into three groups. They are: AEV (avian erythroblastosis virus)-type consisting of strains R and BSA (55,56), which are probably identical; MC29 (avian myelocytomatosis virus)-type consisting of MC 29, CM11, OK1O 15 and MH2 strains; and AMV (avian myeloblastosis virus)-type strains consisting of the BAl/A strain of AMV and of E 26 (57). AEV: The AEV-type strains can induce an acute erythroleukemia and anemia, one to two weeks after inoculation into chickens. II“ the inoculation is intramuscular, most of the strains can also induce sarcomas at the site of injection. The target cells transformed by AEV have been found to display distinct phenotypes of differentiation (51,52). Cells transformed by AEV bear striking similarities to precur— sor cells of erythrocytes as revealed by their expression of high levels of histone H5 (found only in erythroid cells of nonmammalian species) and erythroblast cell surface antigen. Moreover, markers for mature eryth- rocytes, for example heme, globin, carbonic anhydrase and erythrocyte cell surface antigen, are expressed at low levels. The expression of these erythroblastic molecular markers in transformed bone marrow cell cultures is found to be identical to those of the transformed cells in zigg. Moreover, cells transformed by the ts 3“ mutant (58) express the same erythroblast molecular markers at the permissive temperature but show an increase in the expression of hemoglobin, carbonic anhydrase and erythrocyte cell surface antigen when shifted to the nonpermissive temperature. More recently the characteristics of the specific target cells transformable by AEV have been determined by a combination of physical and immunological methods (60). Specific antisera have been developed which can distinguish between the several erythrocyte precur- sors at different stages of differentiation (61), viz. CFU-M (colony forming units in marrow), BFU-EC (burst forming units - erythrocytic), CFU—E (colony forming units - erythrocytic), erythroblasts and erythro- cytes, in that order (62,63). It was found that the BFU-E are target 16 cells for infection by AEV. Since transformation by AEV either in vivg or in xitgg gives rise to erythroblast-like cells it is possible that the initially transformed BFU-E can undergo some maturation to the CFU-E or erythroblast stage. Further work is needed to demonstrate this hypothe- sis in 3232' In addition to transformation of hematopoietic cells, AEV can transform cloned chicken embryo fibroblasts (in this case, cell cloning was essential to eliminate hematopoietic target cells that were present in chicken. embryo cell cultures). .AEV-transformed fibroblasts are similar to RSV transformed fibroblasts in many ways (59). Morphological- ly, both transformed cells show the disappearance of actin cable, a decrease in Large External Transformation Specific (LETS) protein and an increase in the microvilli at their surface. Biochemically, they are agglutinable by lectin and Show an increase in the rate of hexose uptake. These cells are capable of anchorage independent growth and inducing sarcomas in yizg. The structure of the AEV genome has recently been elucidated by molecular cloning (6A). The AEV genome is about 5.1 kb long (carrying one LTR). At least 50% of the sequence, flanked by about 1 kb of gag sequence and 0.” kb of env sequence, is the AEV specific oncogene erb. Transfection of the cloned DNA ligated to the DNA of RAV-1 (a helper virus) leads to the production of AEV virus capable of transforming both fibroblasts and bone marrow cells. Erb, the AEV specific gene sequence, is defined by the absence of homology with the genomes of other avian retroviruses like src, myc (MC 29) or myb (AMV). Several lines of evidence indicate that the erb gene in AEV may be composed of two functional domains: (see Fig. A) 17 .Ao>.mmv memo oonmwansa sogm oopamom .>m¢ no oaocom on» we coaumonaxm .: mesmHm 9i $2.. E as; Gino. $<$?).<vpaeac__oea "mecca embuac:_ H->m< .cowam—suozw umoa m_m>Lmu:w Lw_=mmc pm preoouosm; an uwgzmmma mew: A|v $5 £30 £8 30:5 28 All 83 3385wa 08 mmmucmucaa one .memxsmFOL5uxcm cwxuezo we pawsao_m>wv we muvumc_¥ .H mL:m_m .(—-—-) 83 % ZO_._.<.._DoOz_a._.mOa mxmw>> v_m_N_:O_mmhmm¢mN_ "4%” .1 4 n “ .lv 0 “ "infill O O 7.1! . \I ~ 2 \ \ — — O , — — .. 00 — plllll o o o nfiofig 8.635 72?. >m< ( L l i i l .nhwlrfnikf -27- .uma_aogspzcm mflmmv mmpaooczwxgm Aumv mam__mu umoo awezn fiofl v mmcpm urmesmF pm news vmuoowcr H->m< Amy ”macaw oweoxsm—mcn pm ucvn uwpowmce >m< A v .meecn uauaac:_ H->m< to mauv_w LaaEm eoo_m .N ae=m_a .eme eEem ecu we meewe eeueewcwce EeLw ezmmwp weELez .w.e .emepm ewsexee— use we megwe eeueewcw H->m< Eeew memeu Lesew .e.e mace; -29- .wege ep eweerewLeag ex m.~ we aceEmeLw eweaecmwm e euececem eweez m=Lw>eLe >m< :e mcwxggee ewe >m< ecu we ewpesecem < .>m< we cweEee new use we ewes mcwxggee eeege eee >m< we eecemee Le eecemeee ecu Lew meemmwu weELe: use Lease Eeew <23 we mwmawec< .m ewemwe T... 28 I h 5. IIIIS A09 w e e e e e 9. 8:808 65:8 mes/Eel >w< ..... 222L— - :m 058% >m<\ 0 fl T225223 28. eel _I anm .1 Sum -31- .AH ecemwe emwe eemv mumeweeesuxge we cewueeewwwece peeLeeee on use .ewseze mzesm pezu :exewze eeueewcw Hu>wuegewwwege new: mcexewce eeueewew Hu>wuegewwweee new: mcexewge eepeewcw Hu>m< Am.H meceev m .mwmeemeweegszge we cmwm e: saw: mwmexeew eweceea— we eewe A—uceeeemeem peg» :exewze eeueewcw Hu>1m.\>m< .m @813“... 12.1 emHzee 9011.25 emwemuzz: wage 018... >m< m Bebem name wee”... . .31. D>n_ “H H3>n_ .< NE we. in. , N... 9... en en.» mN_ . *5 . w .x. a 1 , . . I...“ .. .l * . N._I*. . . . ®_ . ONI. f .J 3.. 0.1.... eN/ . t , . 4m“ mfil ... . ..m o . .._........ mm. .. _. ...... . We, 6v own”. 1.... emit a... fin u. .. 0% 00.! QJ/hz. mmw e.l.e.llfll ee. ee. . eeT 0.N....- . eeLN- 9. .61 fl ./ .... fl Bow emuxN mmem. 9. Bow elv. mmeml .395 9. $83.. / \ / \ / \ /_\ /_\ _I Ecm _ m/oom _ I /Eom _ mo0m _I Eom _ mo0m _ I Eom _ meow v :3020 m c9650 N :10 _ c9830 <.2mx3mi_OmI._.>mm T >_._..w<1m......e1.. _-><1 a .mucwEmeLw uwwwuwem Logan . Mewcwsngmv uo: ..o.z mmwmeno—wx cw wcmwwz Lewaee—ee .ex memewnegcpxwe .mm .mpxeegspxcw .om .N-<2m cw :emeL men one we wwe; usmw. ecu op mcweeeemegeee <21 Nu<2m we eceweeem e EeLw pcesmegw :ewuewwpmew Hmeem e we ezoe ween—meegu xew: e .mwe .cwesee new emcee esp we cam “now. esp op meeeemeggee :emeL ex e.o mwgu Lespemew .ucesmegw Hwe>e\wzaem ex <.o ece Newsweww cewuewgpmeg HIEem ex m.o we <2aeeeuewmcegp xew: we eceuxwa e .mnge .52. fl MM mmecwesm 2e eepgeeeg <22: ex H.e ece m.m esp meeeece pen» cweEee -35- new esp we meceeewm weewce ecu op meeeemeecee mwcw .ucesmeww :ewuewLume. Hume ex m.o e we m< we :weeee ewe-> ecu we umee memmeeeeecm pesp :ewmeL ewwweeem >m< ex m.~ use we 4< Lew ewwweeem mmeeee <2oe new: <22 ewEecem we mwmxwec< .m wwsmw. PVU JI PVU1 IE PROBES USEDI ‘35 a ’ lAEV specific 2nv§§l rbLT TErbR Psib Pg: Bom (LTR)B | ‘ASK 92089.0”: 4- . P290952 1 Ch__l____cken I m g pol g env §_5_l ErbT grbL grbR LII? Gog 50223 Kb ECEB Kb ecee .9 aces Kb ecéB Kb 'KF‘E? ’2—40 5 -I06 “hr-IGB "" “240 “(2 Q ““425 “- {-332 " “532-0 '2-3.0 - - -3.l .. —2.o - w... -20 ""' “-245 -‘ I 2 34 5'6 7 8 9 IO Chicken 2 $2» 5..., EC EB _K_b ac @352 I. [2 34 56 78 9l0 I mle. m¢Nlllw AU.NI.t:. AUNT. _.MI. .v .U .... . no M.N.,... w /nn Owl‘ ofiV.’ _N|...... 0.. Hr QVNI.’ QVNIII 0.? I." 44n— JJQ 44n— ATE /\ /\ m/\ /\ wee 1.5 em. Em. mg 0_ m m N w 0 ¢ m N _ 1:. AHNI:I.I .QNlu; —. II.-- .H ll... 0 0*...- m 1*. I .. a 8...... _ . eeTo ll QVNI... Con—WWI. e. meow elv. Mmewfl mwewfl ewew / x E... 19m 49m .90 m 19.98 -38- .m egzmww we ecemew wee .wegm ece wage ”awe >w< we Newpeewesc HoH esp ee mcweceemeeeee .mee we ewuecwx .e weemww w....e. me: we... mN_ I. ' Iv mam—II” I nlthc 9. km.» 322....me 5.2th.» wew\.1oee .9. meow 9e. 8m .9. 8m -40- .meeemww meew>eee cw eeucemeee mece eEem esp ewe ewe; ee>we>cw mcexewce ecw .mwwepee Lew weeeeeeeee weweeaweeexw eem .wwmw .eeEecw .e we eecpee egg e» mcweweeee Leeee emewewweeegpwe epee eeppeem ece :ewpecwseucee <29 e>eEeL op eewwweee .meemewu ecu Eeww eeeeewpxe we: <22 Lowe—wee weuew .cexewce .xe mm—wee peee 2wwee .mw MmepAeeecpxge .um .ewaexee—eecuxee cew>e :w cewmmeeexe ecem age we mwmxwece gown pee .w eeemww . mm. 0 20 .9... 0 me . m 20 . .9... o . .9... a mm m 0.0 o ceeam o. mew N we .. me .e . .e o .9... 2628.213. e>_we.ew_.e..n. &.< we aw. eee ee e.ee~.eweewe eeewe we eewbeee .memeeewwx cw eemmeeexe .ege: :zezm ece e2» eucw eeeweEee ewe; wees :ewpewwuee. ewes» eee peeuxe eage— e ea eeeeewwe>e mecewe e3» emezw .wN;e we euwm stem esp cw eeeewe we: Awfi ecew .ue eeemwwv pceameew HIEem ex e.m esw .mezpmx we eewm .meee eee e. eeeewe me: .e. eeew .ee eeeewev eeeEeeew .meee e. ..N esw .e :exewze Eeew epcesmeew ewwweeem Lesa» we wees :ewuewepmem .w ewemww mflwmx 10'1 _ _om . .em .v_ 1. E... mew mew -44- REFERENCES Anderson, S.M., Hayward, W.S., Neel, B.G. and Hanafusa, H. (1980) Avian erythroblastosis virus produces two mRNA's. J. Virol. 36, 676-683. Beug, H. and Graf, T. (1980) Transformation parameters of chicken embryo fibroblasts infected with the t534 mutant of avian erythroblastosis virus. Virology 100, 348-356. Beug, H., Kitchener, G., Doderlein, G., Graf, T. and Hayman, M. (1980) Mutant of avian erythroblastosis virus defective for erythroblast transformation; deletion of the erb portion of P75 AEV suggests function of the protein in leukemogenesis. Proc. Natl. Acad. Sci. U§A_ZZ, 6683-6686. Crittenden, L.B. (1975) Two levels of genetic resistance to lymphoid leukossi. Avian Diseases 12, 281-292. DeLorbe, W.J., Luciw, P.A., Varmus, H.E., Bishop, J.M. and Goodman, H.M. (1980) Molecular cloning and characterization of avian sarcoma virus circular DNA molecules. J. Virol. 36, 50-61. Fung, Y.K., Fadly, A., Crittenden, L.B., Kung, H.J. (1981) On the mechanism of retrovirus-induced avian lymphoid leukosis: deletion and integration of the proviruses. Proc. Natl. Acad. Sci. USA 18, 3418-3422. Gazzolo, L., Moscovici, C., Moscovici, M.G. and Samarut, J. (1979) Response of hemopoietic cells to avian acute leukemia viruses: effects on the differentiation of the target cells. Cell 16, 627-638. Gazzolo, L., Samarut, J., Bouabdelli, M. and Blanchet, J.P. (1980) Early precursors in the erythroid lineage are the Specific target cells of avian erythroblastosis viruinn vitro. Cell 22, 683-691. Graf, T. and Beug, H. (1978) Avian leukemia virses: interaction with their target cells in vivo and in vitro. Biochim. Biophys. Acta 516, 269-299. Graf, T., Ade, N. and Beug, H. (1978a) Temperature-sensitive mutant of avian erythroblastosis virus suggests a block of differentiation as mechanism of leukemogenesis. Nature 275, 496-501. Graf, T., Beug, H., von Kirchbach, A. and Hayman, M.J. (1980) Three new types of viral oncogenes in defective avian leukemia viruses. II. Biological, genetic, and immunochemical evidence. Cold Spring Harbor Quant. BiOIO, V0]. 53, 1225'12340 Hanafusa, H.C., Halpern, C.C., Buchhagen, D.L. and Kawai, S. (1977) Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants. J. Exp. Med. 146, 1735. -45- Hayward, W.S., Neel, B.G. and Astrin, S.M. (1981) ALV-induced lymphoid leukosis: activation of a cellular one gene by promoter insertion. Nature (London) 229, 475-480. Lai, M.M.C., Neil, J.C. and Vogt, P.K. (1980) Cell-free translation of avian erythroblastosis virus RNA yields two specific and distinct proteins with molecular weights of 75,000 and 40,000. Virology 100, 475-483. Lucas, A.M. and Jamroz, C. (1961) lfl Atlas of Avian Hematology. Agri. Monograph gg, U.S. Department of Agriculture. Neel, B.G., Hayward, W.S., Robinson, H.L., Fang, J. and Astrin, S.M. (1981) Avian leukosis virus-induced tumors have common proviral integration sites and synthesize discrete new RNAs: oncogenesis by promoter insertion. Cell 23, 323-334. Neiman, P., Payne, L.M. and Weiss, R.A. (1980) Viral DNA in bursal lymphomas induced by avian leukosis viruses. J. Virol. 34, 178-186. Pawson, T. and Martin, G.S. (1980) Cell-free translation of avian erythroblastosis virus RNA. J. Virol. 34, 280-284. Payne, G.S., Bishop, J.M. and Varmus, H.E. (1981) Multiple arrangements of viral DNA and an activated host oncogen (c-myc) in bursal lymphomas. Nature, in press. Payne, G.S., Courtneidge, S.A., Crittenden, L.B., Fadly, A.M., Bishop, J.M. and Varmus, H.E. (1981) Analysis of avian leukosis virus DNA and RNA in bursal tumors: viral gene expression is not required for maintenance of the tumor state. Cell 23, 311-322. Purchase, H.R. and Burmeister, B.R. (1978) Neoplastic diseases. Leukosis/sarcoma group. In_Diseases of Poultry, M.S. Hosfad, B.W. Calnek, C.F. Helmboit, W.M. Reid and H.M. Yoder, Jr., eds. (Ames, Iowa: Iowa State University Press), pp. 418-468. Purchase, H.E., Gilmour, D.G., Romero, C.H. and Okazaki, W. (1975) Nature .219. 61-62. ' " Roussel, M.S., Saule, C., Lagrou, C., Rommens, C., Beug, H., Graf, T. and Stehelin, D. (1979) Three new types of viral oncogene of cellular origin specific for haematopoietic cell transformation. Nature 281, 425-455. Royer-Pokora, B., Griesen, S., Beug, H. and Graf, T. (1979) Mutant avian erythroblastosis virus with restricted target cell specificity. Nature (London) 282, 750-752. Sheiness, D., Vennstrom, B. and Bishop, J.M. (1981) Virus-specific RNAs in cells infected by avian myelocytomatosis virus and avian erythroblastosis virus: modes of oncogene expression by defective leukemia viruses. Cell 2;, 291-300. -46- Stehelin, D. and Graf, T. (1978) Avian myelocytomatosis and erythroblastosis viruses lack and transforming gene src of ASVs. Cell 13, 745-750. Stehelin, D., Saule, S., Roussel, M., Sergeant, A., Lagrou, C., Rommens, C. and Raes, M.B. (1980) Three new types of viral oncogenes in defectrive avian leukemia viruses. 1. Specific nucleotide sequences of cellular origin correlate with specific transformation. Cold Spring Harbor Quant. Biol., Vol. 44, 1215-1224. Thomas, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 11, 5201-5205. \ Therwath, A. and Scherrer, K. (1978) Post-transcriptional suppression of globin gene expression in cells transformed by avian erythroblastosis virus. Proc. Natl. Acad. Sci. USA 15, 3776-3780. Tsichlis, P.N. and Coffin, J.M. (1980) Recombinants between endogenous . and exogenous avian tumor viruses: Role of the C region and other portions of the genome in the control of replication and transformation. J. Virol. 33, 238-249. Varmus, H.E., Quintrell, N. and Ortiz, S. (1981) Retroviruses as mutagens: Insertion and excision of a nontransforming provirus alter expression of a resident transforming provirus. Cell 25, 23-36. Vennstrom, B., Fanshier, L., Moscovici, C. and Bishop, J.M. (1980) Molecular cloning of the avian erythroblastosis virus genome, and recovery of oncogenic virus by transfection of chicken cells. g;__ Virol. _3_6.. 575-585. Yoshida, M. and Toyoshima, K. (1980) In vitro translation of avian erythroblastosis virus RNA: identification of two major polypeptides. Virology 100, 484-487. Proc. Natl. Acad. Sci. USA Vol. 78, No. 6, pp. 3418—3422, June 1981 Biochemistry On the mechanism of retrovirus-induced avian lymphoid leukosis: Deletion and integration of the proviruses (RNA tumor virus/B-lymphocyte tumor/proviral DNA/M029 oncogene) YUEN-KAI T. FUNG*, ALY M. FADLYT, LYMAN B. CRITTENDENT, AND HSINc-JIEN KUNc*i *Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824; and *U.S. Department of Agriculture, Science and Education Administration, Agriculture Research, Regional Poultry Research Lab, East Lansing, Michigan 48823 Communicated by Norman Davidson, March 2, 1981 ABSTRACT There is considerable evidence that infection by avian lymphoid leukosis viruses can lead to tumor development in the target organ of the host. The mechanism by which virus-in- duced oncogenic transformation occurs, however, is not clearly understood. As a first step toward deciphering this process, we have characterized the proviruses of the lymphoid leukosis viruses in DNAs extracted from the leukotic and metastatic tumors by using restriction enzyme digestion and filter hybridization analysis with radioactive probes specific for the infecting genome. Our results indicate (1') that lymphoid leukosis tumors are clonal in or- igin; (ii) that there are multiple sites in the cellular genome of the target tissue where the virus DNA can integrate and that, in the majority of the tumors, at least one such site of each tumor is ad- jacent to a cellular sequence related to the oncogene of MC-29 virus; and (iii) that deletions and other structural alterations in the proviral DNA may facilitate tumorigenesis. The oncogenic retroviruses can be separated into at least two classes that appear to induce neoplasms by different molecular mechanisms. The more extensively characterized group in- cludes viruses that induce rapid neoplasms, encode genes for cell transformation (probably of host origin), and are often de- fective, requiring a helper virus for infectivity or replication (1, 2). The second group induces neoplasms that have long latent periods, have no known genes coding directly for cell transfor- mation, and are not defective in replication. Among these, some appear to have the potential for inducing several types of neo- plasms (1, 2). The first class of viruses, although of basic interest in studies of in vitro cell transformation, are probably laboratory products, while the second class of viruses is likely to be re- sponsible for the majority of naturally occurring retrovirus-in- duced neoplasms. Viral induction of avian lymphoid leukosis (LL) is an excellent model of neoplasm by a virus of the second group. The steps leading to mortality with LL include the in- fection of the target cell in the bursa of Fabricius, the transfor- mation of the target cells not earlier than 3 to 4 weeks of age, the development of the grossly visible bursal tumor at 10-16 weeks of age, and the metastasis to visceral organs leading to massive lymphoid tumors and death, usually after 16 weeks of age (3). The present studies are aimed at characterizing the newly integrated exogenous proviruses in LL tumor cell DNA to pro- vide insight into the molecular events that lead to the devel- opment of LL. MATERIALS AND METHODS Cell Culture, Viruses, and Biochemicals. A RAV-l virus stock, purified by three cycles of propagation at limiting dilu- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3418 tions, was used. Infection of chicken embryo fibroblast cultures was carried out at a multiplicity of 0.1, and the infected cells were passaged at least four times before DNA extraction. The media of such cultures contained a high level of reverse-tran- scriptase activity (4). For the synthesis of cDNA probes, con- centrated Prague C virus, purified by repeated banding in su- crose gradients, was used (5). DNA polymerase I, DNase I, and restriction endonucleases were purchased from commercial sources, and [a-32P]dCTP was from ICN. Induction of Lymphoid Leukosis. Day-old chickens of a cross between RPRL (Regional Poultry Research Lab) lines 1515 and 72 were inoculated by the intra-abdominal route with 105 in- fectious units of RAV-l. The chickens were free of common avian pathogens and reared in plastic canopy isolators to 12 weeks of age and then moved to semi—isolated cages. From 120 through 150 days, the birds were palpated for bursal enlargment twice weekly. Sixteen birds were killed; tumorous and repre- sentative nontumorous tissues were taken for DNA extraction. All tissue samples were immediately transferred to vessels con- taining liquid nitrogen and then stored at -70°C until use. For experiments to study the provirus in bursal tissue at preneo- plastic stage, a portion of the bursa was surgically removed 4 weeks after virus inoculation. DNA Extraction and Enzyme Digestions. Frozen tissues were homogenized in a glass barrel with a loose Teflon pestle in 40 vol of 10 mM Tris'HCI, pH 7.5/ 5 mM EDTA. Protease K (25 ug/ ml) and NaDodSO4 (1%) were added to the homog- enate. After incubation at 37°C for 2 hr, the solution was ad- justed to 0.1 M NaCl and extracted with phenol/ chloroform. The DNA samples were concentrated by EtoH precipitation. Digestions of DNA with restriction endonucleases were con- ducted at 37°C for 2 hr. The digested DNAs were analyzed on 0.8% agarose gels and then transferred to nitrocellulose paper and hybridized with appropriate radioactive probes as de- scribed (6). Hybridization Reagents. The radiolabeled nucleotides in all of the following probes were derived from [a-32P]dCTP. (i) cDNA3., which carries the 3'-terminal sequences (S200 nu- cleotides) of the viral genome, was synthesized by using the avian myeloblastosis virus polymerase on $88 poly(A) contain- ing RNA and oligo(dT)12_18 (P-L Biochemicals) as primer. Oligo(dT)-primed cDNA3. was then purified by chromatogra- phy twice on oligo(dT)-cellulose after hybridizing to poly(A) (6). (ii) cDNA5., which represents the 5’-terminal 101 nucleotides of the viral genome, was synthesized by using detergent-acti- vated virion as described (7) and purified by isolation of the 101- Abbreviations: LL, lymphoid leukosis; LLV, lymphoid leukosis virus; M Dal, megadalton(s); LTR, long terminal repeat; ev, endogenous viral; TS, tumor specific; CSV, chicken syncitial virus. 1To whom reprint requests should be addressed. Biochemistry: Fung et al. mer from a 10% polyacrylamide gel (7). (iii) cDNArep. was syn- thesized in the same way as cDNA5. except that the gel-puri- fication step was omitted. This probe, enriched in cDNA5., car- ries 280% sequences of the entire genome. It is capable of detecting all three Sac I-derived endogenous virus fragments corresponding to the major loci as described by Astrin et al. (8). In addition, cDNArep. also detects a 2.5-megadalton (M Dal) end fragment (see Fig. 13, lanes 1 and 2), which preferentially hybridizes to cDNA5.. (in) DNA probes specific for the onco- gene of MC-29 (avian myelocytomatosis virus strain 29) (I, 9) were prepared by nick translation (10) of a plasmid clone, pMyc- Pst, supplied to us by D. Sheiness and]. M. Bishop (University of California, San Francisco). pMyc—Pst, which carries princi- pally the putative oncogene, was derived by subcloning a Pst fragment of a DNA clone carrying the entire MC-29 genomic sequence. RESULTS AND DISCUSSION Viral Etiology and Development of Lymphoid Leukosis. Twenty-nine day-old (1515 X 72) chickens were inoculated with avian lymphoid leukosis virus (LLV), RAV-I. All birds either died of or were killed bearing lymphomas by 253 days of age. Tissues were taken from 16 ofthe birds for DNA extractions and histopathological examinations. Among these 16, all except 1 contained lesions in the bursa of Fabricus; 5 also developed sec- ondary spleen or liver tumors. Thus, in our experimental sys- tem, a near 100% incidence of bursal lymphoma was obtained after virus inoculation. Such a high lymphoma incidence, to- gether with the presence of RAV-l proviruses in all the tumor samples (see below), is consistent with a viral etiology for this disease. Strategies for the Identification of Exogenous Provirus. The studies described here are principally based on digestion anal- yses with Sac I and EcoRI and hybridization with the sequence— specific probes cDNArep., cDNA3., and cDNA5., cDNArep. car- ries sequences representing the entire RAV-l viral genome. cDNAg. and cDNA5., on the other hand, are specific for the 3’ and 5’ terminal sequences of the viral RNA genome (see Ma- terials and Methods). The sequences contained in cDNA3. and cDNAS. (shown in Fig. 1A as boxed 3 and 5) together comprise the long terminal repeat (LTR) present at both ends of the pro- virus. As the 3’-terminal region ($200 nucleotides) of the RAV- 1 genome does not share much homology with any endogenous viral (ev) sequence in chicken chromosome (11, 12), we have used CDNAsr extensively to distinguish the infecting RAV-l DNA from ev sequences. Most chickens ofa (1515 X 72) cross have three ev loci, ea 6, co 1, and (31; 2.§ We have used Sac I digestion to document the presence of exogenous proviruses in tumor DNAs and to iden- tify their integration patterns. This is based on the following considerations: First, Sac I has a single cleavage site in RAV-l proviral DNA, and the fragment sizes are determined not only by the location of this site in the viral genome but also by the nearest enzyme cleavage site in the flanking cellular sequence (Fig. 1A). Therefore, Sac I digestion can provide information concerning the integration site of exogenous proviral DNA. Second, as shown by Astrin and coworkers (8, 18), Soc 1 diges- tion of normal chicken DNAs gives a relatively simple frag- mentation pattern of the ev sequences; additional bands cor— responding to the newly integrated exogenous provirus in the tumor DNA can be readily identified. On cleavage of the ge- nomic DNA with Sac I and hybridization with cDNAmp. the ev sequences are shown as four hands of .\I, 13. 5.9. 3.7, and § Among the 10 ('llill‘ilt‘lt‘l'l7,(’(l birds, numbers 1—153 carry all three (31’ loci. Numbers H—lti luck (’1‘ 2. Proc. Natl. Acad. Sci. USA 78 (1981) 3419 Sac I A EcoRI E R l 1.4 EcoRI 25 EciRIoJ co I 5 cDNA 5' cIDNA 5'2 B Sac I EcoRI/rep* Mr rep" 3’ In Vitro In Vivo Mr ’—3.3 ,E 4,. '—2.5 I . - U» D +4.4 FIG. 1. Restriction enzyme cleavage maps of a colinearly inte- grated RAV-l provirus DNA and identification of tumor-specific (TS) proviral DNA. M r in MDal. (A) Cleavage maps ofEcoRI and Sac 1. Open triangles indicate Eco RI sites not present in the 21) sequences. represents the LTR, which is located at both termini of the viral DNA and carries the 3’- and 5'-terminal sequences of the RNA genome. The wavy line denotes the flanking cellular sequences. The bars indicate the EcoRI fragments detectable by CDNA5. (B) Restriction enzyme digestion analysis of proviral DNA. The DNA samples were extracted from bursa tumor 10 (lanes 2 and 4), from the nontumorous thymus (lanes 1 and 3) of the same bird, from the in vitro RAV-l-infected (lane 6) or uninfected (lane 5) chicken embryo fibroblasts of line (1515 X 72), and from the bursal tissues of a bird inoculated with RAV-I 4 weeks earlier (lane 8) and of an uninoculated bird (lane 7). They were digested with Sac I or EcoRI and analyzed on 0.8% agarose gels and by Southern blotting hybridizations with cDNArep. and cDNAav. 2.5 MDal. In the example shown in Fig. 13, both nontumor (lane 1) and tumor tissue (lane 2) DNA display these four bands. DNA from the tumor displays two additional bands (Mr 8 and 4.0 MDal), which we refer to as tumor specific or TS bands. The exogenous origin of the TS bands was established by hybrid- ization with cDNAa. which detects only RAV-l DNA. The spec- ificity of this probe is shown by the complete absence of ev-re- lated fragments in the DNA from nontumor tissue (lane 3). Hybridization of the tumor DNA with cDNA; (lane 4) shows two distinct bands with size identical to the TS bands detected by cDNAmpe In contrast to Soc 1, there are several cleavage sites for EcoRI in the viral genome, which therefore allows us to analyze the internal structural arrangement ofthe exogenous proviral DNA (Fig. 1A). More important, ev sequences lack the two outer EcoRI sites (indicated by open triangles), which are found only in the exogenous proviral DNA. Consequently, either the 1.4- or the 0, T-M Dal fragment specifically indicates the presence of ev sequences in cellular DNA, as seen by comparing the DNA pattern of a RAV-l infected culture of chicken embryo fibro- blasts with that ofan uninfected culture (lanes 5 and 6). The 1.4- .\lDal fragment (indicated by triangle) is present only in the 3420 Biochemistry: F ung et al. A Sac 1/3I c l o are?4gi u in "" :i' "— i: O' ' infected sample (lane 6). Indeed, this specific exogenous viral marker enabled us to demonstrate that, in >90% of the RAV- 1 inoculated birds, extensive infection of the bursa tissue had occurred as early as 4 weeks after inoculation; a typical example is shown in lane 8, where the 1.4-MDal fragment can be seen in the 4-week bursal DNA of the inoculated bird. This band, however, is absent in the bursal DNA of an uninoculated control (lane 7). Newly Acquired Provirus in Tumor DNA and Clonality of the Tumors. As discussed above, Sac I digestion in conjunction with cDNA3. hybridization provides a sensitive means for iden- tification of the integration pattern of the newly acquired pro- viruses. A survey of DNA of all bursal tumors by this analysis shows that each tumor DNA displays at least one TS band (Fig. 2A), providing strong evidence that all tumors acquired at least all or a portion of one exogenous provirus. It is noteworthy that DNA samples taken from bursal tissues of birds at preneoplastic stages, when assayed by the same method, do not have any TS band, although extensive infection of the target tissue by exogenous viruses can be documented (Fig. IB; unpublished results). These data suggest that the ini- tial infection of the target tissue by RAV-l results in the inte- gration of proviral DNA at many sites in the cellular genome of a large number of cells. The fact that TS bands can be iden- tified in all tumors at the terminal stage indicates that each tu- mor results from selective growth of a homogeneous population of cells (which are characterized by a common proviral DNA structure). The origin of the tumors, therefore, is probably clonal. This conclusion is further supported by the observation that DNAs isolated from multiple tumor nodules located on the same bursa display TS bands distinct from one another, indi- cating that these different tumor nodules are derived from in- dependently infected and transformed cells. An example is given in Fig. 28; the two bursal tumor nodules (B1 and B2) of bird 10 have entirely different Sac I-TS band (indicated by dots) patterns when compared with each other or with the normal thymus tissue control (lane T). These observations are consist- ent with the results of others (14—16), which also indicated that A EcoRI/5' M CI i 2 3 4 5 6 78L9L9 IO M |2|3 I4|516c2M '29—.- 2. 5 «In-o". -fiur - M It... ---—- 22:“ -- 9 an--. -' . 3.3—.- d -- w * a . - ' ~-' . a 2.5 ::-—q~ .‘3 m ' *"CM 13:: a. ‘ * *’* . * * WU |.4—> ; > > «a w <1 4.4-4 * 2345 678L9L9|O|ll2|3|4|5l6 Mr I Q-I2.0+ .. _.'..,_, ‘ "’ . ..6.3~ Proc. Natl. Acad. Sci. USA 78 (1981) FIG. 2. TS proviral DNA as identified by Sac I digestion. (A) cDNA3. hybridization with the DNA samples isolated from bursal or liver (L) tumors. Lane C (control) represents the normal thymus DNA of bird 1. (B) cDNA,ep. hybridiza- tion with the DNA samples from bursal nodules 1 (Bl) and 2 (B2) and normal thymus (T) of bird 10. Dots indicate the TS bands—i.e., fragments detected in the tumor tissue but not in the normal tissue of the same bird. MI in MDal. B Sac I/rep* T BI 32 -I3.0-*'.j 7, —5.9—Il-- , _3_7_- ,.. '. —2.5— LL tumors are consequences of clonal growths of transformed cells. The data in Fig. 2 also show the size variation of TS bands in different tumors, suggesting that integration in a number of sites can lead to the development of a tumor. However, another equally plausible, but not mutually exclusive, possibility is that deletion within the proviral DNA contributes to size variation. Frequent Deletion of the Provirus in Tumor DNA. Evi- dence for the deletion of viral sequences from some of these proviruses was provided by experiments in which EcoRI- cleaved tumor DNA was hybridized with cDNA; probe. Fig. 1A shows that cDNAS. can specifically detect the 1.4-.\/lDal EcoRI fragment near the left end, which carries the entire gag (group-specific-antigen) sequence. As discussed above (Fig. 13), the 1.4-M Dal gag-containing fragment can be readily de- tected in the undeleted RAV—l provirus found both in in vitro infected cells and in the bursal tissue ofinoculated birds at pre- leukosis stages. By contrast, in many tumor DNAs (e.g., 2, 3, 5, 9L, and 12 in Fig. 3A), the 1.4-MDal fragment (triangle) is completely absent. A similar conclusion was reached from hy- bridizations with cDNArep or probes specific for the gag se- quences and from Soc 1 digestion analysis (data not shown). These data thus demonstrate that some of the RAV—l provirus in the LL tumors have undergone extensive structural alteration. Multiple Integration Sites of the Proviruses in Tumor DNA. Hybridization of EcoRI-cleaved tumor DNA with cDNA; also detects the right-end viral-cell junction fragment and provides reliable information concerning the integration site of proviral DNA (Fig. 1A), because the Mr of such fragments cannot be influenced by the potentially extensive deletion(s) in the viral genome. To identify the junction fragments, individual tumor DNAs were compared with DNAs from normal tissues (e.g., thymus or muscle) ofthe same animals. The representative sam- ples of normal tissue DNAs shown in lanes C1 and C2 of Fig. 3A serve as controls for tumor DNA samples in lanes 1—13 and 14—16, respectively. In both controls, only the fragments cor- responding to the endogenous viruses were detected: there are seven EcoRI-co fragments in C1 DNA, including the very faint B EcoRI/MC C 7 8L 9L H '2 '6 FIG. 3. Deletion and integra- tion of the proviruses as analyzed by EcoRI digestion. (A) cDNA5. hy- bridization with DNA samples of bursal or liver (L) tumors devel- oped in birds 1—16. Normal thymus controls, Cl and C2, are from birds * * 9 and 16. (B) pMyc-Pst hybridiza- Z'fi: tion with representative tumor «,4 DNA samples. Triangles indicate the 1.4-MDa1EcoRI fragments and stars represent the right~end viral- . cell junction fragments. M r in MDal. ‘58—. ‘. -4.3< Biochemistry: Fung et al. Table 1. Identification of fragments Mr of right~ end cell-viral junction, EcoRI 1.4-MDal Bird Sample MDal fragment 1. Bursa* 1.8,1 1.3, 0.9 +, A 2. Bursai A 3. Bursa 2.3, 1.71 A 4. Bursa 1.8’r + 5. Bursa 2.8, 18* A 6. Bursa 2.01 + 7. Bursa 2.0”r + 8. Liver 2.8, 2.6, 1.85”r + 9. Bursa . 1 + Liver 1 2.0,)” 1.7 A Liver 2 2.0,’r 1 7 A Liver 3 2.0,T 1 7 A Liver 4 2 0,1 1 7 A 10 Bursa 1 ND ND Bursa 2 2 4,1 1.7, 1 5 A 11 Bursa 1 7* + Liver 1 7+ A Spleen 1. 71 A 12. Bursa 1. 75' A 13 Bursa~t + 14. Bursa 1.81 + 15 Bursa 2.4,’r 1.8,* 1.71 + 16. Bursa 1.81 + Right-end cell-viral junction fragments were identified by cDNA5.. * Bird 1 carries three proviruses; two of them carry deletion in the gag gene, and the other appears to carry an intact EcoRI 1.4-MDal agmen . 1 Also detectable by pMyc-Pst. 4‘- Although the detections of the right-end junction fragments by cDNA5. in birds 2 and 13 are not obvious, TS fragments hybridizable to pMyc-Pst are present in these tissues. Birds 2 and 13 carry c-myc containing TS fragments of 1.8 and 2.4 MDal, respectively. +, The left-end internal EcoRI 1.4-MDal fragment is present; A, the EcoRI 1.4-MDal fragment is absent; ND, not determined. 1.7-MDal band, which is weakly detectable by cDNA5.. C2 DNA has a similar EcoRI cleavage pattern, except that the two small fragments (1.9 and 1.7 MDal) of ev 2 are missing. When the tumor DNAs were compared with these controls, new frag- ments of different sizes appeared. Those fragments indicated by stars, were identified as right- end cell- viral junction frag- mentsl and their sizes are given in Table 1. (Identification of some of the new fragments7 that migrate at positions close to the ev fragments—e. g. the 1.7 VlDal band—was aided by the sig- nificantly higher intensity ofthat band seen in tumor tissue over the corresponding ev fragment observed in normal tissue DNA of the same bird.) The size heterogeneity of the end fragments indicates multiple integration sites. However, it appears that the right-endjunction fragments in the size range 1.7—2.5 MDal are more common than others. It is also noteworthy that, in several cases, the tumor DNA carries more than one TS end fragment and, hence, more than one provirus. These multiple RAV-l proviruses possibly resulted from multiple virus infec- tions ofthe progenitor cell ofa monoclonal tumor. Alternatively, these samples may represent semiclonal tumors in which sev- 11 For those samples which carried deletions in the 1.4—MDal fragment, it is important to rule out the possibility that these new bands ofnovel sizes aIe de11\ cd from the gag- containingl. 4 \lDal internal fragment by st1uctur1l alterations. This was accomplished by fu1the1 h\brid- ization of the 1st bands “ith D\-\ piobes specific foi gag region. All of thei)e right- 1nd fr .Ll 111e11tsassignedaboyefailed to hybiidize to such a pm) Proc. Natl. Acad. Sci. USA 78 (1981) 3421 eral tumor clones coalesced together, as has been suggested for certain terminal LL tumors, based on histopathological evi— dence (17). Linkage of the RAV-l Provirus with the MC-29 Related Endogenous Sequences. Recent studies by Hayward et al. (18) strongly implicate a cellular sequence related to the oncogene of the acute leukemia virus, MC-29, in LL virus leukemogene- sis. The progenitor sequence of MC-29 oncogene (designated as c—myc) has been shown to be highly conserved and present in the genomes of all vertebrates (9). We wished to determine whether the infecting RAV-I DNA is physically linked to the c—myc in the LL tumors characterized in this study. To examine this possibility, a cloned DNA pMyc-Pst that specifically carries the MC-29 oncogene sequence was used as a molecular hy- bridization probe. Representative samples for pMyc-Pst hy- bridizations to EcoRI-cleaved tumor DNAs are shown in Fig. 3B. In normal tissue (lane C), only one high M, band corre— sponding to the c—myc locus is detected; in the tumor tissues (lanes 7, 8, etc), additional bands (indicated by stars) are also observed. The sizes of these additional bands are primarily in the 1.7—2.5 MDal range and match well with the corresponding viral—cell junction fragments assigned by hybridization with cDNAs. in Fig. 3A. These results indicate that, in these LL tu— mor DNAs, the c-myc gene (on one of the two chromosomes) is joined with the RAV-l provirus. Based on this analysis, we could demonstrate that, in all tumors in which the right-end junction fragment can be clearly detected by cDNA5., linkage between the RAV-I provirus and the c-myc sequence exist (see Table 1). In most of the samples in which multiple RAV—l pro- viruses are present, a single one is linked to the c-myc sequence. In one case (i. e. , bird 15, Table 1), all three proviruses are linked to the c-myc. We take the most straightforward interpretation and suggest that bird 15 bursal tumor consists of three coalesc- ing tumor clones and each carries a RAV-l provirus integrating next to the c-myc gene, but at a slightly different position. On the Mechanisms of Oncogenic Transformation. The mechanism by which LLV induces oncogenic transformation is especially intriguing because there is no evidence indicating that LLV codes for an oncogenic product. It has been postulated that specific integration of the LLV DNA into a site near a host oncogene might promote the expression of the oncogene (19). This possibility is particularly attractive in view of the fact that the two LTRs flanking the viral genome contain characteristics of promoters for eukaryotic transcription (20, 21) and that the sequence in the left-end LTR participates in the genesis of viral mRNAs (22, 23). Similarly, the right-end LTR may promote the transcription of downstream cellular sequences (24). The recent identification of novel mRNA species in LLV induced tumors, which carry both LTR-related sequences and sequences pos- sibly of host origin supports this hypothesis (15, 16, 18). The relationship of specific integrations to oncogenic trans— formation. Hayward et al. (18) have recently reported that, in the LL tumors, LLV proviruses are integrated next to the c-myc genes and that enhanced expression of MC-29 sequences are observed (18). These authors have suggested that insertion of the LLV provirus promotes the expression of the c-myc gene, thereby triggering the oncogenic transformation. Our data c011- firm some oftheir observations. We find that, in most of the LL tumors described here, at least one RAV-l provirus of each tu- mor is covalently joined to the endogenous myc locus; however, as seen by the various sizes of the RAV-l-oncMCv joining frag— ments, the exact integration sites of RAV-I proviruses are not always identical in individual tumors. These results suggest that integration of RAV-I at one of several sites near the c-myc gene is conducive to transformation. Recently, we have extended this analysis to the LL-like tumors induced by chicken syncitial vi- 3422 Biochemistry: Fung et al. ruses (CSV). We have previously shown that CSV, a member of the reticuloendotheliosis virus that bears no genetic rela- tionship to LLV, is capable of inducing LL with similar latency and pathology (25). In this case too, we have been able to dem- onstrate linkage between the c—myc the CSV provirus in all tu- mors characterized (unpublished results). As CSV DNA and RAV-l DNA, including their LTRs, share very little sequence homology with each other (26, 27), the finding that they are both integrated at positions next to the c-myc gene in LL tumors strongly implicates this gene and, possibly, adjacent sequences in the transformation of lymphocytes. The detailed mechanisms whereby the integration of either RAV-l or CSV promotes the expression of the c—myc gene have yet to be elucidated. The significance of the viral deletions to oncogenic transfor- mation. One striking finding is the detection of extensive dele- tions of proviral DNA in at least 40% of the tumors analyzed. It is possible that deletions of the viral genome that disrupt the transcriptional program of viral RNA facilitate the transcription of the downstream cellular sequences. Perhaps the transcrip- tion of viral RNA from the left LTR extending into the right LTR may affect the initiation at the right LTR. A disruption of the transcriptional program caused by a deletion in the proviral DNA may expose the right LTR and allow efficient transcription of the downstream putative oncogene. The following observa- tions are consistent with the importance of the LTR in the trans- formation process: (i) all tumor tissues analyzed in this study contain at least one LTR sequence (identified by cDNA3. and cDNAs. probes) and (ii) one tumor (5) harbors extensively de- leted proviruses which possess very little, if any, viral sequences other than the LTRs (unpublished data). Alternatively, the deletion of viral sequences may play a role in the selective growth of the tumor clones. Those cells in which the expression of viral antigens is eliminated by deletion may therefore be rendered less immunogenic and able to escape the host immune response. Histopathological examination shows that, at the onset of the disease, there are many microscopically observed enlarged bursal follicles (considered to be the trans- formed cell clones) (28, 29). Immune selection may account for the finding that only a limited number develop into tumors. Irrespective of the role of deletion of provirus in the tumor- igenic process, our data show that the presence of a complete provirus is not required at the terminal stage of the tumor. This finding lends further support to the hypothesis that the onco- gene(s) involved in the maintenance of cells in the transformed and tumorous state is of cellular rather than of viral origin. We are grateful to Drs. D. Sheiness and J. M. Bishop for providing the pMyc-Pst DNA clone. We thank Drs. C. Payne and H. E. Varmus for communicating data before publication, Mr. Lenny Provencher for excellent technical assistance, and Dr. S. Dube for helpful discussions. We also thank Drs. M. Fluck, E. Fritsch, and J. Dodgson for helpful comments and Ms. S. Uselton for assistance in manuscript preparation. This work was supported by a grant from the Michigan State University Foundation to H.-J.K. and in part by Interagency Agreement l-cp-4- Proc. Natl. Acad. Sci. USA 78 (1981) 0214 with the Division of Cancer Cause and Prevention, National Can- cer Institute. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. Graf, T. 8: Beug, H. (1978) Biochim. Biophys. Acta Rev. Cancer 516, 229—259. Robinson, H. (1978) Curr. Tapics Microbiol. Immunol. 83, 1—36. Crittenden, L. B. (1980) in Viruses in Naturally Occuring Can- cers (Cold Spring Harbor Laboratories Cold Spring Harbor, NY), pp. 529—546. Robinson, H., Swanson, C. A., Hruska, J. F. & Crittenden, L. B. (1976) Virology 69, 63—74. Kung, H. J., Bailey, J. M., Davidson, N ., Vogt, P. K., Nicolson, M. O. 8: McAllister, R. M. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 827—834. Shank, P. R., Hughes, 8., Kung, H. J., Guntaka, R. V., Bishop, J. M. 8: Varmus, H. E. (1978) Cell 15, 1383—1395. Friedrick, R., Kung, H. J., Baker, B., Varmus, H. E., Goodman, H. M. 8: Bishop, J. M. (1977) Virology 79, 198—215. Astrin, S. M., Robinson, H. L., Crittenden, L. B., Bluss, E. C., Wyban, J. & Hayward, W. S. (1979) Cold Spring Harbor Symp. Quant. Biol. 44, in press. Sheiness, D. K., Hughes, S. H., Varmus, H. E., Stubblefield, E. 8: Bishop, J. M. (1980) Virology 105, 415—424. Maniatis, T., Jeffrey, A. 8: Kleid, D. C. (1975) Proc. Natl. Acad. Sci. USA 72, 1184-1188. Neiman, P. E., Das, S., MacDonnel, D. 8: McMillin-Helsel, C. (1977) Cell 11, 321-329. Coffin, J. M., Champion, M. & Chabot, F. (1978) ]. Virol. 28, 972—991. Tereba, A. 8: Astrin, S. M. (1980) ] . Virol. 35, 888—894. Neiman, P., Payne, L. N. & Weiss, R. A. (1980)]. Virol. 34, 178—186. Neel, B. G., Hayward, W. S., Robinson, H. L., Fang, J. 8: As- trin, S. A. (1981) Cell 23, 3234334. Payne, G. S., Courtneidge, S. A., Crittenden, L. B., Fadly, A. M., Bishop, J. M. 8: Varmus, H. E. (1981) Cell 23,,311-322. Purchase, H. G. 8: Burmester, B. R. (1978) in Diseases of Poultry (Iowa State Univ. Press, Ames, IA), 7th Ed., Chapt. 15, pp. 437—438. Hayward, W., Neel, B. C. & Astrin, S. M. (1981) Nature (Lon- don) 290, 475—480. Tsichlis, P. N. 8: Coffin, J. M. (1980) ] . Virol. 33, 238—249. Yamamoto, T., Jay, C. 8: Pastan, I. (1980) Proc. Natl. Acad. Sci. USA 77, 176—180. Yamamoto, T., de Crombrugghe, B. 8: Pastan, I. (1980) Cell 22, 787—797. Weiss, S. R., Varmus, H. E. 8: Bishop, J. M. (1977) Cell 12, 983—992. Melon, P. 8: Duesberg, P. H. (1977) Nature (London) 270, 631—634. Quintrell, N., Hughes, 8., Varmus, H. E. & Bishop, J. M. (1980) J. Mol. Biol. 143, 1363—393. Witter, R. L. 8: Crittenden, L. B. (1979) Int. ]. Cancer 23, 673—678. Shimotohono, K., Mizutani, S. & Temin, H. M. (1980) Nature (London) 285, 550—554. Kang, C. 8: Temin, H. M. (1973) J . Virol. 12, 1314—1324. Cooper, M. D., Payne, L. N ., Dent, P. B., Burmester, B. R. 8: Good, P. A. (1968)]. Natl. Cancer Inst. 41, 373—389. Neiman, P., Payne, L. N. 8: Weiss, R. A. (1980) in Viruses in Naturally Occuring Cancers (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 519—528. 11111111111911119111111111111911191111199111119111111111911191111