Avian leukemia viruses oncogenes and genome structure

Biochimica et Biophysica Acta, 651 (1982) 245-271 245 Elsevier Biomedical Press

BBA 87100

AVIAN LEUKEMIA VIRUSES

O N C O G E N E S A N D G E N O M E S T R U C T U R E

T H O M A S G R A F a and D O M I N I Q U E STI~HELIN b

a Insti tute o f Virology, German Cancer Research Center, Im Neuenheimer Feld 280, 6900 Heidelberg (F. R. G.)

and b Institut Pasteur de Lille, I N S E R M U186, 15 rue C. Gukrin, 59019 Lille (France)

(Rece ived Oc tober 1st, 1981)

Contents

I. In t roduc t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

II. Oncogen ic spec t ra of DLVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

A. Chicken-der ived DLVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

B. The turkey-der ived REV T s t ra in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

III . T rans fo rma t ion specif ici ty of DLVs in vi t ro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 A. Hema topo ie t i c target cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

B. N o n h e m a t o p o i e t i c target cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

IV. Gene t i c content and genome s t ruc ture of DLVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

A. AEV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 B. A M V - t y p e viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

C. MC29- type viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

D. REV T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

V. Express ion of R N A and pro te ins in DLV-infec ted cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 A. AEV: two viral m R N A s ; one gag-onc prote in and a second onc(?) prote in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

B. A M V : two viral m R N A s ; one or more pu ta t ive onc pro te ins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

C. MC29: one viral m R N A ; one gag-onc pro te in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 D. OK10: two viral m R N A s ; a gag-pol-onc prote in and poss ib ly an onc prote in . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

VI. Gene t ics of DLVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

A. Mu tan t s in t r ans fo rming funct ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

B. Recovered viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

VII. Cel lu lar or ig in of DLV oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 A. Conse rva t ion of c-onc genes th roughou t evolu t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

B. C h r o m o s o m a l local iza t ion of c-onc genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 C. Structure of c-onc genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

D. Express ion of c-onc genes in no rma l cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

VIII . Mechan i sms of t r ans fo rmat ion by ALVs and DLVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 A. ALV- leukemogenes i s : The p romote r inser t ion hypothes is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

Abbrev ia t ions : ASV, av ian sa rcoma virus; ALV, avian leukosis virus; DLV, defect ive l eukemia virus; v-onc gene, viral t ransfor-

ming gene (oncogene) ; c-onc gene, ce l lu lar oncogene; AEV, av ian e ry throblas tos i s virus; AMV, av ian myelob las tos i s virus;

M C 2 9 , m y e t o c y t o m a t o s i s v i rus s t r a in 29; R E V , re-

t i cu loendothe l ios i s virus; C E F and QEF, ch icken and quail , respectively, embryo f ibroblasts , LTR, long terminal repeat ; RSV, Rous sa rcoma virus; RAV, Rous associa ted virus; MAV,

myelob las tos i s assoc ia ted virus.

0 3 0 4 - 4 1 9 X / 8 2 / 0 0 0 0 - 0 0 0 0 / $ 0 2 . 7 5 © 1982 Elsevier Biomedica l Press

246

B. DLV-leukemogenes is : Hema topo ie t i c target cell specificity and possible role of c-one genes . . . . . . . . . . . . . . . . . 263

1. A E V and the c-erb gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

2. A M V and the c-myb gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

3. MC29 and the c-myc gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

4. REV T and the c-rel gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

C. T r a n s f o r m a t i o n of nonhematopo ie t i c cells by DLVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

IX. Origin of D L V s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

A. Reverse t ranscr ip t ion of encaps ida ted viral and cellular R N A s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

B. Viral p r o m o t e r insertion; reverse t ranscr ip t ion of encaps ida ted R N A s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

C. Recombina t ion of integrated provi rus with a c-onc gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

D. In tegra t ion of proviruses a round a c-one gene; deletion of internal viral sequences . . . . . . . . . . . . . . . . . . . . . . . . 267

X. Out look . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

I. Introduction

Avian oncogenic retroviruses have been divided into three major groups [1]:

1. Avian leukosis viruses (ALVs). Most strains of these viruses induce predominantly lymphoid leukosis within 10 months after inoculation into susceptible birds. They also cause other types of disease (such as osteopetrosis, anemia, nephrob- lastomas, erythroblastosis and, occasionally~ sarcomas or endotheliomas), but do not transform cells in culture [2,3]. The genomic RNA of ALVs contains three structural genes (gag, pol, env), all of which are needed for virus replication. There appears to be no additional gene which might serve as an oncogene [1,4,5]. D N A sequences homologous to ALVs are found in the genome of many avian species which are part of complete or incomplete 'endogenous' viruses [4,6]. ALVs (also designated as nondefective leukemia viruses [2]) serve as helper viruses for replication-defective transforming viruses.

2. Avian sarcoma viruses (ASVs). These viruses induce sarcomas with a short period of latency and transform fibroblasts in vitro [1]. In addition to virus structural sequences the RNA of these viruses contain different types of transformation- specific sequences [94].

3. Defective avian leukemia viruses (DLVs). These viruses induce different types of acute leukemias within weeks after inoculation. They also cause sarcomas and carcinomas. All strains transform hematopoietic cells in vitro. In addition. most strains transform fibroblasts in culture and a

few appear to transform epithelial cells [2]. Their RNA contains ALV-related sequences as well as transformation-specific sequences, called v-one genes [94]. All virus strains of this group are defective for replication and it is for this reason that they are called 'defective leukemia viruses" or 'DLVs ' [2].

Comprehensive reviews on avian retroviruses in general, genetics and cell transformation have been published by Bishop [4], Vogt [5]~ Hunter [7] and Hanafusa [1]. The biology of ALVs and DLVs has also been reviewed previously [2,3].

In this article we will review the field of avian leukemia viruses, with emphasis mainly on DLVs. The following aspects will be discussed in detail: transformation specificity; genetic content and genome structure; viral RNA and protein expression; genetics; cellular sequences homologous to viral oncogenes; mechanism of transformation; and speculations about how DLVs might have arisen. Recent findings concerning the mechanism of leukemogenesis by ALVs will also be discussed.

!1. Oncogenic spectra of DLVs

I1A. Chicken-derived D L Vs

The eight available DLV isolates can be divided into four groups, depending on their transformation specificity in vivo and in vitro (Table I). The histories, the types of tumor from which they were isolated, as well as the oncogenic spectra of avian erythroblastosis virus (AEV)-type strains, avian myeloblastosis virus (AMV)-type strains and avian

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myelocytomatosis virus (MC29)-type strains have been reviewed previously in detail [2].

A total of about ten AEV-like isolates, four AMV-like isolates, and six MC29-1ike isolates have been described in the literature. Of these, one or two AEV, two AMV- and four MC29-type strains still exist. It is not clear whether or not the ES4 and R strains of AEV represent independent isolates. So far, no biological or biochemical differences have been detected between these two strains and we will, therefore, refer to them by the collective designation of AEV.

Some DLV strains which were isolated from hematopoietic neoplasms (such as AEV-R) continue to induce a disease type similar to the origi- nal one. Other DLV strains (such as members of the MC29 group) do not regularly cause hematopoietic neoplasms, but induce predominantly other types of tumors, such as kidney and liver carcinomas and possibly also endotheliomas. The oncogenic spectrum of a particular retrovirus is, therefore, not always a sufficient criterion to assign the virus to a particular DLV group.

l iB . The turkey-derived R E IA r strain

In this review we include a fourth group, with REV x as a prototype strain. The family of the reticuloendotheliosis viruses (REVs) consists of several C-type retrovirus strains isolated from ducks, turkeys and chickens. They belong basically to the group of ALVs, but are unrelated to the chicken avian leukosis /sarcoma viruses as judged by nucleic acid homology and antigenicity (for a review, see Ref. 9). REVs crossreact antigenically with mammalian C-type retroviruses [10-13]. Re- cently, sequences related to REV have been detected in normal chicken tissues [146]. This suggests that, as for classical ALVs, REV-related endogenous viruses exist in the germ line of chickens. It is, therefore, possible that REVs originated from a mammalian retrovirus integrated into the germ line of a chicken.

The REV-T strain was isolated from a turkey with lymphoid leukosis-type lesions [13]. It causes the rapid onset of a fatal disease in turkeys, chickens and quails, characterized mainly by nodular lesions in the liver and spleen (reticuloendotheliosis) [14,15]. In a more recent study, cells from one

such-tumor have been found to be of B-cell origin [16]. Stocks of REV-T contain a replication-defective component (designated REV r), which is capable of inducing cell transformation in vitro [17], as well as a helper virus (designated REV-A) [147]. Other members of the REV family do not transform cells in vitro, although at least four strains have been described which induced a proliferative disease at the time of their isolation [9], but which now appear to have lost their oncogenic potential (quoted in Ref. 18). The loss of oncogenic potential may be explained by the observation that (at least in REV T) the defective transforming component is easily lost during passage in fibroblast cultures [19,20]. Strains of REV with low oncogenic potential, probably consisting only of helper-type viruses, cause predominantly lymphoid leukosis about 4 months after infection [158].

111. Transformation specifici~ of DLVs in vitro

I l i A . Hernatopoietic target cells

The most widely used tissue for in vitro transformation studies of hematopoietic cells is the bone marrow of 1 3-week-old chicks [21,22]. Em- bryonal yolk sac and spleen cultures have also been successfully employed [23-25]. Hematopoietic cells transformed in vitro by each of the eight chicken-derived DLV strains have been characterized extensively with regard to the expression of a number of transformation parameters. As summarized in Table I, cells transformed by AEV resemble erythroblasts [21], cells transformed by AMV-type viruses resemble myeloblasts, and cells transformed by MC29-type viruses resemble macrophages [21,22,26]. Cells transformed in vitro were indistinguishable from cells transformed in vivo [21], with the possible exception of MC29-type viruses: the MC29 and CMII strains are capable of causing a myelocytomatosis in vivo (neoplasm consisting of immature cells of the granulocytic branch of the myeloid lineage), but they transform hematopoietic cells with the phenotype of macrophages in vitro [21,22]. The hematopoietic target cells of these viruses express lineage-specific differentiation antigens corresponding to those detected in the corresponding transformed cells [24,128].

Thus, DLVs transform predominantly certain types of committed hematopoietic progenitor cells.

In recent studies, cells transformed in vitro by REV T were found to be non-erythroid and non- myeloid in nature and, depending on the clone, to express B- or T-lymphoid (or both) cell surface antigen(s) [27,163]. It is interesting that, in contrast to cells transformed by AEV-, AMV- and MC29-type viruses, most hematopoietic cell clones transformed by REV T do not undergo cellular senescence [ 16,27,163].

The observation that different DLV strains transform specific types of hematopoietic cells can be used as a means to classify these viruses. For example, the E26 virus is a strain which was originally described as an erythroblastosis virus on the basis of cytological data on leukemic blood cells [28]. It was later found to transform in vitro hematopoietic chicken cells which exhibit the phenotype of myeloblasts [21]. In addition, leukemic cells transformed in vivo and grown in culture also exhibited several parameters of myeloid differentiation. This led us to propose that the E26 strain is a member of the AMV group [2]. The classification scheme for DLVs proposed on the basis of their biological properties [2] has now been confirmed fully at the molecular level. All DLV strains with similar transformation specificity in vitro have been shown to share a specific type of oncogene (see Section IV) and to synthesize similar types of putative transforming proteins (see Section V).

Recently, doubts have been raised about the concept of lineage specificity of DLVs. Thus, in- jection of E26 virus into newborn quails leads to the proliferation of erythroid in addition to myeloid cells; Moscovici, C., personal communication, and Radke, K., Beug, H. and Graf, T., unpublished data). We have now also found that E26 (but not AMV) induces in chicks an increase in the number of immature erythroid cells (histone-5-positive) in the peripheral blood, thus confirming studies by others [26]. However, in repeated experiments, the only growing cells which we could recover under standard tissue culture conditions are myeloblasts (Radke, K., Beug, H. and Graf, T., unpublished data). These observations may explain the discrepancy in the classification of the virus men- tioned above. They also suggest that some DLVs are capable of affecting more than one type of

249

hematopoietic cell * As estimated by in vitro transformation assays

(Refs. 22, 24, 29; and Graf, T., unpublished re- suits), 2. 1 0 6 bone marrow cells of a 2-week-old chick contain a maximum of approximately 250 target cells for AEV, 1000 for AMV, 10000 for MC29 and 50 for REV T. The values given for AEV and REV T represent minimum estimates since the relatively low titers of these viruses limit the number of transformed cells they induce.

The target cells of DLVs have so far only been characterized indirectly, since it is not yet possible to obtain purified populations of these cell types. The parameters examined include adherence, phagocytic capacity, buoyant density, volume and differentiation-specific cell surface antigens [22,24,29-31]. From these studies it was concluded that the target cell of AEV in the chick hematopoietic system is an immature erythroid cell, possibly at the level of an early erythroid progenitor cell [128,24,29]. Although present data suggest that the target cells of AMV and MC29 viruses are different, the former being less mature than the latter [24], the possibility that the two populations overlap is not ruled out.

The observed hematopoietic target cell specificity of DLVs could simply be due to the fact that a given DLV strain cannot infect (and, therefore, cannot transform) nontarget cells. This does not seem to be the case, since AEV replicates in macrophage cultures and MC29 replicates in erythroblasts as well as in myeloblasts. In addition, the putative transforming proteins are expressed in nontarget cells [34]. Due to lack of pure populations of uninfected target cells, it was necessary to analyze MC29 replication in erythroblasts already transformed by AEV or in myeloblasts transformed by AMV, and, therefore, these experiments are still subject to criticism. These experiments also do not rule out the possibility that DLVs are expressed in nontarget cells at levels insufficient to induce transformation.

Experiments with AEV have led to the concept that at least some DLVs interfere with the differ-

* It r e m a i n s to be seen w h e t h e r o r no t E26 h a r b o r s a n

e ry th ro id - spec i f i c o n c o g e n e d i f f e ren t f r o m its v-myb gene. It

will a lso be in t e res t ing to d e t e r m i n e if E26 t r a n s f o r m s one o r

two types o f h e m a t o p o i e t i c t a rge t cells.

250

entiation of their hematopoietic target cells. Several mutants of AEV which are temperature-sensitive for transformation were isolated [32,88]; erythroblasts transformed by these mutants at 36°C can be induced to differentiate towards erythrocytes when shifted for 3-5 days to 42°C [32,33,88]. Superinfection of these cells with wild-type AEV, but not with MC29, partially abolishes their capacity to differentiate [34].

In summary, from the data discussed in this section, the following conclusions can be made: 1. Hematopoietic cells transformed by each type of DLV strain display a different phenotype of differentiation. 2. The DLV target cells in chick bone marrow belong to the same lineage as the cells transformed by the corresponding DLV strain. 3. In the case of AEV, and possibly AMV-type viruses, these cells are arrested in their differentiation. 4. With the provisos made above, DLVs are capable of replicating and of expressing their transforming protein not only in target cells but also in nontarget cells.

IlIB. Nonhematopoietic target cells"

Most DLV strains transform chicken and quail embryo fibroblasts (CEF and QEF) in vitro (for chicken-derived DLV strains see Ref. 2; for REVT: Ref. 35). AMV is unable to transform fibroblasts of both species whereas the E26 strain, although apparently unable to transform CEF, is capable of transforming QEF [36]. It is interesting that CEF transformed by AEV or by MC29 assume mor- phologies which are distinguishable from RSV- transformed CEF and that they differ from the latter in the expression of a series of transformation parameters [37]. Specific phenotypes of transformation, which differ from RSV-transformed cells, have also been observed recently in rat fibroblasts transformed by AEV and MC29 viruses (Quade, K., personal communication).

It is unclear whether or not DLV strains are capable of transforming epithelial and endothelial cells. Although it has been reported that MC29. and possibly AMV, transforms embryo liver cells in vitro [2,38], the nature of the cells which are transformed has not yet been defined using cell

type-specific markers of differentiation. This is also true for the foci of epithelial-like cells induced by MC29 in cultures derived from whole chicken embryos [39]. The importance of using differentiation markers to identify the cell types which are targets for DLVs is underscored by the observation that fibroblasts transformed by MC29 occasionally assume an epitheloid shape (Graf., T. and Beug, H., unpublished data).

IV. Genetic content and genome structure of DLVs

For many years the presence of helper viruses in stocks of acute leukemia viruses hampered the investigation of the genetic content of DLVs. Re- cently, however, the molecular biology of DLVs made great advances when it became possible to isolate so-called nonproducer cells. Such cells are infected and transformed by a DLV in the absence of helper virus and contain in their DNA the DLV provirus. Another important advance was made when it was shown that DLVs do not contain the src gene of ASVs [162,150,108].

Four different methods were used to analyze the RNA genomes of DLVs:

1. Liquid hybridization experiments were performed with labeled cDNA probes made to ALV structural genes (gag, pol, env), to the 5' end sequences ( 'strong stop' cDNA), and to the 3' end of the ALV genome. In addition, RNAs were hybridized to cDNAs complementary to the genomes of prototype DLV strains, AEV, AMV, MC29 and REV v, but unrelated to ALV sequences (cDNA erb, mvb, mvc and rel). The viral genome and its corresponding provirus were also studied using the 'Northern ' and Southern blotting techniques. Hybridization techniques make it possible to determine the presence of a given sequence in the RNA, but do not yield information as to its relative localization in the viral genome [164].

2. Fingerprints of oligonucleotides obtained after Tl-ribonuclease treatment of RNA were used to compare unknown sequences with known standards. When applied to the analysis of poly(A)-containing RNAs of different sizes this technique can also be used to map given sequences within a genome [ 106,107].

3. Electron-microscopy (performed on duplexes between DLV RNA and ALV DNA) was used to

m a p precisely segments re la ted to A L V s within D L V genomes.

4. The avai lab i l i ty of molecular c loning technol- ogies has pe rmi t t ed more deta i led analysis of the o rgan iza t ion of D L V genomes, inc luding the de- t e rmina t ion of their nucleot ide sequences.

These techniques y ie lded essent ial ly s imilar and of ten complemen ta ry results, which have been compi l ed in Tab le II. A l though some poin ts are still unclear, ei ther because of missing in fo rmat ion or of confl ic t ing results repor ted by different labora tor ies , we have a t t emp ted to summar ize this i n fo rma t ion in the provis iona l genome maps shown in Fig. 1. In this figure, each DLV genome is d i rec t ly c o m p a r e d to an A L V - t y p e genome. A typical avian leukosis virus (like RAV-1 or RAV-2 or td B77) has a genome length of 7.5-8.5 kbases with the fol lowing structure: A 5' leader sequence (L) is fol lowed by the gag gene which codes for core pro te ins of the virus; the pol gene which codes for the reverse t ranscr ip tase and the ent~ gene which codes for envelope protein(s) . The 3' end of the genome, also known as the 'C region' , is fol- lowed by a s t retch of approx. 0.2 kbases of poly- (A).

The genome st ructure of ind iv idua l D L V strains reflects the fact that they p r o b a b l y arose by re- c o m b i n a t i o n be tween an A L V - t y p e virus and a cel lular oncogene (see Sections VII and IX). As a result , all DLVs have a cel lular inser t ion sequence which subst i tu tes for some of the virus s t ructura l gene sequences ( the dele t ion of some viral genet ic i n fo rma t ion is also the reason why they are defective for repl icat ion) . A compar i son of the cel lular inser t ion sequences of di f ferent DLVs reveals that the eight ava i lab le s t rains fall into four groups: A E V (erythroblastosis virus) has an inser t ion se-

quence t e rmed v-erb; A M V (myeloblastosis virus) - type s trains have an inser t ion sequence

te rmed v-myb; MC29 (myelocytomatosis)-type viruses have an inser t ion sequence des igna ted v- myc ( former ly cal led mac); and REV v (re- t i cu /oendothe l ios i s virus) has an inser t ion sequence cal led v-rel ( former ly cal led ret)*. It is l ikely that in all cases the cel lular insert codes for the t r ans fo rming abi l i ty of the virus (see Sect ion

* The nomenclature used follows the proposal of Coffin et al. [31].

~ ' I~NOME STRUCTURE OF A V I A N L E U K E M I A . VtRU.,S4ES

L gig pol env c ~A

A L V k b ~ , ~ ~ ; ~ , r ; ' ~ ' '

i t i erbA erbB . . . . . . . . A E V " ~ c ~ , , ~ - ~ - ~

A M V

E 2 6

'c 'from MAV

, [ n ~ +other sequences]-" , ~ '

' t M C 2 9 ~ ' ( ~; m y c ,

| i i i J

', t i m y c ,

O K I O ~ ' ' , j ~ - - : - - - - - ~ - , - t i a J - ' - ' - - ' . . . . . . . - . . . . - - - -

: i ,-- '" M H 2 ~ I ' i ~ m y c ;' "'" + other sequences ~( P(P-xFI

251

R E V A , kbQ

R E V T : . ~ . . . . . . Fe( Y . . . .

Fig. 1. Genome structures of avian leukemia viruses. The structures presented were deduced from the numbers shown and references cited in Table II. Each DLV genome is compared to an ALV genome (REV-A for REVT). Areas of deletion are indicated in the ALV genome by a wavy line. Insertions are indicated in the DLV genome as a heavy line. Parentheses indicate ambiguities due to variations in the values obtained by different authors and techniques. Some viruses (CMII, REVT) appear to have undergone a deletion/substitution event plus an additional deletion. Others (E26 and MH2) might contain additional insertion sequences. Open bars indicate the similar- ity in the specific structure of the 3' end of AMV with that of its natural helper virus, MAV.

VI) and that it de te rmines the t r ans forming specif ici ty of the par t i cu la r DLV strain: DLVs fall into the same four groups as de te rmined by sequence analogies as when classif ied on the basis of their t r ans forming specifici t ies (see Section lII) .

IVA. AEV

The genome of this virus is 5 .3 -6 kbases in size. Al l three viral s t ructura l genes ei ther have par t ia l de le t ions (e.g., gag, env) or are comple te ly absen t

bo

TA

BL

E I

I

GE

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TIC

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NT

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Ref

eren

ce n

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in

pare

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ses.

Whe

reve

r po

ssib

le,

valu

es s

how

n (g

iven

in

kbas

es)

have

bee

n co

mpu

ted

sepa

rate

ly f

or s

eque

nces

rep

rese

ntin

g po

rtio

ns o

f th

e vi

ral

geno

me

corr

espo

ndin

g to

5'

lead

er (

L)-

gag,

po

l, en

v an

d 3'

(or

'C

' re

gion

, in

clud

ing

the

U3

sequ

ence

s).

{, n

um

ber

s co

rres

pond

ing

to s

ever

al g

enom

e po

rtio

ns t

aken

to

geth

er;

*, R

esul

ts o

f Sa

ule,

S.,

Rae

s, M

.B.

and

St6

heli

n, D

., u

npub

lish

ed d

ata.

The

pol

yade

nyla

ted

stre

tch

(app

rox.

200

bas

es)

foun

d at

the

3'

end

of r

etro

vira

l R

NA

s ha

s no

t be

en i

nclu

ded

in

the

valu

es s

how

n. N

um

ber

s av

aila

ble

for

AL

V w

ere

obta

ined

us

ing

RA

V-I

, R

AV

-2 o

r td

B77

. L

iqui

d hy

brid

izat

ions

wer

e do

ne u

nder

str

inge

nt

cond

itio

ns (

S1 n

ucle

ase)

tha

t do

not

dis

crim

inat

e be

twee

n di

verg

ed o

r m

issi

ng s

eque

nces

for

a g

iven

vir

al R

NA

.

Vir

us

Vir

al R

NA

G

enet

ic c

onte

nt a

s de

term

ined

by:

Liq

uid

hybr

idiz

atio

n T

l-ol

igon

ucle

otid

e E

lect

ron

Mol

ecul

ar c

loni

ng

or b

lott

ing

fing

erpr

ints

m

icro

scop

y or

seq

uenc

ing

of v

iral

DN

A

AL

V

tota

l R

NA

8

-9

8.5

8.0

7.37

6 ~

5'-g

ag

2-3

2.

5 {

5.3

0.37

9 (5

')+

2.09

9 (g

ag

) ~

pol

2 3

[4,6

9]

2.5

[43,

68]

[44]

2.

684

" en

v 2

-3

2.5

1.6

1.61

6 a

3'

0.2-

0.8

0.9

0.75

0.

598

a

tota

l R

NA

5.

3 [4

5];

6.0

[46]

5.

5 6.

0 5.

3 Y

-ga

g

5' l

eade

r+0.

8 ga

g [4

6]

1.0

1.6

5' l

eade

r+ 1

.5 g

ag

pol

unde

tect

able

un

dete

ctab

le

[47]

un

dete

ctab

le

[48]

un

dete

ctab

le

[45]

env

0,4

[46]

~

1.5

{ 1.

1 (

0.4

3'

0.1

" er

b-A

; e

rb-B

3.

7 [4

2,46

] 3.

0 3.

3 2.

7

tota

l R

NA

7.

2 [4

9];

7.8

[50]

7.

5 7.

1 [5

3,54

] 5'

-gag

2.

5 [4

9]

2.1

[521

[5

31

pol

1.8

* 3.

2 (

5.5

env

unde

tect

able

[4

6,51

]

unde

tect

able

un

dete

ctab

le

[53]

3'

?

kb,

rela

ted

to M

AV

[4

9]

0.7,

rel

ated

to

MA

V

?, r

elat

ed t

o M

AV

[5

31

rnyb

2.

1 [4

6]

1.5

0.9

[53]

E26

to

tal

RN

A

6.2

5'-g

ag

5' l

eade

r+0.

7 ga

g po

l un

dete

ctab

le

[46]

en

v 1.

4 3"

?

myb

1.

4 of

myb

+

[52]

AE

V

AM

V

MC

29

tota

l R

NA

5.

2 [5

5];

6.1

[46]

5.

7 [4

3,47

] 5.

10

5'-g

ag

5' l

eade

r + 1

.5 g

ag

[46]

1.

2 [4

3]

1.2

pol

unde

tect

able

[4

6]

unde

tect

able

[4

3]

unde

tect

able

[5

7]

env

1.6

[46]

(

2.5

[43]

(

2.3

3'

0.13

[5

6]

myc

1.

8 [5

5]

2.0

1.8

CM

II

tota

l R

NA

6.

2 6.

0 [5

9]

5"-g

ag

5' l

eade

r + 1

.2 g

ag

[46]

1.

5 [5

9]

pol

dete

ctab

le o

n bl

ots

* tw

o sp

ots

[43]

en

v 1.

7 2.

5 w

ith

3' o

f R

NA

[5

9]

myc

1.

8 2.

0 [5

9]

OK

I0

tota

l R

NA

8.

0 8.

6 8.

0 5'

,gag

2.

5 2.

5 (

4.7

pol

2.1

[41,

46,7

7]

1.3

[60]

[4

1]

env

1.0

{ 2.

1 (

1.0

3'

0.23

m

yc

1.6

2.5

1.5

MH

2 to

tal

RN

A

6.1

5.7

[43,

61]

5"-g

ag

5' l

eade

r + 1

.2

[46]

1.

5 [6

0]

pol

unde

tect

able

*

unde

tect

able

[6

1 ]

env

0.2

[60]

3'

0.

12 *

{

2.5

myc

1.

2 2.

0 [6

0]

RE

V v

tota

l R

NA

5.

7 [ 1

8,62

] 5.

7

Y-g

ag

{

3.0

[18]

(

2.7

[64]

po

l

env

? ?

3'

1.0

[l 8

] 1.

0 re

l 1.

7 [ 1

8]

1.6-

1.9

5.5

[86]

3.

5 [8

6]

1.0

[86]

1.9

[58]

, 1.

8 [8

6]

a V

alue

s ob

tain

ed f

rom

seq

uenc

ing

RSV

. T

he s

rc s

eque

nces

(1.

577

kb)

have

bee

n su

btra

cted

. D

ata

from

Sch

war

tz,

D.,

Tiz

ard,

R.

and

Gil

bert

, W

., un

publ

ishe

d da

ta.

254

(pol) . The missing sequences are replaced by the insertion sequence e-erb (2.7 3.7 kbases) which is composed of two domains defined by the expression of two mRNAs coding for two proteins (see Section V). The insertion of the erb sequence seems to have occurred between gag and env.

1VB. AMV-type viruses

AMV: The genome of AMV (7.1 7.8 kbases) contains the entire gag gene and part or most of the pol gene. It is, therefore, not surprising that noninfectious AMV particles are synthesized by 'nonproducer ' myeloblasts, containing the AMV genome without the helper virus [52]. The erie gene is undetectable in AMV, and is probably replaced by the cellular insertion sequence v-mvb (2.1 kbases by liquid hybridization) known to map in that region [53]. The 3' end of the AMV genome is unusual, since it contains sequences probably derived from its natural helper virus (MAV) that at its 3' end harbors nucleotide sequences unrelated to the common ALVs (Refs. 49, 52, 53; and St6helin, D., et al., unpublished data). It is not known whether or not the v-mvb- and MAV-related specific sequences are contiguous. The discrepan- cies in the size found for the v-mvb sequence may stem from differences between individual stocks of AMV which have been passaged in different laboratories over a period of many years. Also, the single molecular clone of AMV studied might not be representative of the entire viral population. In addition, it is not known whether or not it is infectious.

E26: The genome of E26 (6.2 kbases) has so far only been analyzed by liquid hybridization (Saule, S., et al., unpublished data). Like AEV, it seems to contain only part of the gag and env genes and to lack completely the pol gene. It contains at least part of the v-myb sequences of AMV and probably harbors additional, so far unidentified, sequences. The relative positions of ~-myb and the additional sequences within the E26 genome have not yet been determined, nor is it known if they are contiguous. The drawing of the E26 genome in Fig. 2 is, therefore, speculative and solely based on the analogy to the organization of the other DLVs.

-

< "4 a Z ~ Z

°o " , ),, g

• \ \ \ \ \ \ e

I = \ ' , , \ ~ , o

I 25 \ \ \ \~ , 6o~

I

I..- I- Zuj '~"~ .~ ~ ! uaZ °

w e,

' I o 1'o loo lo6o •

Chlcke n ~e~t "~

PHYLOGENETIC DISTANCE (xlOeyem'=)

Fig. 2. Stability of c-one genes during evolution. Aliquot~ of DNAs extracted from different species having diverged increas- ingly long ago with speciation ',,,'ere hybridized under the conditions described by Roussel et al. [65]. The values obtained were standardized arbitrarily for comparative purposes to 50% for the chicken, which was also taken as the origin of the scale indicating phylogenetic distance, eDNA erh (O), eDNA mvc (A), eDNA ,0,b (C)). cDNA src (~) . Also shown are results obtained with 3H-labeled cDNAs representing genes known to be conserved throughout the evolution of vertebrates: eDNA glo for globin ( X -- X ), eDNA ova for ovalbumin ( ~. . . . . . . x) . (& . . . . . . . &) indicates the average evolution of unique sequence DNA of the species tested under similar experimental conditions (SI nuclease resistance of hybrids with ]4('-labeled chicken unique sequences DNA, standardized to 100el for chicken DNA).

1 VC. MC29-tvpe viruses

MC29: The genome of MC29 (5.1-6.1 kbases), contains partial gag- and end-related sequences, and lacks the pol gene. The cell-derived sequence e-myc (1.8-2 kbases) is inserted between the gag and ene sequences as a continuous stretch.

CMII: The genome of CMI1 (6 6.2 kbases) is very similar to that of MC29. It contains partial gag and env genes and lacks most of the pol gene. Its cell-derived sequence v-rove (1.8 2 kbases),

quite similar to that of MC29, is probably inserted between the gag and pol sequences [43].

OK10: The genome of OK10 (8-8.6 kbases) is the largest of the known avian DLV strains. It contains a complete gag gene, most of the pol gene (apparently nonfunctional) and part of the env gene. Noninfectious particles are produced by OK10-transformed nonproducer cells [60]. The specific sequence v-myc (1.5-2.3 kb) is located at or near the pol-env junction as a continuous stretch (Sergeant, A. and St6helin, D., unpublished data).

M H 2 : M H 2 is, together with E26, the least studied of the DLV strains. Its genome (5.7-6.1 kbases) contains partial gag- and env-related sequences and a specific sequence v-myc (1.2-2 kbases) which, as judged by liquid hybridization, is more distantly related to that of MC29, CMII and OK10 viruses [65]. As for E26, there might be additional sequences present. The latter observation, made with the technique of liquid hybridization, may also explain the discrepancy in the size of the right-hand end of the genome as determined by Tl-ribonuclease fingerprinting. The localization and order of the insertion sequences shown in Fig. 2 is speculative.

IVD. REV. r

The genome of REV v (5.7 kbases) contains gag and pol sequences related to REV-type ALVs. The specific sequence, v-rel (approx. 1.6 kbases), appears to be inserted as a deletion-substitution within the env gene, which is still partially present. In addition, it contains a large deletion in the gag-pol region which is not contiguous with the v-rel sequences.

Several general conclusions can be drawn from the results reviewed above: 1. The DLV-specific cell-derived sequences are inserted within the helper virus genome at different positions, and, thus, no 'hot spot' for these recombinations seems to exist. 2. In all cases, both the 5' end (including some gag sequences) and the 3' end of the ALV genome are conserved. 3. The insertion sequences replace variable amounts of helper-related sequences, and the order of ALV-related genes seems to be maintained in all DLV strains.

255

4. When the whole gag gene is present, as in AMV and OK10, viral particles are synthesized in the infected cells.

V. Expression of RNA and protein in DLV-infected cells

The observations that DLVs transform their target cells within a matter of days and that their genomes contain specific sequences which corre- late with their biological specificity suggest that they use virally coded proteins to transform their target cells. Several new lines of evidence support this view (for a recent review, see Ref. 66). In some cases, both the putative transforming proteins and their corresponding mRNAs have been identified and characterized. These studies involved the analysis by liquid hybridization and 'Northern' blots of the RNA of DLV-transformed nonproducer cells using 32P-labeled cDNA probes homologous to various viral genes. Proteins synthesized in the t r a n s f o r m e d cel ls w e r e l a b e l e d wi th [35S]methionine or with [14C]arginine-lysine and immunoprecipi ta ted with antibodies against structural proteins or with antibodies to the v-erb gene product p75 of AEV [67] (unfortunately, transformation-specific antibodies detecting the v-myb and v-myc gene products are not yet available). To determine their coding potential, DLV RNAs were also translated in a reticulocyte cell- free system. Proteins synthesized in vivo and in vitro were then compared with the known structural proteins, using tryptic peptide-mapping techniques.

The results obtained by these different ap- proaches (Table III and below) show that DLVs use at least four different strategies of expression.

VA. AEV: two viral mRNAs; one gag-onc protein and a second onc(?) protein

The genomic size mRNA of AEV codes for a 75 kDa protein that is immunoprecipitated by anti- gag sera and by specific sera from chicks which survived an erythroblastosis. According to tryptic peptide analysis, this mRNA encodes p 19 and part of p27 gag protein as well as a unique portion which corresponds to the product of the 5' portion of the v-erb sequence (the v-erb-A domain). AEV

TA

BL

E

III

EX

PR

ES

SIO

N

OF

V

IRA

L

RN

As

AN

D

PR

OT

EIN

S

IN

INF

EC

TE

D

CE

LL

S

+ in

dica

tes

the

synt

hesi

s of

a g

ene

or

spec

ific

seq

uen

ce i

n a

pro

bab

ly c

om

ple

te f

orm

: ( +

),

par

tial

seq

uen

ce o

nly

: ,

no

t d

etec

tab

le:

p, p

rote

in:

pr,

pre

curs

or

pro

tein

; gp

,

gly

cop

rote

in;

nd,

no

t d

on

e; ?

, p

rod

uct

pre

dic

ted

fro

m t

he R

NA

, b

ut

no

in

terp

reta

ble

res

ults

av

aila

ble

yet

. x

I, S

aule

, S.

an

d S

t6he

lin,

D.,

un

pu

bli

shed

dat

a:

x 2,

Hay

man

, M

.,

un

pu

bli

shed

dat

a; x

3,

Beu

g, H

., H

aym

an,

M.

and

Gra

f, T

., u

np

ub

lish

ed d

ata.

Vir

us

Siz

e of

R

elat

edn

ess

to

Ref

eren

ces

Pro

tein

S

yn

thes

ized

in:

R

efer

ence

s G

enet

ic

mR

NA

(s

ize

of

kD

a)

assi

gn

men

t

(S)

5'

gag

po

l en

v 3'

on

e ce

ll-f

ree

livi

ng

syst

em

cell

s

AL

V

34

+ +

+ +

+ 4

.68

.69

.13

3,1

39

21

+ -

+ +

-

A E

V

30

+ ( +

)

- ( +

)

+ +

42

,45

,46

.47

.48

23

+ --

-

( + )

n

d

+ 42

,70

AM

V

32

+ +

+ +

+ 49

.50.

52,5

4

20

+ -

--

--

nd

+

49.5

1

E26

30

+

( + )

MC

29

30

+

( + )

CM

II

30

+ (+

)

OK

l0

34

+ +

25

+

MH

2

30

+ (+

)

22

+ -

RE

V T

29

+

( + )

(÷

- (+

~+

) (+

(+)

(+

- (+

(+)

+?

+ ( +

)

46,5

2

+

+

43,4

6,47

,55

+ +

43,5

5

+ +

46,6

0,56

nd

+

56.4

1

+ (+

) 46

nd

(+

) x

j

+?

+ 18

pr7

6

+ +

gag

pr

180

+ +

63

ga

g-p

ol

gp

85

. 37

+

+ en

c

p75

+ +

71.7

2.73

.143

ga

g-er

b-A

p4

0

+ 74

,75.

76,1

43

erb

-B

p65

+ u

np

ub

lish

ed,

x:

erb

-B

pr7

6

+ +

78.7

9 g

ag

pr

180

+ +

78

ga

g-p

ol

p32,

p48

, p

56

+

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provirus also synthesizes a subgenomic mRNA of 23 S which has a spliced structure. Polyadenylated fragments of AEV RNA synthesize a 40 kDa protein in vitro that has no gag determinants and is also unrelated to p75, and, thus, probably repre- sents the product of the v-erb-B domain. Recently, antibodies that have been obtained from rats injected with AEV-transformed cells precipitate in AEV-transformed cells not only p75 but also a 65 kDa protein. This 65 kDa protein is closely related to the 40 kDa protein synthesized in vitro (Hay- man, M., unpublished data) and it is likely to represent the correctly initiated product of the v-erb-B gene.

VB. AMV: two viral mRNAs; one or more putative onc proteins

The 32 S genome size mRNA of AMV encodes the gag precursor protein pr76 and may also code for the prl80 gag-pol precursor synthesized in ALV-infected cells. No fusion protein involving gag and v-myb sequences has been detected so far. The 20 S mRNA found in infected cells probably codes for one or several v-myb-type proteins. From preliminary data it appears that E26 does not correspond to this scheme. Rather, its strategy of transcription might resemble that of MC29. Inter- estingly, some E26-transformed myeloblast cell strains express as few as 10 copies of E26-specific RNA molecules per cell. This low level of RNA is apparently sufficient to maintain the transformed phenotype. A 130 kDa candidate gag-myb protein has been detected but remains to be characterized.

VC. MC29: one viral mRNA," one gag-onc protein

In MC29-transformed nonproducer cells only one viral mRNA, which is genomic in size, has been detected. A gag-myc 110 kDa fusion protein was identified by immunoprecipitation from both in vivo and in vitro translation experiments. This protein contains the p19 and p27 gag-related proteins as well as unique tryptic peptides unrelated to the structural gene products of ALVs, presumably corresponding to the v-myc gene-coded product. CMII also codes for a gag-myc type protein (90 kDa) and probably uses a similar strategy of expression. The unique (myc) tryptic peptides of

257

p110 MC29 and p90 CMII are very similar to each other but differ from the unique (erb) peptides of p75 AEV [87].

VD. OKIO: two viral mRNAs; a gag-pol-onc protein and possibly an one protein

The 34 S genomic mRNA of OK10 produces the pr76 gag gene product, but no prl80 gag-pol polyprotein. Instead, nonproducer cells contain low levels of a 200 kDa polyprotein which appears, as judged from tryptic peptide analyses, to correspond to a gag-pol-myc fusion protein. It remains to be shown that p200 accounts for the transforming ability of OK10. Alternatively, it is possible that the subgenomic 23-25 S mRNA which has been detected in OK10-transformed nonproducer cells and which has a coding capacity sufficient for a 50 kDa protein contains the viral oncogenic functions. MH2 is, so far, less well-characterized, but preliminary evidence suggests that it uses a strategy similar to that of OKI0 (Saule, S. and St6helin, D., unpublished data).

Although still incomplete, these studies allow several preliminary conclusions: 1. The various DLV strains use different strategies to express their cell-derived sequences. 2. There is no simple correlation between the strategy of expression and the target cell specificity of transformation of a given DLV strain, e.g., MC29 and OK10 viruses both transform similar target cells but use different strategies of expression, while OK10 and AMV viruses seem to be similar in their modes of expression but transform different target cell types. 3. The non-gag portion of the protein synthesized by a given DLV strain correlates with the transforming specificity of the virus. 4. The observations that the gag gene portion of transforming proteins can vary widely in different strains and that some strains seem to code for a transforming protein which lacks a gag portion suggest that gag peptides play no key role in the transforming potential of DLVs.

Each DLV strain probably utilizes the same strategy of expression in the different species and target cells which it infects. For example, AEV- transformed chicken erythroblasts and fibroblasts as well as AEV-transformed rat fibroblasts all

258

synthesize the two AEV m R N A species (30 and 23 S) at a similar ratio, although the level is about l0 times lower in rat than in chicken cells [42]. In addition, hematopoietic non-target cells infected with AEV or MC29 synthesize apparently unaltered gag-onc fusion proteins [34].

VI. Genetics of DLVs

Recent work on the genetics of DLVs has made it possible to assign a role of AEV- and MC29- coded proteins in transformation. These studies will be discussed below.

VIA. Mutants in transforming functions

The data reviewed so far provide no direct evidence that the various transformation-specific DLV sequences are required for the maintenance of the transformed state. The characterization of the first DLV ts mutant isolated, ts34 AEV, has shown that the continuous production of a DLV gene product is necessary for the maintenance of erythroblast transformation [32]. This ts mutant is also defective in inducing an erythroblastosis in chickens [32], animals known to have a body temperature exceeding 41°C (nonpermissive temperature for the ts mutant). Four additional ts mutants of AEV have now been isolated and these appear to be basically similar to ts34 AEV [88].

All ts mutants of AEV studied so far are not only ts for erythroblast transformation but also ts

for at least some parameter of fibroblast transformation [88,92]. Since it is unlikely that they all represent double mutants, this indicates that either one of the two v-erb genes alone or a combination of both are required for the maintenance of erythroblast and fibroblast transformation.

Recently, a first ts mutant of AMV was isolated (Moscovici, C., personal communication). Despite efforts in several laboratories, particularly with MC29, it has so far not been possible to isolate ts

mutants from any of the other DLV strains. The isolation of nonconditional mutants defec-

tive for transformation is hampered by the fact that, unlike some ASV strains, DLVs are replication-defective. Transformat ion-defect ive DLV mutants cannot, therefore, simply be isolated by screening the progeny of a mutagenized DLV stock

for the presence of replication-competent, non- transforming derivatives. However, the finding that most DLVs transform not only hematopoietic cells but also fibroblasts [2] makes possible an ap- proach in which mutants are sought which have lost the ability to transform one cell type but not the other. Such a 'host range' mutant was recently isolated from AEV: this mutant (td359 AEV) has lost its ability to transform erythroblasts but still transforms fibroblasts [89]. td359 AEV synthesizes an altered p75 protein which lacks three of about 53 lysine-arginine tryptic peptides. The missing peptides belong to the v-erb-A portion of the molecule [90]. The 40 kDa protein synthesized in vitro, presumably from the v-erb-B gene of the AEV genome, is unaltered [91]. It is not yet known whether the mutant synthesizes an altered 65 kDa protein.

Transformation-defective mutants have also been isolated from MC29. The three mutants studied so far exhibit a drastically decreased ability to transform chicken macrophages, but are unaltered in their capacity to transform fibroblasts [93]. All three mutants express a gag-mvc fusion protein which is smaller than that of wild-type MC29 virus [93] and lacks some of the myc-specific

tryptic peptides (Ramsay, G. and Hayman, M.J., pe r sona l c o m m u n i c a t i o n ) . O l igonuc leo t ide analyses support this finding and also demonstrate that the deletions seen at the protein level are due to deletions in the RNA and not to premature termination signals for protein synthesis (Bister, K., Ramsay, G., Hayman, M.J. and Duesberg, P.H., personal communication).

VIB. Recovered viruses

Hanafusa and colleagues [ 107,167] first demonstrated that transformation defective RSV mutants with partial deletion in the src gene can be 're- paired', that is, competent transforming viruses (r-RSV) with properties similar to wild-type RSV can be recovered from chickens injected with the mutants. Recently, such recovered viruses were also obtained from td359 AEV by in vivo passage of the virus (Graf., T., unpublished data) and are designated rAEVs. Like wild-type AEV, the two rAEVs obtained efficiently induce an erythroblastosis; one of them synthesizes a p75 protein

which is of wild-type size, the other is slightly larger (Beug, H. and Graf, T., unpublished data). In addition, a wild-type revertant of a tdMC29 mutant was recently isolated after in vitro passage of the mutant. This virus has regained its macrophage-transforming potential as well as some of the missing myc-speci f ic peptides (Graf, T., Ramsay, G. and Hayman, M.J., unpublished data).

Vii. Cellular origin of DLV oncogenes

The availability of DLV transformation-specific cDNA probes makes it possible to search for the presence of homologous sequences in the DNA of normal cells. As shown below, such homologous cellular sequences have been found for all four types of DLV oncogene and, thus, are analogous to the src gene of Rous sarcoma virus [95]. They are designated as 'c-onc genes' to distinguish them from the viral oncogenes or 'v-onc genes' [31].

VIIA. Conservation o f c-onc genes throughout evolution

Before discussing the evolutionary stability of c-onc genes, endogenous sequences related to ALVs will be briefly reviewed. The DNA from most chickens contain ALV-related sequences which, in some instances, code for a complete nondefective endogenous virus termed RAV-O [6]. These endogenous viral sequences (ev) are found in domestic and wild fowl at dispersed genetic loci, but are not detectable in non-avian vertebrates and in some exotic chicken species [96], nor in domestic chickens bred to be free of endogenous virus [97]. Thus, these sequences may represent remnants of retroviruses which became integrated into the germ line. Alternatively, it is possible that they have evolved from transposable elements, as proposed recently by Temin [98]. A third possibility is that they were formerly ubiquitous, but that in some birds they were eliminated during evolution. Whichever interpretation proves to be correct, the fact that animals lacking ev sequences develop normally argues against an indispensable role of the sequences during development or cell differentiation.

As found first for the src gene of Rous sarcoma virus [95], DNA homologous to the transforma-

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tion-specific sequences of DLVs is also present in the DNA of normal host cells. DNA homologous to the v-erb [65], v-myb [65] and v-myc [65,99] genes are found in all vertebrate species tested at one to two copies per haploid genome. The homology decreases with increasing evolutionary distance from the chicken, but species which are about 500 million years apart from birds still show detectable hybridization, even under stringent conditions [65] (Fig. 2).

Unlike the ev genes, these sequences are, therefore, comparable to highly conserved genes such as the globin or ovalbumin genes and probably play an important role in development, differentiation or growth. In contrast, although DNA sequences homologous to the v-rel gene are present in the DNA of turkeys and (with a significantly lower homology) in chickens and quails, they are undetectable in the DNA of any mammalian species tested [100]. It is, therefore, unclear whether the v-rel sequences represent a true cellular gene or whether they were derived from ev-like sequences, as appears to be the case for the transforming sequences of the defective component of Friend murine leukemia virus [10]. It has to be pointed out, however, that the cDNA used in the work with REV v [100] was prepared differently than the cDNA specific for v-erb, v -mvb and v-myc, and this may account for some of the differences seen in the evolutionary distribution of these sequences.

VIlB. Chromosomal localization o f c-onc genes

Unlike mammalian karyotypes, avian karyotypes consist of two major classes of chromosomes: 15-16 pairs of macro-chromosomes of 'normal' size and 40-60 indistinguishable mini- chromosomes. To localize c-onc genes, macro- and mini-chromosomes have been separated by density gradient centrifugation and then hybridized by the technique of Southern to DLV-specific sequences using cDNA probes. In these studies, using the MSB-I cell line, the c-myc gene was found to be located in the macro-chromosome fraction [103]. Using an in situ hybridization technique with iodine-labeled specific cDNAs and primary chicken cells, the c-erb, c-myb and c-myc genes were found to be located on a small macro-chromosome (number 10-13 for c-erb and 12-16 for

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c-myb and c-myc) [102] (Tereba, A., personal communication). It is not entirely clear whether the c-erbA and c-erbB genes are both located on the same chromosome. In no case could a linkage between a c-onc gene and an ev gene be demonstrated.

VIIC. Structure of c-onc genes

Following the cloning of AEV [45], MC29 [58] and AMV [53] viral genomes in prokaryotic vectors, some of the DLV-related c-onc genes were also cloned recently. Portions of the chicken c-erb genes were isolated in a series of phage clones from a chicken D N A library and have revealed some basic information about the organization of these sequences [104,165]. The c-erb-B domain contains 11 introns spanning approx. 25 kbases of DNA. The erb-A gene contains at least 3 introns and a total size of more than 2.5 kbases. It is not yet clear whether both genes are located adjacent to each other and in the same order as in the AEV genome. Other studies have indicated that the organization of the chicken c-myc and c-mvb genes is probably much simpler than that of the c-erb sequences and that these genes are contained within a few kbases of cell D N A (Bishop, J.M., personal communication). Sequences homologous to chicken v-erb, v-myb and v-myc genes have also been detected recently in D N A from human cells by screening libraries of prokaryotic vectors. The structures of these sequences and their expression in human tumors are being intensively investigated in a number of laboratories.

VIID. Expression of c-onc genes in normal cells

Using cDNA probes specific for the v-erb-A and v-erb-B sequences of AEV, four species of mRNAs have been detected in normal cells at the level of expression of approx, one copy per cell. As judged from 'Northern ' blot analysis, two of these (4.5 and 3.0 kbases) are derived from the c-erb-A domain and two (12 and 9 kbases) are derived from the c-erb-B domain [104]. It is not clear whether the smaller RNA molecules represent splice products of the corresponding larger ones [104]. In contrast, a single 2.8 kbase c-myc RNA [103] and a 4.5 kbase c-myb RNA [105] have been

found in normal cells. It is likely that these RNAs code for proteins since they are found on poly- somes.

The evolutionary stability of the cellular erb, myb and myc genes together with the observed hematopoietic target cell specificity of DLVs suggested that DLV-related cellular oncogenes serve some basic function in the regulation of growth or differentiation of normal hematopoietic cells [46,65,94,109]. It was, therefore, of great interest to determine whether or not these sequences are specifically expressed in hematopoietic cells. The first study on the expression of a DLV-related c-one gene was published by Chen [105], using liquid hybridization techniques. This author found that the c-myb gene was expressed at about 20 copies in normal bone marrow cells, at about 10 copies per cell in embryonic bursa and yolk sac, but was not expressed in nonhematopoietic tissues (about one copy per cell). A variety of hematopoietic and nonhematopoietic tissues were also examined by in situ and liquid hybridization in the laboratory of one of us (D.S.) for the expression of c-erb and c-rnyc genes. The data indicate that c-erb gene is highly expressed (about 50 copies per cell) in about 0.1% of normal bone marrow cells [110]. As found by liquid hybridization, c-myb and c-myc sequences are expressed at about 10 copies per cell in fractions of bone marrow cells enriched for immature cells. In addition, they are expressed at variable levels in transformed hematopoietic cell strains of different lineages [110].

The data available so far suggest that the c-onc genes can be expressed in hematopoietic cells that are not target cells for the homologous DLVs. For example, macrophage populations are known to contain a high proportion of target cells for MC29, fewer targets for AMV and none for AEV [22,24], yet all three types of oncogenes are expressed at low levels in this cell population [110]. Similarly, erythroblasts transformed by AEV express a high level of c-myc m R N A [110], although viruses containing the v-myc gene are not known to transform erythroid cells. In this case, it could be argued that the transformation by AEV induces the expression of the c-myc gene in erythroblasts. Data obtained with transformed fibroblasts indicate, however, that transformation does not generally lead to an

increased expression of DLV-related c-onc genes [l l0l .

VIII. Mechanisms of transformation by ALVs and DLVs

In analogy to the picture which is emerging for the sarcoma viruses it is likely that DLVs act as transforming agents by reintroducing into the host cell a cellular oncogene now under the control of a viral promoter. The increased expression of this gene in certain cell types may then lead to changes in growth behaviour or differentiation, resulting in the acquisition of a neoplastic phenotype.

The observations that ALVs induce neoplasms only after a long latency period, that they do not transform cells in vitro, that their genomes do not contain a cellular insertion sequence, and that they do not encode proteins other than those required for virus replication, have made it difficult to analyze the mechanism by which these viruses transform cells. Several possibilities could be con- sidered.

1. The expression of structural or replicative genes may lead directly or indirectly to cell transformation, e.g., it has been suggested that the env

protein of certain murine leukemia viruses acts as a mitogen [111].

2. The integration of an ALV provirus into host cell DNA may cause mutations which may alter the expression of cellular genes required for growth or differentiation.

3. The inserted ALV provirus may activate a cellular oncogene by introducing a viral promoter into appropriate regulatory sites of target cells. Recent evidence suggests that such a mechanism may be responsible for the induction of bursal lymphomas by ALVs. This evidence will be reviewed below.

VI I IA . A L V-leukemogenesis." the promoter insertion

hypothesis

Before entering the discussion of this section it is necessary to review some of the structural fea- tures of an integrated ALV provirus. Integrated ALV proviruses contain the three virus structural genes (gag, pol, env ) flanked at each end by identi- cal sequences of approx. 350 base pairs designated

Cel l DNA In tegra ted provi rus

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Cel l DNA

LTR gag - pol - env LTR c - o n c 5' 3 '

U3 RU5 U:~ RU5 / J / /

RU5 RU5 e - ortc

Viral RNA Vi ra l /ce l l . RNA

Fig. 3. Model for the activation of a c-onc gene by the integration of an ALV provirus [40].

as long terminal repeats (LTRs, Fig. 3) [112]. The LTRs consist of a unique sequence derived from the 3' end (U3), a unique sequence derived from the 5' end (U5) of ALV genomic RNA and of a short terminal repeat of about 20 nucleotides (R) which is present at both ends [4,112-114]. The 3' end of genomic RNA is similar in many virus strains and is also designated as the 'C region' [107]. This region is composed of R, U3 and additional noncoding sequences between U3 and env. The proviral DNA is larger than the parental RNA, U3 being repeated at the 5' end and U5 being repeated at the 3' end of the molecule. Sequencing and in vitro transcription studies indicate that the U3 region contains a promoter-like sequence [115,159]. The U3 region in the left LTR is, therefore, thought to direct the transcription of progeny viral RNA. The LTR of exogenous viruses is approx. 75 base pairs longer than that of endogenous chicken viruses [116] and this is probably due to differences in the C region between these two classes of ALV-type viruses [ 113].

The existence of a correlation between the presence of a C region characteristic for endogenous viruses (C n) and their low growth potential as well as a correlation between the presence of the C region from exogenous viruses (C*) and a high growth potential [117] suggested a role of the U3 portion of the C region in viral transcription. The differences in the U3 region may also account for the fact that exogenous chicken viruses are oncogenic, whereas chicken viruses of endogenous origin (such as RAV-O) are not oncogenic. That this difference in oncogenic potential is due to differences in the env gene is ruled out by studying

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recombinant viruses which have the same env gene but differ in C [ 118]. Generally, recombinants with a C x sequence are capable of causing lymphomas, whereas recombinants with a C n sequence are non-oncogenic [118]. Recently, however, a recombinant of the latter type was found which, although it had lost its strong lymphomagenic potential, was still able to induce other types of neoplasms with a long latency period (Robinson, H., personal communication). This recombinant differed from other non-oncogenic strains in a region outside U3, suggesting that the noncoding sequences between env and U3 play a role in determining the target cell specificity of ALVs.

Results from several laboratories have now also directly suggested a role of the LTR in ALV- lymphomagenesis. In these studies, D N A and RNA from ALV-induced bursal lymphomas were analyzed by blot hybridization using cDNA 5' (complementary to the first 101 nucleotides of the 5' end of ALV genomic RNA, corresponding to R and U5) and cDNA rep (corresponding to the total ALV genomic RNA). The results can be summarized as follows:

1. Although ALVs are known to be capable of integrating at many sites, possibly at random [114,120], most proviruses were found to be integrated at a limited number of sites in the DNA of bu r sa l lymphomas [122,123]. The finding that the cells in different lymphomas of the same bird showed the same pattern of viral integration [40,122,162] suggests that the lymphomas arose clonally as a consequence of a rare event.

2. Lymphomas from different animals showed, as a rule, different patterns of integration. Many tumors contained several proviruses, but some contained only one [40,122]. Several tumors showed similar sites of provirus integration [40,122].

3. In some tumors the structural genes of ALVs were largely, if not totally, absent, indicating that viral products are not required for the maintenance of transformation [40,122]. The absence of complete proviruses may be explained by an im- munoselection for non-virus producing neoplasms.

4. All the tumors examined contained at least one copy of the LTR [40,122].

5. All lymphomas contained species of RNA which did not hybridize to cDNA rep (they contained too few virus-specific sequences), but which

still reacted with cDNA 5'. These RNAs are expressed at high levels in the lymphomas and are thought to represent transcripts initiated in a viral LTR and continued into an adjacent cellular gene [40].

6. Of about 35 lymphomas examined, 30 were found to contain the LTR integrated at four specific locations upstream of the c-myc gene [124] (Astrin, S., personal communication). These tumors expressed 80-330 copies of c-myc RNA per cell (as compared to 2-5 copies in normal adult bursas) [124]. None of the other c-onc genes tested (src,

fps, erb and mvb) appeared to be expressed at increased levels. The integration of a provirus next to the c-myc gene in ALV-bursa tumors has recently been confirmed in other laboratories [122,123,127]. In addition, it appears that in REV- induced lymphomas (long latency) proviruses also integrate next to the c-myc gene [146].

These results have served as a basis for a general hypothesis [40,122] designated 'oncogenesis by promoter insertion', illustrated in Fig. 3. The basic assumption of this hypothesis is that viral LTR sequences promote the transcription of c-one genes. Since the integrated provirus is flanked by LTR sequences at both the 5' and the 3' end, transcription may initiate at the right-hand end LTR and continue into the adjacent cellular sequences. Al- ternatively, transcription may originate in the left- hand LTR, proceed over the viral structural genes and read into cellular sequences, passing termination signals which are assumed to exist in the right-hand LTR. The finding in ALV lymphomas of mRNA molecules containing R-U5 viral sequences plus cellular sequences (both lacking gag,

poL env) suggests that the right-hand LTR is frequently used as a promoter.

The activation of c-myc sequences by a viral LTR in ALV-induced lymphomas provides an at- tractive explanation for the long latency period of ALVs. At present, however, there still remain a number of inconsistencies in the observations obtained by different laboratories:

1. In some ALV-induced bursal lymphomas an LTR is located either downstream from the c-myc

gene or upstream, but in the opposite direction. Nevertheless, the c-mvc sequences were expressed at high levels in all tumors examined [122] (Payne, G. and Varmus, H., personal communication).

These findings are at first glance difficult to recon- cile with the promoter insertion model. It is interesting to note, however, that insertion of an LTR sequence at the 3' side of the transforming gene of Moloney sarcoma virus was sufficient to activate the transforming activity of this gene, as determined by transfection assays [125].

2. Cooper and Neiman [126], using a transfection assay, showed that DNA from ALV-induced lymphomas transformed NIH 3T3 cells at high efficiencies. However, these authors did not detect viral LTR sequences or c-myc sequences in the recipient cells [126,127]. This observation raises the possibility that the expression of c-mvc sequences is needed only for the initiation and not for the maintenance of transformation, and that the lymphoma cells contain transforming DNA other than mye. Such a possibility is supported by the way in which ALV-lymphomas develop: Al- though ALV-induced nodules can be seen in the bursa of infected animals within a few weeks, typical lymphomas appear only after a latency of several months. It is, therefore, possible that ALV provirus integration induces a c-myc expression (or maintains the expression of c-myc if it is already active) and that this leads to the development of nodules. Secondary events may then be necessary for tumor progression and for the outgrowth of lymphomas.

3. Hematopoietic cells transformed in vitro by myc-containing DLVs exhibit a macrophage- but never a lymphoblast-phenotype [21,22] (see Sub- section IIIA). Furthermore, MC29-type viruses cause carcinomas and myelocytomatosis but not lymphoid neoplasms [2]. A simple explanation would be that MC29 viruses cannot be expressed in bursa cells, although they are known to be expressed in other nontarget hematopoietic cells (see Subsection IIIA). Another explanation is that the animals die of other tumors before lymphomas reach a detectable size. It is also possible that there are qualitative differences between the c-myc and v-mvc gene products which determine the target cell specificity of the virus.

4. In about 15% of the lymphomas examined by Hayward et al. [124] the rnyc gene was not found to be induced. It remains to be seen whether or not some of the other known cellular oncogenes are activated in these tumors.

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In recent experiments it was found that erythroblasts induced by ALVs contain proviral DNA next to the c-erb gene (Kung, H.J., personal communication). It will be interesting to determine whether or not both c-erb genes are activated in these leukemic cells.

VIIIB. DLV-leukemogenes&: hematopoietic target cell specificity and possible role of c-onc genes

The essential findings made in connection with the hematopoietic target cell specificity and the mechanism of leukemogenesis by DLVs can be summarized as follows: 1. Each strain of DLVs transforms predominantly one type of hematopoietic target cell and different types of DLV strains show different target cell specificities. 2. Each type of DLV contains a specific v-onc gene. 3. For each v-onc gene there exists a homologous c-one gene. 4. DLVs and their transforming proteins are expressed not only in target cells but can also be expressed in nontarget cells, suggesting that the host restriction of viral transforming activity occurs at a posttranslational level.

Since it seems possible that the only common feature between the erb, myb, myc and rel genes is their transducibility by retroviruses and their con- sequent ability to transform hematopoietic cells, it seems appropriate to discuss the mode of action of each DLV prototype virus separately. This auto- matically also leads into a discussion of the pattern of expression of the cellular oncogenes in different cell types and of the possible function of these genes in normal hematopoiesis.

VII1B-1. A E V and the c-erb gene Infection of hematopoietic cells with AEV leads

to a block in the differentiation of immature erythroid cells and, thus, to the formation of leukemic erythroblasts (see Subsection IIIA). We have proposed earlier that the c-erb gene product(s) is an erythroid differentiation protein specifically expressed in AEV target cells [2,34]. In this model, the v-erb product(s) was assumed to be nonfunctional and to act by inhibiting the c-erb gene product(s) competitively. Alternatively, an over-

264

production of functional v-erb gene product(s) might stimulate the target cells to grow, thus indirectly inducing a block in differentiation. Both these possibilities lead to the prediction that the c-erb gene(s) is selectively expressed in AEV target cells. The data available so far agree with this prediction: expression of the c-erb gene(s) is low in populations of all normal tissues examined, but is high in a small minority of normal bone marrow cells [110]. The proportion of cells which express this gene (approx. 0.1%) corresponds roughly to the number of AEV bone marrow target cells [24] and to the number of normal erythroid burst-forming cells, the presumptive target cells of the virus [128]. It will be interesting to determine whether or not the cells expressing the c-erb gene(s) are indeed immature erythroid cells.

VIIIB-2. A M V and the c-myb gene Infection of hematopoietic target cells with

AMV leads to an outgrowth of myeloblast-like cells. These cells appear to be blocked in their differentiation because they often tend to differentiate spontaneously towards mature macrophages [21]. The target cells of AMV seem to be less mature than those of MC29, but it is not excluded that these two populations overlap [22,24]. The question of the target cells of AMV is somewhat controversial, and one study suggests that AMV induces a dedifferentiation of macrophages [130]. Mutants temperature sensitive for transformation would be helpful to shed more light on this prob- lem.

Although it is likely that AMV transforms by a different mechanism than MC29, it is striking that the pattern of c-myb gene expression in normal hematopoietic cells is quite similar to that of the c-myc gene, but completely different from that of the c-erb gene [110]. Thus, similar speculations can be made for this gene as for c-myc (see below). In contrast to the c-myc gene, none of the ALV-induced lymphomas examined so far showed an enhanced expression of the c-myb gene, indicating again that these two genes may have different roles in the cell.

VIIIB-3. MC29 and the c-myc gene Infection of hematopoietic cells with MC29

leads to an outgrowth of macrophage-like cells

[21,22]. It is not clear whether or not the virus blocks differentiation. A preliminary antigenic characterization of MC29 target cells suggests that they are less mature than the transformed cells [24], but these studies will have to be repeated using monoclonal antibodies. Expression of the c-myc gene seems to be less tightly controlled than the c-erb gene, as c-myc is expressed in immature hematopoietic cells of various lineages [110]. The expression of c-onc gene products in nontarget cells is not unique to MC29. The c-abl gene related to the Abelson leukemia virus-transforming gene is also expressed in nontarget tissues (for a review, see Ref. 129). The expression of the c-myc gene in hematopoietic nontarget cells [110] is incompatible with our earlier model [2,34] and with the above speculations about the mode of action of AEV. The pattern of c-myc gene expression suggests that its product is required for the growth a n d / o r development of a variety of hematopoietic cell types. If so, then target cell specificity of the virus could be determined by the expression pattern of the hypothetical substrate molecule, e.g., substrate molecules of the myc protein might be present in MC29 target cells, but absent in nontarget cells. Unfortunately, at present, there is no candidate substrate molecule for myc protein whose distribution could be studied.

VIIIB-4. R E V T and the c-rel gene Infection of hematopoietic cells with REV v leads

to an outgrowth of very immature lymphoid cells [27]. Whether in these cells the virus induces a block in differentiation or whether it immortalizes aberrant cell types which were blocked in differentiation prior to infection remains to be tested. The distribution of c-rel gene expression in different normal tissues has not yet been studied.

VII1C Transformation of nonhematopoietic cells by DL Vs

In discussing possible mechanisms by which leukemia viruses transform, it is necessary to stress that the term 'cell transformation' has a completely different meaning in fibroblast than in hematopoietic systems. The main criterion for in vitro transformation of hematopoietic cells is their growth under culture conditions where normal cells

would die or grow at only low rates. It is unclear whether or not they differ also in other properties, since a direct comparison of the properties of transformed cells with that of pure hematopoietic target cell populations is not possible. In contrast, in vitro transformation of chicken fibroblasts is defined by the expression of a number of 'transformation parameters', such as morphological and cytoskeletal changes, changes in growth parameters, production of plasminogen activator, increase in hexose transport, etc. [131]. This definition is possible since not only transformed but also normal fibroblasts grow in tissue culture and they can, thus, be directly compared. For epithelial cells, as for hematopoietic cells, because of the difficulties still encountered in culturing normal cells, the definition of the term transformation is at present still mainly based on the induction of cell proliferation.

Is a single protein sufficient for the transformation of different types of target cells by one DLV strain2 For MC29 the answer is probably yes. For AEV, it is not yet completely ruled out that it codes for two distinct transforming proteins (see Subsection VIA). Nothing is known for the other DLV strains in this regard. Assuming that a single transforming protein transforms fibroblasts in addition to hematopoietic cells, again two possibilities can be distinguished. (a) Dosage model. A decreased activity of the transforming protein might be sufficient to transform one target cell type but not the other. (b) Domain model. Differ- ent domains of the transforming protein (some of which may overlap) are required for the transformation of different target cells.

The dosage model predicts that transformation of different target cell types can only be dissociated in one way, whereas the domain model predicts that it can be dissociated both ways. All the t d mutants of MC29 isolated so far, as well as td359 AEV, are defective in their ability to transform hematopoietic cells, but are still fully competent (if not more efficient) in their fibroblast- transforming ability. This could be taken as an indication, as has been suggested for the Abelson leukemia virus, that less transforming activity is required for fibroblast- than for hematopoietic-cell transformation. This appears not to be true for AMV since this virus is apparently unable to

265

transform fibroblasts and yet it transforms myeloblasts. More td mutants will have to be isolated before one of the above alternatives can be excluded. It is also possible that they are not mutually exclusive and that different transforming proteins act by different mechanisms.

It is not clear whether the ability of MC29 to transform epithelial cells correlates with its ability to transform macrophages a n d / o r fibroblasts. In this regard it would be interesting to know whether or not the td mutants of MC29 are capable of transforming epithelial cells.

As discussed before (Subsection VIIIB), a possible explanation for the activity of DLV-transforming proteins in hematopoietic cells is their homology to putative hematopoietic differentiation proteins. Unfortunately, with this concept it is difficult to explain how DLVs transform fibroblasts or epithelial cells. It is not known whether DLV- transforming proteins react with the same substrate molecules in fibroblasts as in hematopoietic cells. It is also unknown whether or not DLV- transforming proteins have a protein kinase activity, such as has been described for ASVs [94,119] and Abelson leukemia virus [129]. It is intriguing that in fibroblasts transformed by AEV, but not MC29, a slight increase in the overall phosphoryla- tion of tyrosine of host peptides [132] and that of a 36 kDa protein [121] has been observed. In addition, candidate transforming proteins of MC29- type viruses are phosphorylated, but not at a tyrosine (Ref. 144, and Ramsay G., personal communication). It would be interesting to know whether DLV onc proteins are part of growth factor / receptor systems, as has been postulated for the transforming proteins of some mammalian and avian sarcoma viruses [134].

IX. Origin of DLVs

From the evidence discussed in this review, it is clear that avian DLVs originated by an event in which cellular onc sequences have been inserted into an ALV-type retrovirus genome. Have DLVs arisen from endogenous or exogenous retroviruses? Since the gag, e nv and U3 sequences differ between different ALV strains, this question can be studied by comparing the corresponding RNA regions of a particular DLV strain with that of its

266

natural helper virus, that of endogenous viruses, and with that of other ALVs. In addition, the g a g

gene products of all these viruses can be compared by tryptic peptide fingerprinting. Such studies have shown that, e.g., AEV [73,136], MC29 [135,136], AMV [49,52] and CMII [59] have probably arisen by recombination with their natural helper virus. It cannot be excluded, however, that DLVs were generated by recombination with endogenous viruses and that they acquired sequences of exogenous viruses in a subsequent recombination event.

Nondefective ALVs and ASVs recombine at relatively high frequencies with each other [5,145]. Recombination was also shown to occur between deletion mutants of ASVs which contained no detectable sequence homology [138]. It is not clear at what stage in the replication cycle of retroviruses recombination takes place. Formation of heterozygote virus particles (each retrovirus contains at least two full copies of viral RNA and these can stem from different parental viruses) seems to be a necessary prerequisite of recombination [140]. This suggests that recombination occurs during reverse transcription [141,142,68]. There is also the possibility that recombination occurs at the level of unintegrated proviral D N A (discussed in Ref. 145). Recombination between different DLV strains has not yet been shown. However, studies by Tsichlis and Coffin [135] indicate that DLVs are capable of recombining with ALVs. The observation that different substrains of MC29 show differences in their g a g portions [ 137] also suggests that DLVs are capable of recombining with their helper viruses. Finally, mutants of RSV with partial deletions in the v - s r c gene are capable of regaining c - s r c sequences by recombination with cellular DNA. This seems to be also possible for DLVs (see Subsection VIB).

From what is known about the genomic structure of DLVs, their RNAs meet the following minimal requirements: a v - o n c sequence is flanked on both sides by virus structural gene sequences. The 5' side contains the R and U5 region plus at least the p19 coding part of the g a g gene. The 3' side contains the R, the U3 and another untrans- lated region as well as, usually, at least the 3' portion of the e n v gene. The R, U5 and U3 regions are thought to be necessary for integration and replication. Sequences around the 5' end of the g a g

gene seem to be essential for packaging of viral RNA into virions [148,149]. Sequences close to the 3' end of the e n v gene might also fulfill a function necessary for replication.

As reviewed in Section II, DLVs originate in nature only at low frequencies. Since it has not yet been possible to generate DLVs under controlled experimental conditions, essentially nothing is known about the molecular mechanisms underly- ing this process. In the following we will discuss a series of theoretical possibilities by which a DLV originates as a consequence of a recombination of an ALV-type virus with a c - o n c gene, giving rise to a DLV-type virus. Each model is illustrated in the diagrams of Fig. 4.

~,~ Reverse Iranscrlpt lon of cncapSld ~teo viral and cOlu lar RNAs

~ t J

B iral promoter Ins~ d,~)n r* v~'r~,~ tr~n~Crlpt,on Of encapsldaled nNA

[ ] If i I

• I I I ¸

L J

C ecomblnat to n e l in legrated provirLJS wi th C o nc g@ne

D ntegrat ion of proviruses around a c one gene deletion of internal vlra[ sequences

. . . . ( , I I , i " ~ ~/ l t "

delet ion delet ion

! A~I mode ls

i Or

i i

Symbols

v,ral DNA [ ] LTR

cell DNA

v,rad RNA

I I I ten I~NA

Ilaaklng DNA sequences

,ntron DNA

~alhway ol recomblnat,on

Fig. 4. Four models by which DLVs might have arisen during evolution.

IXA . Reverse transcription o f encapsidated viral and cellular R N A s

In this model it is assumed that viral and cellular m R N A are coencapsidated into virus particles. Recombination occurs during subsequent ret- rotranscription of the RNAs into cDNAs. From studies with a mutant of Rous sarcoma virus it is known that cellular RNA can be packaged into virion particles [148]. This RNA can also be ret- rotranscribed into D N A by viral reverse tran- scriptase [149]. Assuming that the reverse tran- scriptase is capable of changing templates in the course of its reaction, a recombination could occur at the transition between RNA and DNA by a copy choice mechanism [141], perhaps involving breakage and repair [68]. Alternatively, recombination could take place during DNA-dependent synthesis. In both cases, two independent recombination events have to be postulated. It is known that c-onc RNA is associated at very low frequencies with ALV RNA [150], but it is unclear whether this RNA is truly encapsidated and retrotran- scribed. The model predicts that DLVs only arise in cells in which c-onc genes are expressed.

IXB. Viral promoter insertion; reverse transcription o f encapsidated R N A s

In this model, it is assumed that DLVs originate in three separate steps. In the first step, a retro- viral provirus integrates upstream of a c-onc gene. In a second step, to obtain transcripts of the type R-U5-X-onc which can be efficiently packaged, the right-hand portion of the provirus has to be de- leted, either before or after integration. In a third step, this hybrid RNA is co-encapsidated with complete ALV RNA molecules. Recombination linking the hybrid molecule with the 3' portion of the ALV RNA might then ensue during reverse transcription in an analogous fashion, as discussed in the first model.

A possible precedent for the type of reaction required for the last step (also required in model 1) has recently been described [151]. Mouse cells can be transformed by transfection with a molecularly cloned D N A of Harvey sarcoma virus. Some of the transformed cells were found to contain a virus D N A whose 3' viral sequences are missing. When

267

superinfected with a helper virus, such cells produced transforming viruses at low frequencies. These viruses stably reacquired the missing 3' viral sequence and resembled the parental wild-type Harvey sarcoma virus [ 151 ].

IXC. Recombination o f integrated provirus with a c-onc gene

In this model it is assumed that c-onc DNA recombines with an integrated provirus by ille- gitimate recombination. To generate a DLV, the provirus must lack some of its internal genes or lose them during or after the recombination process. Alternatively, viral sequences could also be spliced out from the complete viral-cellular transcript after recombination has occurred. A remote possibility exists that c-onc genes correspond to transposable elements. On structural grounds, this does not seem to be the case for the v-src and v-mos genes, the only onc genes whose complete sequences are available so far [160,161]. However, as long as the sequence of the cellular homologues is not known, this possibility cannot be ruled out. It is also possible that insertion-substitution of a c-onc gene and a provirus occurs by chromosome translocation. In this context, it is interesting that chromosome rearrangements are frequently observed in leukemias and other neoplasms [152,153].

IXD. Integration o f proviruses around a c-onc gene," deletion o f internal viral sequences

Sequencing of various types of avian and mammalian integrated proviruses has revealed that they have a structure resembling typical transposable elements [154-156]. On the basis of these findings, Temin [98] has recently postulated that retroviruses have arisen from ancestral transposable elements capable of forming an LTR-like structure upon integration. He has also postulated that rapidly transforming retroviruses might have arisen by a transposition of two proviruses around a c-onc gene, with subsequent deletion of the internal LTRs and virus structural gene sequences. Although a transposition of retroviruses has not yet been demonstrated, the model could still be used as follows: it is conceivable that two retroviruses integrate at random around a c-onc gene.

268

Subsequently, either by deletion of internal viral sequences or by an aberrant splicing of a viral-cellular-viral read-through RNA, a molecule might be generated which has the structure of a DLV genome. The complete process would require at least four recombinat ion/delet ion events and does not seem to be very likely.

X. Outlook

Since the publication of a recent review on avian leukemia viruses [2], a great deal has been learned about some of the basic questions related to virus-induced leukemogenesis. Some of the most important findings can be summarized as follows: acute leukemia viruses transform their target cells with a set of leukemia-specific oncogenes. The viral oncogenes have been acquired from the host cell during evolution. The corresponding genes from the host cell (c-onc genes) can be activated by the nearby integration of an ALV-type retrovirus with a strong promoter, thus resulting in leukemia.

With these fundamental findings, several new questions have been raised: In the case of AEV, how did two independent cellular inserts become incorporated into the virus? Do both play a role in transformation? What is the biochemical function of the different types of viral and cellular oncogene products? Do leukemia-specific cellular oncogenes correspond to hematopoietic differentiation genes? Does the mechanism of transformation differ for different oncogenes and for different target cells (e.g., hematopoietic cells and fibroblasts)? Are similar oncogenes, as defined in the avian virus system, also involved in non-virally induced leukemogenesis, such as in humans?

The answers to some of these questions promise to be as exciting as the fascinating insights which have been gained during the last few years on the mechanism of virus-induced leukemogenesis.

Acknowledgements

We thank Hartmut Beug, Michael Hayman, William Hayward, Hartmut Hoffman-Berling, Patricia Kahn, Kathryn Radke, Harriet Robinson, Simon Saule, Harold Varmus, Bj6rn VennstrtSm and John Wyke for comments and discussion.

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Avian leukemia viruses oncogenes and genome structure

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