Temperature sensitive mutants of avian sarcoma viruses

Biochimica et Biophysica Acta, 417 (1975) 91-121 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 87015

TEMPERATURE

SENSITIVE

MUTANTS

OF AVIAN

SARCOMA

VIRUSES

JOHN A. WYKE

Department o f Tumour Virology, hnperial Cancer Research Fund Laboratories, P.O. Box 123, Lhwoht's Inn Fields, London ( U.K.) (Received November 6th, 1974)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

91

II.

Isolation of Avian Sarcoma Virus ts Mutants

. . . . . . . . . . . . . . . . .

94

Ill.

The Use of ts Mutants in Studying Avian Sarcoma Virus Replication . . . . . . . . A. Definition of Early and Late Functions . . . . . . . . . . . . . . . . . . . . B. Mutants in Early Functions . . . . . . . . . . . . . . . . . . . . . . . . .

96 97 99

C. Multiple Mutants in Early and Late Functions . . . . . . . . . . . . . . . . . D. Mutants in Late Functions . . . . . . . . . . . . . . . . . . . . . . . . .

100 103

The Use of ts Mutants in Investigating Cell Transformation . . . . . . . . . A. Effects of Temperature Shift . . . . . . . . . . . . . . . . . . . . . . B. Transforming Functions Expressed in Restrictive Conditions . . . . . . . C. The Effects of Mixed Infection . . . . . . . . . . . . . . . . . . . . .

. . . .

105 106 108 109

D. Complexity of Transforming Functions . . . . . . . . . . . . . . . . . . . . E. Strain Differences in Transforming Functions . . . . . . . . . . . . . . . . .

110

IV.

V.

VI.

. . . .

. . . .

110

The Use of ts Mutants in Genetic Studies . . . . . . . . . . . . . . . . . . . . A. The Viral Genome: Haploid or Polyploid? . . . . . . . . . . . . . . . . . . B. Recombination Studies with Avian Sarcoma Virus ts Mutants . . . . . . . . . .

112 113

C. A Model for the Viral Genome

. . . . . . . . . . . . . . . . . . . . . . .

114

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

117

Prospects

Acknowledgements

I 11

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

118

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

119

I. INTRODUCTION C o n d i t i o n a l l e t h a l m u t a n t s h a v e p r o v e d v a l u a b l e in i n v e s t i g a t i n g t h e b i o l o g y o f b a c t e r i o p h a g e s a n d they are being increasingly used in studies o n animal viruses. T h e first r e p o r t e d c o n d i t i o n a l l e t h a l m u t a n t s o f R N A

tumor viruses were isolated

a b o u t 6 y e a r s a g o [1 ] a n d s i n c e t h a t t i m e i n t e r e s t i n R N A t u m o r v i r u s b i o l o g y h a s

NR

ts N Y 68

td B E 1

ts N Y 10

ts N Y 19 ts L A 22 to ts L A 29, ts I.A 31 to ts L A 35 ts L A 30m

FU-19

Ts 68

Ta

Ts 10

Ts 19

ts 335 ts 338

ts L A 335 ts L A 338m

NR

TI--T6

ts 2 2 - - t s 35

ts L A 33~m ts L A 336m

New mutant designation a

t:: 75 t.s 149

designation

Original mutant

PR

SR

BH

SR

SR

SR

t~77

Virus strain from which mutant was derived a

Free virus Transformed cells Transformed cells

N-methyl-N-nitroN-nitrosoguanidine 5-fluorouracil 5-fluorouracil

5-azacytidine

N-methyl-N-nitroN-nitrosoguanidine

Transformed cells

Free virus

5-bromodeoxyuridine Cells during first 12 h of infection

Transformed cells

Material exposed to mutagen

5-azacytidine

Mutagen used

5

2.5

3

Not appreciably affected

NR

0.1

3

Approximate virus survival after mutagenesis b, ! ' / .

2¢

NR

17

2

1

2

I

Approximate incidence ts mutants b, %

13,14,15

12

10,11

9

7,8

5,6

1

Reference

Listed in approximate chronological order of publication. " New mutant designation and strain abbreviations are according to the proposals of Vogt et al., [19]. Abbreviations are: B77, Bratislava strain 77 of avian sarcomav rus; BH, Bryan high titre strain of Rous sarcoma virus: PR, Prague strain of Rous sarcoma virus: SR, Schmidt-Ruppin strain of Rous sarcoma virus; m, denotes known or suspected multiple mutant; NR, new designation not recorded. b Figures in the two penultimate columns are calculated from data given in the references quoted in the last column. N R , data not recorded. c This figure is the incidence in mutagenised stocks, not the recovery after selection for mutants.

ISOLATION OF T E M P E R A T U R E SENSITIVE M U T A N T S O F A V I A N S A R C O M A V I R U S E S

TABLE I

I'O

ts L A

ts L A

337

672

ts

ts

672

337

343m

PR

PR

PR

ts O S

ts O S

ts O S

0260

0122

0538

ts

ts

ts

538

122

260

SR

SR

B 77

ts 20, ts 21 tsLA20, tsLA21 B77 ts 339, ts 340 ts L A 339, ts L A 340

ts L A

343

ts

Transformed cells

Free virus

5-bromodeoxyuridine Cells during first 24 h of infection 5-azacytidine Transformed cells

U!traviolet light

Transformed ceils

5-azacytidine 5-azacytidine

Transformed cells

Free virus

5-azacytidine

Ultraviolet light NR

NR

NR

NR

NR

NR

NR

NR

NR

NR

NR

I

NR

NR 14

18

18

18

16,17

16

15

94 grown enormously. It is therefore now appropriate to assess how studies on conditional lethal mutants of these viruses have contributed to this expanding knowledge, and to suggest how such studies might continue to provide valuable information. The conditional mutants of RNA tumor viruses, like those of other animal viruses, are all temperature sensitive (ts). In common with other mutants of this type they are useful for several reasons. Mutants can be maintained under permissive conditions and can theoretically be isolated with defects in any of the virus functions. It should therefore be feasible to map the whole virus genome. Moreover, mutations in different virus genes may be functionally identified by complementation tests and, by utilising shifts between the permissive and restrictive temperature, the mode of action of any mutated gene could be determined. These advantages are all being exploited in work with ts mutants of avian sarcoma viruses. This article will restrict itself to these well-studied viruses and will emphasise recent work in those areas where the use of conditional lethal mutants is particularly appropriate. Thus, after considering the isolation of ts mutants of avian sarcoma viruses, 1 will attempt to review critically their use in elucidating the physiology of virus replication and transformation, and their potential for mapping the virus genome and studying genetic interaction among viruses and between viruses and their host cells. Other studies on cornavirus physiology and genetics including work on ts mutants prior to April 1973, have been reviewed recently [2,3] and will not necessarily be discussed in detail here.

II. ISOLATION OF AVIAN SARCOMA VIRUS

ts

MUTANTS

There is very little evidence on the spontaneous incidence of ts mutants in avian sarcoma virus stocks. Temin [4] found ts mutants of various physiological types occurring in Schmidt-Ruppin strain Rous sarcoma virus at an overall incidence of 3 ~ . However, these mutants, though easy to detect, were apparently difficult to maintain and it seems likely that non-leaky spontaneous mutants with low reversion rates would be detected far less readily. All the mutants in current use were isolated following chemical or physical mutagenesis of virus or virus-infected cells, and Table I lists those that have been reported in the literature. The mutagen was generally directed against the viral RNA, but it is presumed that the marked mutagenic effect of 5-bromodeoxyuridine early in virus infection [10] is due to its substitution in the proviral D N A synthesized at this time. In addition to the mutants listed in Table I, Balduzzi [20] has isolated about 20 mutants of Bryan high titre strain of Rous sarcoma virus following mutagenesis with 5-bromodeoxyuridine and Bookout and Sigel [21 ] have isolated mutants after 6°Co-'( irradiation of Schmidt-Ruppin Rous sarcoma virus. Virus survival after mutagenesis was reduced in all cases, sometimes drastically, and ts mutants were detected among the survivors either by random cloning and testing or by negative selection procedures which favoured survival of mutants

95 with specific properties [13]. There are not yet enough data to compare the efficiency of mutant induction with degree of virus killing by different agents. All mutants reported to date are heat sensitive with a permissive temperature of 35-37 °C and a non-permissive temperature of 40~,1 °C. Mutagenesis leading to a high number of lethal mutations has the disadvantage that it would also tend to produce a higher proportion of multiple conditional lethal mutations among the survivors. Multiple mutations in separate functions would yield mutants with the complex physiological behaviour shown by a number of avian sarcoma virus t s mutants. Some of these mutants have already been shown to have defects in more than one function (see Section III) and it is likely that other mutants of this type also possess two or more lesions, complicating their physiological and genetic analysis. Multiple mutations in the same function would yield a mutant with less leakiness and a lower reversion rate than a virus with only the corresponding single mutation in that function. Such multiple mutants would not only be enriched by any selection procedure, but it is also likely they would be inadvertently favoured by workers isolating their mutants "non-selectively" by cloning and testing. The stability of such mutants may, in fact, make them desirable for certain physiological experiments, but they may be unsuitable for use in genetic work. Thus, although there is no firm evidence to date for any mutants carrying multiple lesions in a single function (see Sections III and IV) one should always beware of this possibility. Spontaneous t s mutants should be far less likely to carry multiple lesions, but most workers have so far preferred to risk the hazards of mutagenesis on the reasonable assumption that it increases mutant yield. Mutant isolation is further complicated by the possible presence of other oncornaviruses in the cells used to grow virus stocks. These contaminating viruses, which may possess many functions in common with those of the agent under study, could mask ts lesions by functional complementation or genetic recombination. The problem is obvious in the case of Bryan high titre strain of Rous sarcoma virus which will not replicate in the absence of a helper virus [22]. However, a more insidious and intractable dilemma is that of endogenous viruses. Chicken cells, the host of choice for most commonly studied avian sarcoma viruses, carry the DNA of an oncornavirus which has considerable sequence homology to the DNAs of avian sarcoma viruses [23-25]. The extent of transcription and translation of the genes of this agent differs in different chicken strains but under appropriate circumstances all the endogenous viral functions can be expressed. Simple tests can detect the group specific antigens and envelope glycoproteins of the endogeneous virus in otherwise uninfected cells [26,27], and in cells lacking these markers of virus expression it seems that the endogenous viral RNA is not transcribed [28,29]. Most mutants have been isolated and studied in cells which do not express endogenous viral functions by these criteria, thus minimising contamination by the endogenous agent, but one cannot assume that the lack of detectable viral proteins invariably implies complete quiescence of the endogenous genome. Recombination with endogenous viral genes or complementation by functions other than the group specific and envelope

96

expresslon

of

1~__~_~L~

'2222,r2~&,

4

"J

Fig. 1. The life cycle of an RNA tumor virus (from Tooze, 1973).

proteins is a possibility that can only be finally ruled out by growing viruses in cells free of genetically related endogenous genomes. Unfortunately cells of this sort, such as those of Japanese quail and duck, are unsuitable for many purposes as they do not support efficient replication of a number of avian sarcoma viruses [30].

Ili. THE USE OF

ts

MUTANTS 1N STUDYING AVIAN SARCOMA VIRUS REPLICATION

The basic steps in oncornavirus replication have been elucidated mainly in studies with non-defective avian sarcoma viruses. By early 1973 most workers would have agreed that Fig. 1, taken from Tooze, [2], summarised in diagrammatic form the salient features of the life cycle of these viruses. On the basis of data which are discussed on pages 585 to 615 of [2], Fig. 1 divides virus replication into several stages, the italicized numbers in the text below referring to numbers in the figure. (1) By virtue of specific glycoproteins in its envelope, the virus binds to membrane receptors and penetrates the cell. (2) Uncoating of the viral RNA genome and translocation to the nucleus follow rapidly, though not necessarily in that order. (3) The RNA-dependent D N A polymerase ("reverse transcriptase"), which is present in the virion, transcribes a complementary ( - - ) D N A copy from the virion 60-70 S ( + ) R N A template, utilizing endogenous primers. This D N A synthesis appears to start before the virus reaches the nucleus [39,105]. (4) Enzyme activities present in the same molecule as the reverse transcriptase degrade the RNA moiety of the hybrid formed and synthesize a + D N A strand in the same sense as the virion RNA, using

97 the residual - - D N A as template. (5) The double stranded DNA provirus integrates into the cell chromosome. Although it is not certain that this integration is essential for subsequent virus replication and cell transformation, recent evidence suggests that this is so [39,96]. It is not known whether cell division is a necessary prerequisite for integration, but transcription (6) of RNA from the provirus template does not occur until the infected cell divides. (7) Newly transcribed + R N A is probably messenger (8) for virt~s-specified proteins in the cell cytoplasm. Some of these proteins control expression of cell transformation, and some are virion components which become associated with + R N A (9) and mature and bud at the cell surface as new virions. Mutants are now known with ts defects at various stages of this postulated life cycle. These mutants are proving useful, firstly in confirming and extending conclusions derived from other investigations and secondly in defining steps in virus replication which were not revealed in other studies.

IliA. De[in#ion of early and late functions The characterisation of these mutants is being achieved by experiments of gradually increasing sophistication. Non-defective avian sarcoma viruses transform the cells in which they replicate, enabling simple tests to define three physiological classes of mutant; transformation defective, replication defective, and those with a coordinate defect in both transformation and replication. The same two simple parameters of cell transformation and production of infectious virus can be further studied upon shifting cultures from permissive to non-permissive temperature or vice versa at various times after infection. These temperature shift experiments subdivide the three basic mutant classes into groups with lesions in "early" and "late" virus functions. Early mutants, once they have established infection under permissive conditions, behave as wild type at non-permissive temperature. Conversely, in experiments begun at restrictive temperature, a subsequent shift to permissive environment fails to elicit any sign of the appropriate virus-specified functions. Thus these mutant functions act only transiently at the beginning of infection, but their action is essential to initiate subsequent virus replication and/or cell transformation. In contrast, shifting cells infected with late mutants to or from permissive temperature at any time after infection results in the expected appearance or disappearance of the virus-specified character under study. Therefore these mutants have defects in functions required continuously throughout infection if transformation and/or replication are to be maintained. The definition of early and late categories on the basis of temperature shift experiments is clearly unsatisfactory, since these tests only reveal whether a function is required transiently or continuously without any real indication of the stage in the virus life cycle at which it acts. One might imagine, for instance, a virus-specific function which is only required for a short time but that time is late in infection. It is therefore preferable to divide early from late functions by a stage in the virus life

98 cycle which is clearly defined in concept and readily detected experimentally. The point after proviral DNA synthesis but before its transcription into progeny RNA is a suitable watershed. The events before this stage, which possibly culminate in proviral integration, are likely to be transitory and may even occur uniquely in the fate of a single input genome. The happenings beyond this point, however, are by their nature more likely to occur continuously and as multiple events in the experience of a given provirus. Experimentally this stage can readily be defined as the point where the progress of avian sarcoma virus infection is arrested in cells which do not undergo DNA synthesis and mitosis. In such stationary cells the DNA provirus is formed and becomes stably associated with the cell [31], but transcription of new virus specific RNA does not occur [32]. Thus infection is arrested prior to transcription from provirus though it is not yet certain whether or not the stable provirus is integrated into the cell DNA. Despite this unresolved doubt, the failure of avian sarcoma virus to proceed with its infection in stationary cells seems a convenient, and probably not arbitrary, experimental way to distinguish early from late functions. So far, all the mutants TABLE 11 PHYSIOLOGICAL CHARACTERISTICS OF AVIAN SARCOMA VIRUS ts MUTANTS " T, defective in transformation; R, defective in replication; - - , no defect. Numbers in parentheses refer to probable separate lesions in multiple mutants. Mutant (ts)

Early defecta

LA 335 LA 337

T+-R

L A 336m

T + R(I)

T(2)

(l) Reverse transcriptase (2) Maintenance of transformation

LA 30m M I 100m

T(I)

T(2)

(1) Initiation of transformation (2) Maintenance of transformation

LA 338m L A 343m

T(I)

T-- R(2)

(1) Initiation of transformation (2) Transcription of RNA from provirus (may be a multiple defect)

LA 334m

T(I) R(2)

(I) Maintenance of transformation (2) Maintenance of replication

LA 339 LA 340

T~R

Maintenance of transformation and replication. Not known if single or multiple

LA 672

R

Reverse transcriptase

T

Maintenance of transformation

T1 to T6; FU-19; N Y 10, N Y 19, NY68; B E 1; LA 20 to LA 29, L A 31 t o L A 35; O S [ 22, O S 260, O S 538

Late defect~

Structure or function affected by ts lesion Reverse transcriptase

99 that were classified as early or late in simple temperature shift experiments have fallen into the same categories when tested for their ability to proceed beyond their ts step upon infection of stationary cultures [33]. These mutants will now be considered in detail in the probable order in which their defects are manifested during infection. Table II summarises their properties, and should aid understanding of the description which follows. IIIB. M u t a n t s in early functions Two mutants L A 335 and L A 337 [13,15] are defective in functions which only

operate in the first few hours of infection but which affect both virus replication and cell transformation. Thus, cells infected at non-permissive temperature (41 °C) show no virus functions on shifting to permissive temFerature a few hours later, whilst a shift from permissive to non-permissive conditions results in an apparently wild phenotype once infection has been established. The infectivity of both mutants is inactivated more rapidly than that of wild type virus by preincubating virions at 41 °C suggesting that the ts defect might be mediated by a virion molecule [15]. The a mutant of BH-Rous sarcoma virus, which neither replicates in nor transforms cells, is known to lack the virion reverse transcriptase [34] and it was no surprise to find the activity of this enzyme in L A 335 and L A 337 was both temperaturesensitive on testing at 41 °C and heat labile upon incubation at this temperature prior to testing [15]. Further studies by Verma et al. [35] on reverse transcriptase purified from the two mutant viruses showed that both the RNA- and DNA-dependent D N A synthetic activities, as well as the R N A ' D N A hybrid nuclease (Ribonuclease H; [36]) were heat labile on preincubation at high temperature when compared with enzyme from wild type virus. Presence of template-primer did not stabilise this lability. The observed temperature sensitivity is associated with heat lability of the smaller a subunit of the reverse transcriptase molecule [35], agreeing with the findings of Grandgenett et al. [37], that all three enzyme activities reside in this subunit. Moreover, Mason et al. [38] isolated wild type revertants from both LA 335 and L A 337 and also performed crosses between the mutants and avian leukosis viruses which yielded recombinants (Section VB) with a stable wild phenotype. The reversion or recombination to wild type was associated in all cases tested with the acquisition of a reverse transcriptase which was no longer temperature sensitive [35]. Mason et al. [38] also failed to find in vivo complementation between LA 335 and LA 337 suggesting that they are probably defective in the same gene product. However, the possibility that L A 337 is also altered in envelope properties [15], a defect that may be consequent upon, or independent of, the lesion in reverse transcriptase, has not yet been pursued. These studies, perhaps the most extensive so far on any avian sarcoma virus mutant, provide the best evidence to date that the action of reverse transcriptase is essential for subsequent virus replication and cell transformation, and that at least part of the enzyme is virus coded. In cells infected by LA 335 and LA 337 at 41 ~C

100 less virus-specific D N A is detected than under permissive conditions [39], the sum of these results suggesting that provirus formation is an obligatory part of the virus life cycle. However, because the mutants are ts for all three activities of the reverse transcriptase enzyme it remains possible that up to two of these functions may not be essential for infection to proceed. IIIC. Multiple mutants in early and late functions

Another mutant, LA 336 [I ], also shows a defect in reverse transcriptase under appropriate circumstances. Like LA 335 and LA 337, initiation of infection at restrictive temperature (41 ~C) leads to a failure of replication and transformation which is irreversible on a subsequent shift to permissive conditions (35 °C). However, unlike the previous two mutants, LA 336 does not behave as wild type once infection has been established at 35~C. Although replication of this virus is unaffected by a late shift from 35°C to 41 c'C, its transformation remains dependent on permissive conditions for its maintenance [14,40]. This defect in maintaining the transformed phenotype resembles that shown by the many transformation defective mutants discussed below and probably results from a second ts lesion in the LA 336 genome [41 ]. It is the early ts defect of LA 336 that has attracted most attention, for when considered in isolation from the late lesion in maintenance of transformation, it is phenotypically indistinguishable from the ts defects of LA 335 and LA 337. The virion of LA 336 is no more heat labile than that of wild type virus [14,40] and early experiments suggested that endogenous reverse transcriptase activity was not temperature sensitive [42,43]. However, the ability of leukosis viruses to complement the ts defect in LA 336 by phenotypic mixing [41 ] suggested that this virus has a mutated virion component. M611ing [44] now finds that the reverse transcriptase of LA 336 is heat labile when preincubated at high temperature prior to testing its activity on exogenous templates. The RNA-dependent DNA polymerase and the ribonuclease H of LA 336 show approximately equal heat lability, a distinction from LA 335, LA 337 and wild type virus in which Verma et al. [35] found the ribonuclease H decays at a higher temperature than the polymerase activity. Moreover, the reverse transcriptase of LA 336 is apparently stabilised by preincubation with template, a treatment which does not affect the heat lability of LA 335 or LA 337. Thus LA 336 may have a reverse transcriptase defective in its affinity for template, and template binding may involve a component separate from that determining the enzyme activities, for Vriis [41] claims that LA 336 can complement both LA 335 and LA 337 to a low, but significant degree. Recent results of Panet et al. [106] are pertinent here, for they suggest that a function of the larger fl subunit of avian oncornavirus reverse transcriptase is to increase the binding affinity of the enzyme for template'primer. In the absence of such binding, because either template'primer or fi subunit are lacking, the enzyme activities of the smaller subunit are more susceptible to heat inactivation. Moreover, in the isolated ~ subunit, in contrast to the aft holoenzyme, the ribonuclease H activity seems no more resistant to heat than the other two enzyme activities. The similarities between these observations and the claimed behaviour

101 of LA 336 suggest that the mutant lesion might lie in the association of the two enzyme subunits, perhaps in the fl subunit itself. The possibility that the smaller subunit is derived by cleavage of the fl subunit [44] increases the likelihood that at least part of both moieties of the reverse transcriptase molecule is virus coded. It is still not clear why earlier attempts to reveal a ts reverse transcriptase activity in LA 336 failed when the same virus was clearly ts in its ability to initiate infection, but of course the ability to synthesize D N A in vitro does not necessarily imply a concomitant ability faithfully to transcribe the whole viral genome. Two other mutants which have a clear early defect are LA 30 [14] and M1 100 [21]. Unlike the three early mutants discussed above, LA 30 and M1 100 replicate normally under non-permissive conditions to yield progeny which are apparently identical to the input virus. However, if infection is initiated at 41 °C there is no transformation and the infected cells rapidly lose the capacity to transform on a shift to permissive conditions. Thus these two mutants are defective in an early function required only to initiate transformation. They are also ts in maintaining the transformed state: as long as the first few hours of infection are at 35°C, the ability to transform the cell is stabilised but its expression remains temperature sensitive on shift to or from 41 °C. If the virions of LA 30 or M I 100 are preincubated at 41 °C they lose their transforming ability far more rapidly than does wild type virus. Although it is not certain whether their replicative capacity is similarly heat labile, this suggests that the function needed for initiation of transformation is mediated by a heat labile virion component whose activity is either irrelevant to, or less crucial for, virus replication. This hypothesis is further supported by recombination studies (see Section V) between these mutants and non-transforming avian leukosis viruses. For instance, all of six recombinants of LA 30 isolated with the host range of the avian leukosis virus parent no longer showed virion heat lability and they were no longer ts in initiating transformation [45]. However, the recombinants remained ts in maintaining transformation and were, in fact, indistinguishable from the many mutants described below which also have this defect. Recombinants of M I 100 behaved similarly [21] and thus these viruses are probably double mutants whose late lesion, like that of LA 336, affects only transformation maintenance. The nature of the early lesion in LA 30 and M1 100 has a number of intriguing implications. The heat labile virion component needed to initiate transformation is presumably encoded in the genomes of the avian leukosis viruses used in the recombination studies [21,45] even though these viruses cannot themselves transform chick fibroblasts. However, since LA 30 and M I 100 can produce infectious transforming progeny at 41 °C the lesion probably does not represent a heat-dependent loss or major conformational change of some part of the mutant genome. For the same reason, a reverse transcriptase defect analogous to those shown by LA 335, LA 337 and L A 336 is also unlikely, though this enzyme has not yet been investigated in LA 30 or M I 100. It therefore seems probable that these mutants can produce a normal provirus, though, as mentioned above, it is not yet certain that all three known functions of reverse transcriptase are needed for infection to proceed and it is

102 still conceivable that a complete double stranded DNA provirus is not an essential replicative intermediate. Infection by L A 30 can progress beyond its ts stage in stationary cell cultures [33], so the defective function is operative before transcription of virus-specific RNA from proviral D N A [32]. This reasoning pinpoints the time of action of the LN 30 ts function at about the same stage in infection as the presumed integration of provirus into cellular DNA, and studies on LA 30 and M I 100 may elucidate this poorly-understood period of the virus life cycle. The idea that these mutants may show defective proviral integration is of heuristic value for several reasons. Oncornavirus virions are known to contain a number of enzymes (reviewed in [46]) some of which, particularly DNA ligases and endonucleases, might be involved in provirus integration. It has usually been suspected that these enzymes were of host cell origin, but it would now be interesting to test their activity in LA 30 and M I 100. Furthermore, integration following exogenous viral infection can now be detected in cells such as quail or duck which do not contain endogenous genomes with homology to Rous sarcoma virus [24,47]. If LA 30 and M I 100 are found not to integrate under non-permissive conditions this has clear implications for the mechanisms of cell transformation and virus replication. If they can integrate at 41 ~C, then the question remains as to the specificity of their integration site at different temperatures. Methods of fractionating cellular D N A would provide an approach to this problem (see, for example [48]), the answer to which would also have a major bearing on the mechanism of transformation. The behaviour of these mutants raises another question which is amenable to experimental test. The loss of ability to initiate transformation which occurs at 41 °C is not reversed on shifting to 35°C even though the virus is replicating [14,21 ]. Thus the progeny virus cannot reinitiate transforming infections under the cultural conditions used in these experiments, and the reason for this is unknown. New infections may be prevented because the cells become stationary and refractory to virus infection [49] and progeny virus may fail to re-infect the cell in which it is produced for the same reason, or because the progeny is non-infectious until it has matured in the virus particle [50] or because input virus induces, by an unknown mechanism, some form of resistance to reinitiation of infection or transformation. In summary, the physiology of LA 30 and M I 100, like that of the two mutants to be discussed next, hints at aspects of avian sarcoma virus infection which have not yet been revealed by studies on wild type virus. Two other mutants, LA 338 and LA 343, resemble LA 30 and M I 100 in that they are ts in a function required early in infection solely for initiation of transformation [14,51]. However, this ts function, unlike that o f LA 30 and M I 100, is not associated with virion heat lability, and though it appears to operate before the block to infection which occurs in stationary cells, its leakiness is such that the data leading to this suggestion are equivocal [33]. What is clear is that LA 338 and LA 343 also show a late reversible defect(s) in maintaining both cell transformation and virus replication [14], and preliminary recombination studies show that these mutants have two or more ts lesions [51]. LA 338 and LA 343 are apparently very similar, though

103 the latter appears to be leakier, and most work has been done on the former. Cells infected by LA 338 at 41 °C are not transformed and yield no virus. On a shift to 35°C the cultures yield virus, even though the ability to transform has been lost. Friis [41] considers that this stabilisation of the subsequent ability to replicate indicates that provirus has been formed at 41 °C but there appears to be no direct evidence for this as yet. Indeed, since L A 338 seems directly comparable to LA 30 as far as early functions are concerned, all the cautions mentioned above with L A 30 apply in considering whether or not LA 338 provirus is formed and integrated. The long lag of 12 h between shift to permissive temperature and appearance of progeny virus [41] suggests that the late LA 338 ts defect(s) is one of the first to operate at this phase of infection. This is in keeping with the observations of Ha[pern et al. [52] that LA 338 infected cells at 41 °C contain reduced levels of virion p27 (gsl) antigen (for virion protein nomenclature, see [53]). Nucleic acid reassociation experiments [54] have shown that infected cells at 35°C contain 10-20 times as much virus specific RNA as cells at 41 °C so it is possible that L A 338 is defective in transcribing viral RNA from proviral DNA. This lack of viral RNA could explain why the L A 338 late lesion(s) affects both transformation and virus synthesis. However, if Hunter is correct in his suggestion [51 ] that the late lesion is in fact two separate defects, one in transformation alone, the other in replication alone, then this neat explanation is less acceptable. Nonetheless, the L A 338 defect demonstrates that viral transcription is controlled not only by cellular [32], but also by viral factors, and it is interesting that L A 338 can be complemented at 41 °C by co-infection with avian leukosis virus or by L A 672, another late mutant described below [41 ]. IIID. Mutants in late functions Individual ts lesions in all late mutants that have been studied in any detail show defects in either viral replication or cell transformation. The L A 334 mutant, like LA 338 and L A 343, at first appeared to be coordinately defective [1 ] but Owada and Toyoshima [55] isolated from LA 334 a nonconditionally transformation-

defective variant, probably a deletion mutant, which was still ts for replication. Moreover, recombination between L A 334 and the avian leukosis virus, Rous associated virus-l, yielded two recombinants which replicated as wild type but were still ts for transformation. Other workers [56] showed that the ts defect in transformation could revert independently of that in replication. These data argue strongly that LA 334 bears at least two distinct lesions, one affecting transformation, the other replication. Before it was appreciated that L A 334 was a multiple mutant, Friis et al. [40] had shown that the replication defect was very late in infection and a shift to per-. missive temperature resulted in the appearance of infectious virus within an hour This rapid virus maturation was claimed to be dependent on protein synthesis [57] but viral RNA [40,55] and viral group specific antigens, including p27 [40,52] were already present in the infected cells at non-permissive temperature (41°C). Virus bearing markers of L A 334 could be rescued at 41 °C by superinfection with avian leukosis virus [40] or avian sarcoma virus [41,55] and this complementation occurred

104 even if the rescuing virus was of the same envelope subgroup as L A 334. Thus at 41 °C LA 334 does not exert virus envelope-mediated interference with superinfection. This is particularly interesting since Ogura [58] has recently shown that the LA 334 infected cells at 41 °C possess about 10 times as many budding virions at the cell surface as equivalent cells at permissive temperature. About a third of these 41 °C buds were of abnormal morphology suggesting that they incorporated atypical or multiple cores, and it was further found that some virus was released at 41 °C, the bulk of it being nonqnfectious. These data all suggest that the replication defect in LA 334 is in a late stage of maturation, possibly involving virion protein or envelope glycoproteins. Further information on this point is equivocal. The Bryan high titre strain of Rous sarcoma virus, which lacks the gp85 envelope component [59] is claimed by Friis [41] to be complemented by LA 334 at 41 °C and Halpern [60] finds that LA 334-infected cells contain gp85 at restrictive temperature. On the other hand Owada and Toyoshima [55] show that rescue of Bryan high titre strain of Rous sarcoma virus by LA 334 at 41 °C is slight and of dubious significance, even though endogenous virus-complemented BH-Rous sarcoma virus does seem to be stimulated under these conditions. Their observations are in keeping with the suggestion of Katz and Vogt [57] that gp85 synthesis is reduced in LA 334 infected cells at 41 °C. However, Katz and Vogt also found that p27 synthesis was somewhat reduced, though detectable, at 41 °C and Friis and colleagues, studying the non-infectious virions released at 41 °C, have suggested that at least one of the mutant defects involves maturation of virion internal proteins [41 ]. it should be remembered that these studies utilised the original doubly-mutated LA 334, so it is not known whether the biochemical defects observed relate to lesions in transformation or replication. A resolution of these discrepancies will be welcome, particularly since it might help to define the roles of the different viral proteins and envelope glycoproteins in infection, interference and transformation. It has been pointed out by Friis [41 ] that the late nature of the LA 334 defect, together with the presence of abortive buds at 41 '~C, resembles the situation reported by Wong and McCarter [61 ] for a spontaneous mutant of Moloney routine leukemia virus. However, the murine leukemia virus mutant apparently does not depend on protein synthesis to produce virus on a shift to permissive temperature, which suggests either that the two mutants do differ in the nature of their lesion or that the interpretation of some of the inhibitor experiments is at fault, A number of other ts mutants with late replication defects have been isolated [41,42], but only one, LA 672, has been studied in any detail, This virus induces normal cell transformation at 41 "C, and both virion group specific and type specific (envelope) antigens are detected in infected cells at this temperature [16]. Indeed, large yields of non-infectious virus particles are produced at 41 °C [16], and these appear to contain all the major virion proteins, and RNA that is indistinguishable from that of wild type virus produced at the same temperature [62]. However, these non-infectious particles contain no reverse transcriptase activity [62]. Virions of LA 672 produced at permissive temperature are not heat labile,

105 and their reverse transcriptase activity is normal. It therefore seems that the defect at 41 °C is introduced during virion assembly and maturation, and could arise in two ways; either through a lesion in the enzyme molecule itself, or through a faulty maturation function. L A 672 can be complemented at 41 °C by avian leukosis virus [16] and by the late mutants L A 338 and L A 334 [41 ]. In contrast, it cannot be complemented by L A 335 and L A 337, and since these two mutants have defects in the reverse transcriptase molecule it seems likely that L A 672 may be similar [62]. Moreover, M611ing [44] finds that the non-infectious LA 672 produced at 41 °C contains a molecule with the antigenic properties of reverse transcriptase even though it is enzymatically inactive. It thus seems likely that the L A 672 defect is in the reverse transcriptase molecule itself, which, on the basis of kinetic temperature shift experiments, may be maturing at a stage of infection a few hours earlier than the presumed envelope defect of L A 334 [16]. This late mutant, with a possible defect in the same gene as that which malfunctions in the two early mutants we considered at the beginning of this section, thus brings full circle our survey of mutants which define various stages of the avian sarcoma virus life cycle. However, the majority of avian sarcoma virus ts mutants which have been isolated do not appear to have any effect on the production of infectious virus, being defective only in transforming the infected cell. They are the subject of the next section.

IV. THE USE OF ts MUTANTS IN INVESTIGATING CELL TRANSFORMATION Martin [5] and Biquard and Vigier [7] were the first to report the isolation of mutants with a ts defect in cell transformation, convincingly demonstrating that transformation is under the continuous control of viral genes. Moreover, since these viruses replicate at restrictive temperature the transforming genes seem irrelevant to replication, a finding in keeping with previous demonstrations that transforming viruses can give rise to transformation-defective derivatives (reviewed in [3]). It seems likely, therefore, that this group of mutants represents defects in only a small portion of the virus genome, even though they are very commonly isolated (Table 1I). Perhaps their ease of isolation is explained by the probable lack of transforming functions in endogenous viruses which might otherwise have complemented their lesions, together with the non-essential nature of their defect: if avian sarcoma viruses are polyploid, as is postulated in the next section, then a recessive conditional mutation would be isolated more readily if it were possible for the other genomes in the virion to be non-conditionally defective in the mutant function. It was readily appreciated that the nature of these mutants makes them ideal tools for controlled studies of cell transformation, since temperature shift experiments result in a rapid acquisition or reversion of the transformed phenotype in mass populations of cells. For this reason avian sarcoma virus ts mutants have been widely used in the last five years to obtain a variety of physiological data on cell trans-

106 formation, much of it probably only distantly related to the primary virus-controlled events. Most of this work cannot be mentioned here, but some of it has been reviewed before [3]. For the present | will emphasise two experimental approaches that one hopes will shed light on the means by which the viral genes elicit cell transformation. The first approach follows the time scale and metabolic requirements for the appearance or disappearance of various transformed cell parameters in temperature shift experiments with mutant infected cultures. This sort of experiment should reveal differences between mutants and hopefully should distinguish events of primary importance in virus-induced transformation from those which are secondary occurrences. The second type of study investigates whether any parameters of transformation are induced at restrictive temperature by mutants under conditions of both solitary and mixed infection. This again should identify differences between mutants and might also show which markers of transformation are invariably associated with one another.

IVA. The effects of temperature shift Temperature shift experiments, with and without metabolic inhibitors, have been performed on a variety of mutants, in each case monitoring only a few of a range of parameters. The results so far appear rather haphazard and it is difficult as yet to fit them into a fully coherent scheme. When shifted from restrictive to permissive temperature, cells infected by mutants of Schmidt-Ruppin and Prague strains of Rous sarcoma virus show the first signs of morphological transformation by two h after shift, though some alterations, presumably secondary, occur slowly and may not be complete for several days [63,64]. If transformation is quantitated by following the increase in sugar uptake, then a 50~o level is achieved by about 5-8 h after shift [9,64,65]. This parameter probably represents increased transport of sugar [66,67] and it roughly accords with morphological change. These mutants are thus indistinguishable in their kinetics of establishing transformation, but the BH-Rous sarcoma virus mutant studied by Bader [11] appears to differ. The appearance of rounded cells and the increase in rate of sugar uptake on a shift down occurs only slightly faster with the BH-Rous sarcoma virus mutant than with the mutants mentioned above. However, a change peculiar to Bryan high titre strain of Rous sarcoma virus transformation, the appearance of cytoplasmic vacuoles, occurs extensively by 20 min after shift, and this change is accompanied by an equally rapid fall in the level of cellular cyclic adenosine 3', 5'-monophosphate (cyclic AMP) [68] and adenylate cyclase activity [69]. When the effect of metabolic inhibition on temperature shift down is studied the results are in accord with the kinetic data. Being late mutants, it is perhaps not surprising that DNA synthesis, where it has been investigated is not necessary for the appearance of transformation [8,9,11,65]. Indeed, cell cultures infected by these mutants at restrictive temperature can be arrested in the G 1 phase of the cell cycle by medium depletion [32,70] and in such cultures simple shift to permissive temperature is enough to initiate cell DNA synthesis [70]. It thus seems that in an established

107 mutant infection, in contrast to a newly initiated infection, expression of late viral functions are not only independent of cell D N A synthesis per se but can in fact stimulate this event. This is not to say, however, that virus-induced transformation is independent of other events which are specific to various stages of the cell cycle, and studies with ts mutants may permit an approach to this question. Inhibition of RNA and protein synthesis apparently blocks the transformationspecific appearance of plasminogen activator in cells infected by Martin's T5 mutant [71]. The same inhibitors also apparently prevent increased hyaluronate synthesis by the BH-Rous sarcoma virus mutant of Bader [11 ]and increased sugar uptake both by this mutant [I l] and some mutants of Prague strain-Rous sarcoma virus [41]. On the other hand, RNA inhibition has no apparent effect on morphological transformation and increased sugar uptake by other Prague strain of Rous sarcoma virus mutants [41] nor by the Schmidt-Ruppin strain of Rous sarcoma virus mutants of Kawai and Hanafusa [9] and Biquard and Vigier [8]. However, morphological transformation and increased sugar transport by these latter mutants are suppressed by inhibition of protein synthesis. Unlike sugar uptake and hyaluronate synthesis, the rapidly shifting BH-Rous sarcoma virus-specific transformation parameters of cytoplasmic vacuolation, and also increased cellular water content [72], are indeFendent of protein synthesis inhibitors when cells infected by the BH-Rous sarcoma virus mutant are shifted down [11,72]. It also seems that in cells infected by the SRRous sarcoma virus mutant of Biquard and Vigier a shift down results in increased cell agglutinability by concanavalin A in the presence of inhibitors of protein synthesis that prevent expression of other transformation criteria [73]. Thus the temperature sensitive block in the expression of these latter parameters seems to be at a posttranslational level However, transformation by the BH-Rous sarcoma virus mutant can be blocked byinhibitors that act at this level, for infected ceils will not transform on temperature downshift in the presence of the cyclic AMP analogue N 6, 2'-0dibutyryl adenosine 3', 5'-monophsphate [68]. It would be interesting to know whether transformation by this mutant is similarly affected by a protease inhibitor which seems specifically able to depress morphological transformation, and the removal of a transformation sensitive surface glycoprotein (LETS protein) in cells infected by a Prague strain of Rous sarcoma virus mutant [74]. Metabolic inhibitors thus seem to affect various transformation-specific functions at different levels of control; transcriptional, translational or post-translational. It might be expected that the primary virus-induced changes are those which appear most rapidly on shift and which are least subjected to metabolic inhibition. These mutants all produce virion RNA under non-permissive conditions, and if the virion RNA is also the virus message [75,76] then one would not expect the viral transforming gene(s) to require transcription on shift down. The most likely situation is that the viral gene products are produced, but are heat labile, or that there is a defect in their translation. On the other hand, it seems inconceivable that the multitude of events associated with transformation can be mediated by RNA already transcribed in the non-permissive cell, and some parameters should have a

108 requirement for new transcription. Applying this reasoning to the available data, the likeliest candidates so far for a directly virus-controlled function are the vacuolation and decrease in cyclic AMP level seen very shortly after shift in cells infected with Bader's Bryan high titre strain of Rous sarcoma virus mutant. However, the vacuolation is a change only seen in this strain of Rous sarcoma virus, and furthermore in the T5 mutant of Schmidt-Ruppin strain of Rous sarcoma virus adenylate cyclase activity decreases only slowly on shift down [77]. The effect of inhibitors on cyclic AMP does not seem to have been investigated in mutants of strains other than Bryan high titre strain of Rous sarcoma virus. In mutants of Schmidt-Rupin strain and some of Prague strain of Rous sarcoma virus, increase in sugar uptake seems to depend only on new protein synthesis, and might be regarded as a primary alteration, but with the Bryan high titre strain of Rous sarcoma virus mutant, this change depends also on RNA synthesis. Although this might represent a second mutation in Bryan high titre strain of Rous sarcoma virus, it would presumably have to be in a function which interacts with the rapidly-shifting component only at a post-translational level. Alternative explanations are: (1) a low level of activity leaks through the metabolic block, sufficient to cause the rapidly-shifting change but insufficient to produce the sugar uptake alteration, or (2) the strain differences in metabolic requirements for increased sugar uptake reflect a real difference in the transforming functions of different virus strains. These three possible interpretations also apply to other data discussed a little later. When shifting from permissive to non-permissive temperature the kinetics of reversion are similar, or sometimes rather slower, than those of the reciprocal shift [9,64,65]. Once again, not all parameters revert simultaneously; for instance, in cells infected by Bader's [11 ] BH-Rous sarcoma virus mutant the cytoplasmic vacuolation reverts in 3-6 h whilst the level of sugar uptake remains high over this period. There is relatively little information on the metabolic requirements for reversion, but that available suggests that cells can revert in the presence of inhibitors of DNA, RNA and protein synthesis [8,11]. Reappearance of some untransformed phenotypic features presumably requires post-translational modification. For example, the LETS glycoprotein referred to above which disappears on transformation will reappear rapidly on shift up [64]. This reappearance occurs when protein synthesis is inhibited, suggesting that a pool of the protein exists in transformed cells. However, since the complete glycoprotein cannot be detected in transformed ceils the pool must consist of a precursor which is modified on shift up [74]. It is probable that other characteristics of transformation will not disappear simply by switching off the viral transforming function in the absence of other metabolic activity. When enough parameters are studied one might expect that the reversion of some of them will be prevented by inhibition of RNA or protein synthesis. IVB. Transforming functions expressed in restrictive conditions Studies on residual transforming functions at non-permissive temperature provide further evidence for heterogeneity among ts mutants and the changes they

109 elicit. Observed differences between infected cells at restrictive temperature and uninfected cells were at first morphological and behavioural. The ability to form colonies in agar suspension is not ts in LA 334 [14,40] whilst the LA 25 mutant induces cell growth to high density at non-permissive temperature without a morphological transformation [14,78]. Becker and Friis [79] have now isolated several Prague strain of Rous sarcoma virus mutants similar to LA 25 in which cell growth to high density at 41°C is associated with increased ability to multiply suspended in agar and a level of sugar transport between that of normal and transformed cells. Balduzzi [20] has isolated similar Bryan high titre strain of Rous sarcoma virus mutants, but in these cell growthto high density is not invariably accompanied by a propensity for growth in agar. Friis [41] suspects these observations may indicate two viruscontrolled transforming functions, one of which affects morphological transformation without affecting growth in suspension. However, unless ts mutants with the reciprocal lesion (in agar colony formation only) can be isolated it will be difficult to exclude the possibility that these differences result from leakiness, the change in morphology requiring more efficient expression of the function than the capacity to multiply in agar. However, if Friis' suspicion is correct, non-leaky mutants with defects in both morphological transformation and agar colony formation may prove to have two lesions, an eventuality which will complicate their use in genetic studies (Section V). It may be relevant to this that Barlati et al. [80] have divided transformation into two stages on the basis of cellular methylene blue incorporation. In a standard focus assay transformed foci incorporate the dye, but there are areas of morphologically normal cells which also take up dye. When cells infected with the Schmidt-Ruppin strain of Rous sarcoma virus ts mutant F U 19 are shifted to permissive temperature they pass through a stage when these dye-incorporating morphologically normal areas appear and only later develop into frank morphological transformation. It would be interesting to see if an ability to take up dye at restrictive temperature is associated with growth to high cell density, growth in agar, or the ability of some mutantinfected cells at 41 °C to support plaque formation by leukosis viruses [9,14]. IVC. The effects o f mixed infection Double infections by ts mutants at restrictive temperature (complementation tests) are another approach to investigating the complexity of viral transforming functions. Kawai et al. [12] divided three mutants of SR-Rous sarcoma virus into two groups by their ability to complement one another in mixed infection and produce transformation at 41 °C. By the same criterion, Wyke [45] divided i 1 Prague strain of Rous sarcoma virus ts mutants into four complementation groups (now called cooperative transformation groups), but was unable to fit three other mutants into this grouping. These results suggested that there were several viral transforming functions. Later Wyke et al. [81] found that transformation at 41°C was not observed unless progeny from the original mixedly infected cells were allowed to infect other cells. At the same time they showed that mixed infection readily yielded wild

110 type recombinants, and suggested that it was these recombinants which were mainly responsible for the transformation at non-permissive temperature. The genetic implications of these findings are discussed in Section V, but their physiological significance is still in doubt. The failure to detect complementation in the original mixedly infected cells could be because no complementation occurs, or because it does occur but it is not efficient enough to overcome the handicap to development of transformed loci which is provided by preventing reinfection. This question has not yet been answered, but if there is no complementation this would suggest either that the mutants are all defective in one function, or there is some bar on different functions achieving complementation in trans. This might occur if the functions are all translated from a single polycistronic message, mutations which disrupt posttranslational processing.

IVD. Complexity of transforming functions Physiological investigations also leave some doubt as to the variety of transforming functions represented by the four Prague strain of Rous sarcoma virus cooperative transformation groups. Mutants from different groups are indistinguishable in the behaviour of their glycolipids [82], LETS glycoprotein [64] and adenylate cyclase activities [83] in temperature shift experiments. On the other hand, Kurth et al. [78] find that the tumor-specific surface antigens are temperature sensitive in two of the cooperative transformation groups (Aupoix et al., [84] having similar findings with the Schmidt-Ruppin strain of Rous sarcoma virus mutant, FU 19), while in the other two groups the antigen is undiminished at restrictive temperature. The same pattern of tumor-specific surface antigen expression is found in mutanttransformed rat cells [85] increasing the likelihood that antigen expression is directly controlled by the virus, and that its variable presence at 41 ~C is not an artefact. However, it is possible that though the antigen is immunological[y detectable at 41 °C in two cooperative transformation groups it may still be functionally inactive in these otherwise phenotypically normal cells. Further evidence for differences between the cooperative transformation groups comes from Friis [41 ] who has studied the metabolic requirements for a single parameter of transformation, increase in sugar uptake. He finds that the increase in uptake is independent of RNA synthesis but requires new protein synthesis in three of the groups. However, for the fourth group (one in which tumor-specific surface antigen is ts) RNA as well as protein synthesis seems necessary to obtain increased sugar uptake on shift down. As with Kurth's data, these results could reflect a functional difference between mutants, or simply a differential leakiness of mutant functions, the correspondence between physiological difference and cooperative transformation group being evidence for the former.

I VE. Strain d~lferences in transforming functions An interesting finding in Wyke's [45] cooperative transformation studies was the failure to obtain meaningful groupings in mixed infection with Prague strain of

111 Rous sarcoma virus mutants and those of either Schmidt-Rupin strain of Rous sarcoma virus or B77. Balduzzi [20] has confirmed the groupings for Prague strain of Rous sarcoma virus but finds that these mutants also do not give a meaningful pattern of cooperative transformation with his Bryan high titre strain of Rous sarcoma virus mutants. He suggests this probably reflects a difference in the transforming functions of these virus strains. Strain differences in transformation can be manifested in other ways. We have already mentioned the peculiarities of BH-Rous sarcoma virus transformation, and other virus strains also produce transformed cells of characteristic morphology [86]. The kinetic parameters of the adenylate cyclase of transformed cells shows a strain specificity [69,77,83] whilst differences in the rate of disappearance on downshift of the LETS glycoprotein [63,64] may result from the different virus strains used by the two groups who studied it. The complexity and strain differences of transforming functions have implications for the nature and evolution of the viral genome. It may be significant that viruses of different strains can undergo phenotypic mixing and complementation for functions involved in replication, as described in Section III, yet cannot cooperate in transforming functions. This observation suggests that though replication may have altered little in the evolution of these viruses, the non-essential transforming functions may have undergone divergent evolution, or may indeed be of different origin, in the various strains. The complexity of these functions is as yet unknown, but becomes of crucial importance when the coding capacity of the virus is considered in the light of the current uncertainty as to the size of the viral genome. Some of these genetic problems are considered in the next section.

v. THE USE OF

ts

MUTANTS IN GENETIC STUDIES

Vogt [87] and Kawai and Hanafusa [88] showed that cells mixedly infected with an avian sarcoma virus and a non-transforming avian leukosis virus of different envelope subgroup yielded some progeny in which the transforming capacity of the avian sarcoma virus and the envelope properties of the avian leukosis virus were stably recombined. This genetic recombination occurred at a high frequency and suggested that if enough markers were available it should be possible to attempt at least a rough recombination mapping of the avian sarcoma virus genome. The isolation of a variety of avian sarcoma virus t s mutants has now provided the markers to make this project theoretically feasible, but because of difficulties, both conceptual and practical, mapping studies are still rudimentary. Conceptual difficulties arise in interpreting the results of mapping experiments with avian sarcoma virus, since these affect, and are affected by, considerations relating to two other closely allied questions of oncornavirus biology; the structure of the virus genome and the mechanism of virus recombination. To appreciate the interdependence of these three problems it is necessary to summarise briefly the biochemical and genetic data bearing on them.

112 VA. The viral genome: haploid or polyploid? The 70-S single stranded virion RNA can be dissociated into several 35-S subunits [89]. It was originally suspected that each of these subunits is unique and thus high frequency recombination, as observed by Vogt [87], could be explained by reassortment of 35-S molecules from different parents into the recombinant progeny. However, studies with cloned virus stocks showed that the 35-S subunits of avian sarcoma virus are uniform, and of consistently larger size, than the subunits of avian leukosis virus or of transformation defective derivatives of avian sarcoma virus [90]. If the subunits are all unique, how (and why) does an essentially simultaneous reduction in the size of each subunit occur in the genesis of a transformation-defective avian sarcoma virus? What is more, the transforming progeny from a mixed infection of avian sarcoma virus and avian leukosis virus possess subunits whose size is typical of those of avian sarcoma virus, even though some of their genetic material was derived from an avian leukosis virus parent [91]. How can this be explained if recombination occurs by subunit reassortment? To account for these observations, Vogt [92] proposed that the avian oncornavirus genome might be polyploid, each subunit representing the complete haploid genome. Recombinants, he suggested, arise from a mixed infection first as heterozygous particles, and Weiss et al. [27] have in fact provided evidence that such heterozygotes exist. When these heterozygotes infect fresh cells recombination, which is of necessity intramolecular, could then occur during, or subsequent to reverse transcription. Of the proviral D N A then formed, an amount equivalent to one haploid RNA (35-S) genome would proceed with the infection, and the nature and size of this provirus would determine the uniform nature of all the subunits in the progeny virions. Only rarely in an infected cell would provirus representing more than one haploid genome equivalent give rise to progeny, and these exceptions would account for the persistence of heterozygotes observed by Weiss et al. [27]. In the past year two pieces of evidence favouring a polyploid genome structure have been obtained by studying the oligonucleotides produced by T 1 ribonuclease digestion of virion RNA. First, several groups [91,93,94] have independently observed that the quantity of oligonucleotides of known chain length obtained after digestion corresponds to a size of about 3"106 daltons for unique virion RNA. This is the approximate size of a 35-S subunit but is a minimum estimate for complexity since it assumes that lhe large oligonucleotides studied are no more frequent in occurrence than sequences in the rest of the viral RNA. Second, a study of several recombinant clones derived from a mixed infection of avian sarcoma virus and avian leukosis virus has shown that different recombinants have subunits of slightly different electrophoretic mobility which give slightly different oligonucleotide fingerprints after digestion [9l]. This was adduced to be evidence for recombination occurring by different unequal crossover events, since results of the same complexity could only be obtained by reassortment of unique subunits if each virion contains at least five such subunits. Further evidence suggesting that the true virion size is that of a 35-S subunit has come from studies on the size of infectious DNA recovered

113 from transformed cells [95], the size of integrated proviral D N A determined by reassociation kinetics [48] and the size of isolated, newly-synthesized provirus [39,96]. However, it should be mentioned that R N A ' D N A reassociation kinetics performed by Taylor et al. [97] resulted in estimates of virion RNA complexity two to three times the size of a 35-S subunit, though these studies, like those on nuclease digestion, are not conclusive. Fan and Paskind [98] reached the same conclusions as Taylor et al. [97] in their studies on murine leukemia virus, but it is possible that the genetic complexity of mammalian and avian oncornaviruses may differ. VB. Recombination studies with Avian sarcoma virus ts mutants

Some of these biochemical data are subject to the criticism that they study total virus nucleic acid, only a fraction of which might represent infectious virus. Genetic studies, apart from their ultimate aim of mapping viral functions, can provide additional corroborative evidence on genome structure and behaviour, since they study only biologically active virus. Seen in this light, even the preliminary genetic studies performed so far are proving valuable. Mason et al. [38] studied recombination between the reverse transcriptase mutants L A 335 and L A 337 and avian leukosis virus whilst Friis et al. [62] performed similar studies on L A 672 and avian leukosis virus. In view of the likelihood that L A 672 is also mutant in reverse transcriptase it is interesting that both studies gave similar results. The ts mutant markers appear linked to the envelope glycoprotein (host range) marker, though about 1 0 ~ of the total recombinants had recombined between these two markers. In a conservative interpretation of these data the authors argued that even if the high proportion (90~o) of recombinants in which these markers remained linked represented reassortment of genome subunits, then at least the lower frequency recombination between these markers was likely to represent another mechanism. In another study, Wyke et al. [81 ] attempted to avoid some of the causes of criticism against earlier recombination experiments by studying crosses between two ts mutants. In this work the viruses were all derived from Prague strain Rous sarcoma virus and were thus more likelyto be genetically homogeneous than some of the viruses used in avian sarcoma virus crosses with avian leukosis virus. Moreover, since both parents could transform under appropriate circumstances, all the progeny of a mixed infection, and not just the transforming progeny, could readily be identified, providing an internal control for reciprocal recombination and the relative contribution of each parent to the progeny. In a cross of this sort between L A 335 and a transformation-defective mutant L A 29 there was again evidence for linkage between the reverse transcriptase and envelope glycoprotein markers, though to a less marked degree than in the experiments of Friis, Mason and co-workers. Wyke et al. also found that recombination occurs among transformation-defective ts mutants in different cooperative transformation groups (see Section IVC) and even between mutants in the same group. In this latter case at least they considered that the result could not be due to subunit reassortment and must involve intramolecular recombination.

ll4 These experiments [81] were also intended to provide information on the means by which these recombinants arise. In the cross between LA 335 and LA 29 harvests from mixedly infected cells were examined for their content of heterozygous particles at a time when only one infectious cycle would have been completed. The rationale behind this was as follows. Viruses with a haploid genome made up of unique subunits would probably give rise to many recombinants by reassortment and might only occasionally give rise to heterozygotes. Moreover they would probably show heterozygosis for one marker without being heterozygous for others, unless, of course, the three markers studied (reverse transcriptase, envelope glycoprotein and transformation maintenance) were all fortuitously determined by loci on the same subunit. if, on the other hand, the viruses were polyploid, heterozygotes might be an obligatory precursor of recombinants, though the postulates of Vogt [92] would predict that most of such heterozygotes would escape detection since the selection of only one haploid genome complement at or after reverse transcription would immediately generate recombinant or parental viruses from the mixed genotype. However, any heterozygote that persisted would, in contrast to the haploid genome model, be heterozygous for all the markers studied. The results surprisingly showed that not only were heterozygotes for all three markers far in excess of partial heterozygotes, but heterozygotes as a whole were far more common than true recombinants. Only in successive cycles of infection were many recombinants generated from these heterozygotes, which notwithstanding showed a tendency to persist (cf. [27]). Interpretation of these data is clouded by the possibility that viral clumps might mimic heterozygotes, but a likely explanation for the results is that the genome is functionally polyploid for the markers studied, though the stability of the heterozygous forerunners of recombinants cannot be explained easily in the replication scheme envisaged by Vogt [92]. Studies on cooperative transformation between transformation-defective ts mutants also showed that transformation at restrictive temperature, which probably involves recombination (Section IVC), was not expressed in the original mixedly infected cell but became apparent only after reinfection [81]. Though other explanations are possible, this also suggests that reinfection is a necessary prerequisite for recombination, a concept in keeping with the formation of polyploid heterozygotes in the first infectious cycle which give rise to recombination in successive infections. VC. A mode~for the viral genome

These findings are explained in a model for virus replication put forward by Cooper and Wyke [99]. This postulates that if the RNA primer for reverse transcriptase [100,101 ] is hydrogen bonded to the viral genome, this would provide difficulties in transcribing the antiprimer region which is not susceptible to ribonuclease H. This problem is solved by the virion's containing more than one genome in a closely coupled tandem so that reverse transcription to form - - D N A can proceed across the subunit boundary (Fig. 2A), recovering the antiprimer region, "'a', by transcription of the distal (right hand) subunit. Once reverse transcription has progressed beyond

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Fig. 2. Model for reverse transcription and integration of a polyploid RNA tumor virus genome (from Cooper and Wyke, in the press). For simplicity this model depicts the genome as diploid, comprising two dissimilar subunits (markers k't and kt'). A-D, Stages of reverse transcription generating a r e c o m b i n a n t - - D N A ; E, three ways in which sequences could be selected for circularisation; E3, F, G, Circularisation and integration of recombinant and heteroduplex provirus ( - - D N A markers kt, + DNA markers k't).

the proximal (left hand) subunit this can be degraded back to the primer region by the ribonuclease H activity of a second enzyme molecule which thus frees the proximal - - D N A to act as template for synthesis of a + D N A strand, this being primed by the 3' terminal of the distal R N A subunit (Fig. 2B). The + D N A may displace the antiprimer + R N A (Fig. 2C) (or this may be removed later), and at the same time its own primer is removed by ribonuclease H to yield a molecule (Fig. 2D) in which the left hand end of the + D N A shows complementarity with the single stranded "a-c" region of the - - D N A . The existence of this homology would allow the + DNA to form a circle (Fig. 2E) which would act as a template to which a similar length of the - - D N A is base-paired. Since the - - D N A contains one copy of the antiprimer region but two copies of the rest of the genome it can base pair with the 4_DNA template in a variety of ways as illustrated in Fig. 2E and in this way recombinants can be generated if the two original RNA genomes were of different constitution. This scheme thus provides mechanisms for antiprimer transcription, for intramolecular recombination and for simultaneous generation of unit size progeny (the sizes of which are determined by the 4- D N A template), which are not available to a haploid segmented genome. It also provides a role for the ribonuclease H activity of the reverse transcriptase molecule. Moreover, the model proposes a means of forming a circular provirus and by analogy with other virus systems this may be a

116 necessary prerequisite for provirus integration. Evidence has now been obtained for a circular provirus of the size predicted by this scheme [39,96] and this provirus may in fact integrate [96] in units equivalent in length to a single 35-S RNA subunit [48]. The model takes no account of the poly-adenosine sequences at the 3' end of the RNA subunits, but the possibility that these sequences may be absent from 3050 ~o of the subunits [107] could have implications for the proposed tandem arrangement of the molecules. A model proposed by Hill and Hillova [102] also postulates that recombination occurs by transcription from one subunit to another, but their proposals differ in other details. The implications of the model, including the possible nature of subunit coupling, are discussed in detail by Cooper and Wyke [99], but one possibility not elaborated there is that this scheme provides a means of maintaining heterozygous viruses for more than one generation following a mixed infection, a phenomenon observed experimentally [27,81] but not easily explicable by the polyploid model of Vogt [92]. Firstly, as shown in Fig. 2E the double stranded provirus has a ÷ D N A representing the proximal RNA genome of Fig. 2A and - - D N A which will, to a greater or lesser degree, be a recombinant between the proximal and distal genomes. Thus a heteroduplex provirus will be integrated in the infected cell and this would be revealed as a partial heterozygote when, at mitosis, daughter cells receive proviruses transcribing progeny of different genotype. However, as mentioned above, genotypically mixed particles observed by Wyke et al. [81] were mainly heterozygous for all markers studied. This would be the case if the virus in fact contained three genomes. The events depicted in Fig. 2 would still occur, but the - ? D N A template might now comprise the proximal and middle subunits and a diploid provirus would be integrated, However, recombination would occur only between the proximal and distal genomes retaining the middle subunit intact in the provirus. It follows from this that the size of the recombinant provirus genome would still be dictated only by the amount of the -~ DNA template which is homologous to the proximal subunit. Thus recombinants of uniform size would still be generated and isolated pure if a selection was applied in their favour, but, in the lack of selection, heterozygosis would tend to persist. Indeed if the proximal and distal genomes were identical, but derived from a different parent than the middle genome, then a heterozygous but non-recombinant provirus would be formed and recombination would be delayed by one generation. It is interesting that about 50 ~ of the heterozygotes detected after a single cycle of mixed infection do not yield recombinants in the next cycle [33]. This model is consistent with the biochemical and genetic evidence favouring genome polyploidy, and provides a rationale for the phenomenon. The triploid version of the model provides, in addition, an explanation for the existence of particles which are heterozygous for multiple markers, but it is at variance with the electron microscope studies of Mangel et al. [103] which suggest that most virions contain only two subunits. Indeed it should be stressed that none of the evidence on which these conjectures are based is as yet entirely convincing. Biochemical data strongly suggest that the virion RNA is made up of subunits, and that these subunits share

117 many sequences in common. However, it remains possible that the subunits also bear unique sequences. Similarly, although biochemical and genetic data very much favour the occurrence of intramolecular recombination they do not rule out the existence of additional recombination by subunit reassortment. The genetic experiments suggesting that heterozygosis precedes the formation of stable recombinants are less strong. It is possible, though less likely, that recombination occurs during the original mixed infection, either during provirus formation, during transcription of new virus RNA from provirus, or even among the new RNA transcripts themselves. The model proposed above makes testable predictions, the outcome of which should be useful in deciding between these various possibilities.

VI. PROSPECTS As this review shows, Avian sarcoma virus ts mutants have provided a wealth of information on the physiology of virus replication and transformation, and they are just beginning to contribute to studies of virus genetics. Much of the work quoted was done in the last three years and in fact it has been necessary to consider much unpublished, and therefore unconfirmed, data in an effort to make discussion clear and interesting. This suggests that the use of these mutants is expanding and it is therefore worth considering how this work should develop. Extrapolation into the future is easier when considering the use of mutants to study virus replication and genetics, fields which have been the interest of small numbers of workers. The existing replication-defective mutants should repay more intense study, particularly those in which the ts lesion affects a stage of infection not revealed by other techniques. At the same time the emerging complexity of virus control during its life cycle suggests that many more mutants should be isolated to fill the gaps which will inevitably be revealed. This work will be tedious but ultimately worthwhile. As far as genetic studies are concerned, it seems likely that the dilemma of the viral genome's structure and complexity will soon be resolved by a combination of biochemical and genetic techniques. This should ease the interpretation of attempts to map viral functions, and one might soon hope for a rough map to provide a genetic basis for understanding viral strategy. A great deal of mapping can probably be done with mutants that are already available, unless problems arise due to unsuspected double mutations. However, mapping studies should also be extended to any newly isolated replication-defective mutants. In considering transformation-defective mutants predictions are more difficult, their usage being very dependent on innovations in the technology of studying transformed cells. From the virological point of view it appears that emphasis should now be less on isolating additional mutants and more on understanding those that already exist. Studies on the differences between mutants in various cooperative transformation groups should be pursued to reveal the complexity of viral transforming functions. However, the primary virus-controlled changes may be revealed

118 more readily by undertaking a detailed study of a single mutant. Such a study would follow the effect of temperature shift, with and without metabolic inhibition, on the expression of a range of parameters. No mutant has yet been subjected to such an intensive study, though the work of Bader and collaborators on his Bryan high titre strain of Rous sarcoma virus mutant and studies on ts N Y 68 by Robbins et al. [63] and other groups are tending in this direction. Even when the viral transforming functions become better understood, their interaction with the host cell will demand study. Recent work with normal rat kidney cells transformed by ts avian sarcoma virus suggested that the host can modify expression of the viral genes. G r a f and Friis [17] found that in one such cell the nonpermissive temperature was 37~'C, the permissive temperature 33°C, whilst in the avian host 37°C was a permissive temperature, with 41 '~C providing restrictive conditions. A lowering in the temperature range of the mutant thermosensitivity did not occur in transformed normal rat kidney studied by Wyke [33], perhaps because they were selected at a higher temperature, but in these cells, as in those of G r a f [104], the change in phenotype which resulted from a temperature shift was far slower than in avian cells [33,85]. G r a f [104] further found that phenotypic revertants of the ts normal rat kidney which became stably transformed at high temperature sometimes yielded wild type virus on rescue, but in other cases the rescued virus was still ts. Thus modification by host cells can grossly alter expression of virus functions, and such phenomena should provide a fruitful area for further study. Transformed mammalian cells may be useful for other studies, since they can be readily cloned to yield homogeneous cell populations. Moreover, the replicating functions of avian sarcoma virus are suppressed in these hosts, and they contain no endogenous viruses with any homology to avian oncornaviruses. Such cells may be particularly suited to studies on the kinetics of appearance and disappearance of transformed cell parameters, though work of this kind has already begun with infected chicken cells. Indeed, irrespective of whether or not these parameters are directly related to viral gene action, avian sarcoma virus ts mutants will probably continue to serve a major use as a tool for "'switching on" and "~switching off" cell transformation rapidly and reproducibly.

ACKNOWLEDGEMENTS Many workers helped in making the review as up-to-date as possible. Dr R. Friis and collaborators, Drs J. Bishop, T. Graf, M. Halpern, R. Kurth, K. M611ing and H. Ogura communicated many new results. I am also grateful to Drs J. Bader, P. Balduzzi, D. Baltimore, J. Bookout, F.. Hunter, W. Mason, P. Robbins, M. Sigel, K. Toyoshima and P. Vigier for allowing me to quote from work of theirs and their colleagues prior to publication. Drs R. Hynes, H. Murphy and J. Bell provided valuable criticism of the manuscript.

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