Complementation of transforming functions by temperature-sensitive mutants of avion sarcoma virus

VIROLOGY

64, 28-36 (1973)

Complementation

of Transforming Mutants

Functions

of Avian

Sarcoma

by Temperature-Sensitive Virus’

JOHN A. WYKE2s 3 Department

of Microbiology,

University of Southern California, School of Medicine, IAvenue, Los Angeles, California 90033 Accepted March

1025 Zonal

1, 1979

Complementation tests have been performed on avian sarcoma viruses which are temperature sensitive in their ability to transform cells. A rapid qualitative test and a quantitative test are both described. Eleven mutants which are defective only in transforming properties have been divided into 4 clear complementation groups, with 3 similar mutants not., as yet, assigned to any of these groups. Two further mutants with coordinate lesions in transformation and viral replication comprise another 2 distinct complementation groups. The problems of interpreting the data presented, and the implications of any int,erpretation, are discussed. INTRODUCTION

whether different behavioral categories represent mutations in separate genes and, The isolation of a number of temperature also, whether a single behavioral group can sensitive (ts) mutants of avian sarcoma virus has been described in previous com- contain mutants with lesions in different munications (Toyoshima and Vogt, 196913; functions. For these reasons complementaWyke, 1973). These mutants have been di- tion tests have been performed between vided into 2 major classes on the basis of representatives of different categories and physiological properties. Mutants in Class on mutants within a single category. Since all T and C class mutant,s are ts in transT replicate at the nonpermissive temperature but are unable to transform the cells forming abilities, successful complementation they infect, whereas Class C mutants are was gauged by the appearance of cell transformation under nonpermissive conditions. coordinately defective in both transformation and replication under nonpermissive MATERIALS AND METHODS conditions. Further physiological studies Viruses and cells. The isolation and prop(Friis et al., 1971; Linial and Mason, 1973; erties of the avian sarcoma virus ts mutants Wyke and Linial, 1973) have subdivided these 2 mutant classes into 6 categories on studied here have been described previously (Toyoshima and Vogt, 196913; Friis et al., the basis of their behavior in temperature 1971; Wyke, 1973; Linial and Mason, 1973; shift experiments. It is important to know Wyke and Linial, 1973). In all cases the 1 Supported by U.S.P.H.S. Research Grant permissive temperature was 35” and the No. C.A. 13213 from the National Cancer Instinonpermissive temperature 41”. Most studtute and Contract No. NIH-NC1 72-2032 within ies involved mutants of the Prague strain the Special Virus Cancer Programme of the Naof Rous sarcoma virus (PR-RSV). tional Cancer Institute. The cultivation and characterisation of 2 Support,ed by a Fellowship from the Leukechick cells followed standard methods (Vogt mia Society of America Inc. and Ishizaki 1965; Duff and Vogt, 1969). 2 Present address : Imperial Cancer R.esearch Only cells which were negative for chick Fund Laboratories, P.O. Box 123, Lincoln’s Inn Fields, London WC2A 3PS, England. helper factor (Weiss et al., 1973) were used 28 Copyright All rights

@ 1973 by Academic Press, of reproduction in any form

Inc. reserved.

OF AHV IS MIJTASTS

COMPLEMENTATION

in growing mutant stocks and performing complementation tests. All cult8ures were maintained in Ham’s FlO medium supplemented with 10 % tryptose phosphate broth and 5% calf serum. To enhance virus absorption, all infections were performed in the presence of 2 /*g of polybrene per milliliter (Toyoshima and Vogt, 1969a). Cultures were grown in either 35mm Falcon plastic petri dishes (for quantitative complementation t,ests and plaque assays) or in Falcon microtest plates No. 3040 (for rapid complomcntation tests). ~ZIUS assays. The focus assay, which was the basis of the complementation tests described, was modified from standard procedures (Rubin, 1960; Vogt, 1969). Plaque formation was assayed by t,he method of VVyke and Linial (1973). RESULTS

Rapid, tests for Complementation between Rous Sarcoma Virus ts Mutants The Class T mutants form a homogeneous category indistinguishable in their behavior in temperature shift experiments (Wyke and

29

Linial, 1973). The only exception is ts 30, in which the virion is ts and there is an early ts function needed for the stabilization of cell transforming potential. Since t’hese mutants are similar in physiological criteria, it is interesting to know whether they can be distinguished on the basis of behavior in complementation tests. With the exception of ts 35 (which was derived from subgroup C PR-RSV), all these mutants belong to PR-RSV subgroup ,4. Quantitative coinfection of cells by viruses in the same subgroup is difficult because of t.he phenomenon of early interference (Steck and Rubin, 1966), and thus complementation between the original mutant isolates may not bc feasible. However, it was found that infection of cells in microplate wells with relatively high inocula of 2 mutants in the same subgroup (m.o.i. about 0.7 for each mutant)) could result’ in clear complemcntation for transformation. The degree of complementation was judged by assessing the area of the microplat,e well that was transformed after 6 days incubation at 41” (Table 1). An obvious objection to this test, apart’ from the impossibility of :rssa!.ing the

TA4l3LE: I

C:OMPI,I;MKNTATIOKFOR TRAKSFORMATION AT 41" BI:TTVJ.;I~NPAIRS OF 7' CLASS fs ~~CTANTR OF PRAGUI.: STR.~IX Itors ~ARCOM.~~'IRUS"

(1The subgroup of each mutant was that in which it was originally isolated (subgroup -4 for Is B-34, subgroup C for ts 35). Complementation was assessed in microplates by mixing 10’ FFU of each virus in a well and adding 1.5 X IO* susceptible chick cells. The wells were overlaid with agar medium at 24 hr and transformation was scored 5 days later on the following scale: 0, no transformation; 1. up to ZOr:/,, of the cell sheet transformed; 2, 2@40$!& of the cell sheet transformed; 3, 40-60y0 of the cell sheet, transformed; 4, 60-80yQ of the cell sheet transformed; 5, 80-100~~ of the cell sheet, transformed. h 7’s 27 and ts 33 had not been isolat,ed at the time these experiments were performed.

30

WYKE

number of doubly infected cells, is the fact that as few as 20 discrete transformed foci could cover 50 % of the well by 6 days. Thus, even though leakiness of the mutants was not significant, it might nonetheless be difficult to distinguish between meaningful and trivial degrees of complementation. On the other hand, this test is economical in both time and materials and gives consistent results. It provides both a useful preliminary to, and confirmation of, the quantitative complementation described below. If a score of 1 or less (Table 1) is regarded as failure to complement, then ts 25, 30, and 34 form one complementation group, ts 24 and 31 another, the remaining viruses being distinct or uncertain. The only anomaly is the apparent failure of complementation between ts 22 and ts 30. If a score of 2 is also regarded as negative complementation, then ts 28 joins the larger of the previously defined groups, and ts 23 and 35 form a third complementation group. Isolation

of Genetically Reasserted ts Mutants

The problems of viral interference can be overcome by using mutants in different subgroups. The subgroup of an avian sarcoma virus can be stably altered by mixed infection with an appropriate leukosis virus followed by isolation of viruses bearing the host range marker of the leukosis virus and the transforming ability of the sarcoma virus (Vogt, 1971b; Kawai and Hanafusa, 1972b). Cells were infected at 35” with mutants ts 22 to 34 (subgroup A) and either RAV-2 (subgroup B) or RAV-49 (subgroup C), all viruses being at an m.o.i. of 0.2. After 3 transfers at 3-day intervals the cells were completely transformed and progeny virus was harvested. Mutants of reassorted host range were isolated by the infectious center technique described by Weiss et al. (1973). Appropriate virus dilutions were plated on 5 X lo5 C/A cells in a 36-mm dish and Mitomycin C (2 pg/ml) was added 24 hours later. After 18 hours of treatment, the Mitomycin C was removed, the cultures were washed 3 times in fresh medium, and 5 X lo5 fresh C/A cells were added. Successful infection of the original C/A cells depended on the pseudotype of the virus derived from the

mixed infection. However, Mitomycin C treatment killed these cells, and unless they were doubly infected they would not subsequently produce pseudotype virus. Thus, only mutant,s with stably reassorted host range would transform the added C/A cells. The cultures were overlaid 4 hours after the fresh C/A cells were added, and foci were isolated and sonicated after 7 days’ incubation at 35”. The sonicate was then subjected to a further selective cloning by the infectious center technique. Virus isolated after the second cloning was tested for host range and temperature sensitivity, but no attempt was made to assess the percentage of reassorted virus in the progeny of the original mixed infection. Three potential recombinants of both subgroup B and subgroup C were tested for each of the 13 mutants. In every case the virus clone was found to have acquired the host range of the leukosis virus while maintaining a ts transforming marker of the original mutant. Moreover, the recombinant mutants all grew to titers similar to those of the original subgroup A mutants. Once again ts 30 was a sole exception. Recombinant clones of this mutant no longer showed the virion temperature sensitivity and early ts requirement of the original subgroup A isolate. The subgroup B and subgroup C recombinants of ts 30 were thus physiologically similar to all the other T class mutants. These data suggest that the original ts 30 isolate was a double mutant, and this phenomenon is being investigated further. Quantitative Complementation Tests between Avian Sarcoma Virus ts Mutants The reassortment of ts mutants into different subgroups facilitated quantitative complementation tests as described by Kawai et al. (1972). A monolayer of susceptible chick cells was either maintained as control or preinfected with a ts mutant at an m.o.i. of 2.5. This was sufficient for the majority of the cells to become infected with at least 1 focus-forming unit (FFU). The superinfecting mutant or wild-type virus was added in appropriate dilutions 2 hr later, and the cultures were overlaid after 24 hr. Prior to

COMPLEMENTATION

OF ASV TABLE

-~

ts MUTANTS

31

2

PERCENTAGE COMPLEMENTATION FOR TRANSFORMATION AT 41” IXTWFXN PAIRS OF T CLASS Is MUTANTS OF PRAGUE STRAIN Rots SARCOMAVIRIW

~~

-

22 23 24 25 26 27 28 29 30 31 32 33 34 35

0 35 80 64 55 / 42 21 32

0 121 45 31 16

0 64 45 100 11 55 33 90 18 83 70 55 37 a 18 20 100 48 19 45 .tO 110 240

0 loo

100

/ 10

1

21

2 72 2 74 34 1 15 105

22

23

24

25

I ~ / ’

0 74 0 9100 50 1

loo loo 11 39 40 131 26

0 43 100 2 loo loo 90 11 29 3 66 11 94 35 27

28

0 87 77 79 55 186 50 29

0 152 0 190 100 5 37 10 230 70 74 30

31

0

10 21 15 32

0

3 0 52 121 0 .~~~_____-..-._33

34

~-

35

ts mutant n Susceptible chick cells seeded in 35.mm dishes were exposed to the preinfecting ts mutant at, an m.o.i. of 2.5. Appropriate dilutions of a superinfecting mutant in a difierent subgroup were added 2 hr later. Cultures were incubated at 35” or 41”, overlaid 24 hr later with agar medium, and incubated a further 5 days at 41” before counting foci of transformation. The numbers of foci produced by the superinfecting mutant at 41” compared to the count at 35” is corrected for mutant leakiness, normalized to the counts of wild-type foci under identical assay conditions and expressed as a percentage. Figures given are the highest values obtained for any combination of mutants and condit,ions in infection. overlaying, cultures were maintained at either 35” or 41”. Incubation at 35” improved subsequent focus production only in infections involving subgroup A k 30, possibly because of the early ts lesion in this mutant. Transformed foci were counted after 5 days further incubation at 41”. Control cultures of superinfecting virus dilutions were maintained at 35” and counted after 7 days. The degree of complementation was expressed as the percentage focus production by the superinfecting mutant at 41” compared to that at 35” after making the corrections detailed in Table 2. To allow for variations between virus subgroups both subgroup B and C mutants were used as preinfecting viruses, and subgroups A, B and C were used for superinfection. Several clones of each mutant were tested within each subgroup. Tests involving subgroup B mutants were often difficult to score, as described below. The degree of complementation between any pair of mutants can vary greatly, and this variation does not appear to depend on which mutant superinfects and which

preinfects the cell layer. Differences between particular mutant clones are the most likely causes of this variation, and such differences would be hard to analyse in view of the large number of mutant pairs involved and t.he large amount of virus used in each preinfection. For reasons that will be discussed, the highest degree of complementation between any pair of mutants, irrespective of which was the preinfecting and which the superinfecting virus, is regarded as the most’ meaningful figure. These figures are given in Table 2, and Table 3 lists the mutant pairs which show high and low degrees of complementation. On the basis of thcsc data 4 complementation groups can be defined (Table 4) comprising 11 of the 14 !!’ class mutanm. Mutants within a group show less than 109% complementation, wit)h the exception of ts 34 which complements is 25, 28, and 30, but not Is 33, to a slightly greater extent. At least some members of any group complement members of the other 3 groups to 90 % or more. The definit’ion of groups I, II and III agrees w&h the qualitative

32

WYKE TABLE 3 INFECTIONS BY ts MUTANTS OF PRAGUE STRAIN RSV WHICH SHOW Low AND HIGH (>70%) COMPLEMENTATION FOR TRANSFORMATION AT 41”a

DOUBLE

Low w-19%

&9%

ts mutant

a Data

pairs

from

Table

23 24 25 25 25 26 27 28 28 30 33

+ + + + + + + + + + +

35 31 28 30 33 28 29 30 33 33 34

22 22 22 23 23 23 25 26 28 28 30 32

+ + + + + + + + + + + +

High 20-2970 -

30 32 35 27 28 33 34 32 32 34 34 33

(<30%)

22 23 24 27 32

+ + + + +

28 32 35 33 34

7 l-SO% 22 25 25 26 29 29 31

+ + + + + + +

24 29 31 27 31 32 35

Sl-90% 23 24 27 29

+ + + +

30 29 32 30

91-100+% 23 23 24 24 24 25 25 25 26 26 26 27 27 27 27 28 29 30 30 31 34

+ + + + + + + + + + + + + + + + + + + + +

24 34 27 32 34 26 27 35 30 31 35 28 30 31 34 31 34 31 32 34 35

2.

data shown in Table 1. The other 3 T class mutants, ts 22, 26, and 32, do not show a consistently low complementation with all the members of any defined group and hence cannot, as yet, be assigned to any group. Complementation tests of this kind were extended to further T class Is mutants. Dr. G. S. Martin kindly provided 5 mutants of Schmidt-Ruppin strain RSV (Subgroup A), and Dr. R. R. Friis supplied 2 mutants of avian sarcoma virus B77 (subgroup C). Clones of these viruses were tested against one another and against the PR strain ts mutants described above. The degree of complementation was in all cases low, and not consistent with the complementation groups already defined. Similar problems were encountered when testing complementation of class T mutants of PR-RSV with the B77 coordinate mutants, t.s 334 and ts 336 (Toyoshima and Vogt, 1969b; Wyke and Linial, 1973). The failure to obtain unequivocal data in these tests may be due merely to peculiarities of the clones

used, but it may reflect previously unexpected incompatibilities between avian sarcoma viruses of different strains. Indeed, when a PR strain class C mutant, ts 338, was used it clearly complemented the class T mutants (Table 5). It is interesting that, in contrast, the PR co-ordinate mutant f-s 335 did not complement any of the T class mutants. Ts 335 has a lesion affecting very early stages of infection, and involving the activity of the viral RNA directed DNA polymerase (Linial and Mason, 1973). It is probable that some early lesions, as in ts 335 and subgroup A ts 30, cannot be complemented by coinfection with another virus under nonpermissive conditions. Plaque Production by Subgroup B ts Mutants Kawai and Hanafusa (1972a) and Wyke and Linial (1973) have described the production of plaques by subgroup B and subgroup D leukosisviruseswhentheyinfect cells confluently preinfected at 41” with certain ts mutants of subgroup A. The 2 plaque-

COMPLEMENTATION TABLE

4

COMPLEMENTATION GROUPS OF CLASS T ts MUTANTS OF PRAGUX STRAIN Rous SARCOMA Vmus

ts mutants

Complementation group 25, 23, 24, 27, 22,

I II III IV Unclassified TABLE

28, 30, 33, 34 35 31 29 26, 32

5

PERCENTAGE COMPLEMENTATION FOR TRANSFORMATION AT 41” BETWEEN PREINFECTING CLASS C ts MUTANTS AND SUPERINFECTING CLASS T ts MUTANTS OF PRAGUE STRAIN Rous SARCOMA VIRUS~

Preinfecting is Mutant (Subgroup C)

Superinfecting ts mutant (Subgroup A)

22 23 24 25 26 27 28 29 30 31 32 33 34

r

335

338

0 0 0 0 0 0 0 0 0 0 0 0

55 100 29 110 100 100 64 26 48 49 100 100 290

0

DSee footnote Table 2

supporting mutants most frequently used in the present study were ts 25 and ts 28. When these mutants were used as the subgroup C preinfecting virus, they supported plaque production by all superinfecting ts mutants of subgroup B, but the subgroup A superinfecting mutants did not produce plaques. This shows that the ability to support plaque production is not affected by the subgroup of the supporting ts mutants. However, t,he property of inducing plaque formation in mutant infected cell layers does seem to be closely associated with the host range of the inducing virus. It is not possible

OF ASV ts MUTANTS

3.3

to stat’e whether the plaques were induced by the subgroup B mutants themselves, by transformation defective derivatives of these mutants or by contaminating RAV-2 which had persisted through the cloning of the subgroup B recombinants. However, 2 observations suggest that the ts virus itself could induce plaque formation. 1. The plaque-forming titer and focusforming t#iter of subgroup B mutants are approximately equal. (Plaque-forming units at 41” are from 20% t.o 100 o/c of the focusforming titer at 35”.) 2. In cases where the preinfecting and superinfecting viruses are in different complementation groups, the plaques often seem to arise from degenerating foci of complementation and retain a ring of transformed cells around their periphery. Plaques take about 7 days t’o appear, in contrast’ to the 3-4 days described in the plaque assays of Kawai and Hanafusa and Wyke and Linial. In this respect they more closely resemble the plaques produced by subgroup B viruses as described by Graf (1972), and the plaques produced by Bryan high t’itcr strain observed by Bader (1972). When a subgroup B virus is used for preinfection the phenomenon is harder t#oassess. In many cases the whole cell monolayer appears to degenerate and fails to take up neutral red after about 7 days at 41”. For these reasons it \vas often impossible to obtain quantitative data from complementation t’csts involving subgroup B ts mutants. 1)ISCUSHION

Complementation for cell transformation upon coinfection wit’h 2 RSV ts mutants has been clearly demonstrated. However, the degree of complementation between any 2 mutants was found to vary when different virus clones were used. The data presented show the maximum degree of complement’ation observed for any particular coinfection. Selection of data in this way must be just,ified, particularly since it’ would tend to maximise the nnmbcr of complementat,ion groups. One explanation for the observed clonal variat,ion in complement’ation could br the variable occurrencr of defective viruscw in

34

WYKE

the stocks used. Rous sarcoma virus isolates have been shown to contain 4 to 17% of transformation defective variants (Vogt, 1971a). They may also contain variants defective in replication as well as transformation, but which nonetheless can exert an early interference. The frequency of variants of this kind would be very difficult to determine. Transformation defective variants and coordinately defective variants may or may not possess the gene functions necessary to complement any particular ts mutant. Thus, for any coinfection with ts mutants, the following possibilities could exist. 1. The preinfecting mutant contains defective viruses which do not complement the superinfecting mutant. If these defective viruses interfere with nondefective members of the same stock, the m.o.i. is essentially lower than calculated and the complementation would show a spuriously low value. 2. The preinfecting mutant contains defective viruses which can complement the superinfecting virus. The meaningful m.o.i. is then higher than anticipated, but since the calculated multiplicity of 2.5 should infect the majority of the cells this would have little effect on the complementation. 3. The superinfecting mutant contains defective viruses which cannot complement the preinfecting mutants. Since the superinfecting virus is greatly diluted, these should have no effect on the data obtained. 4. If the superinfecting mutant contains defective viruses which can complement the preinfecting mutant, then a spuriously high degree of complementation would be obtained. The combination of these 4 possibilities would be unique for any 2 virus clones, and thus variations in data could have very complex reasons which would be elucidated only by very lengthy analyses. In practice, it is perhaps remarkable how often the maximum degree of complementation observed is at or near the 100% level (Tables 2 and 5). Moreover, the data given in these tables, with the exception of ts 34, are typical of the majority of experiments performed, and only in relatively few cases was there less complementation. The reduced complementation, which may be as low as 20 % of t,he maximum values obtained, usually oc-

curred in experiments using clones of low titer, and might have resulted from defective viruses in the preinfecting mutant. A high degree of complementation would be obtained only when at least one mutant clone of a pair tested was essentially free of such defective viruses and would thus provide a competent preinfection. The only mutant which showed more than 100% complementation in several crosses was ts 34 when it was used as the superinfecting mutant These high values could be explained by the fourth possibility mentioned above, and it is tempting to apply the same reasoning to the relatively high complementation values obtained between ts 34 and some other members of complementation group I. If ts 34 clones of this type were used for preinfection then possibility (2) above would predict that the complementation would not be affected. Indeed, it was found that the bulk of tests employing ts 34, whether as preinfecting or superinfecting mut,ant, showed complementation near 100 %. The fact that most mutants showed a ceiling complementation of around 100% thus suggested that lower values were often spurious, and only consistently low degrees of complementation are indicative of true complementation groupings (Tables 3 and 4). A consistent pattern of complementation has emerged, but it has not proved possible to classify 3 out of 14 mutants. This may reflect true features of the ts lesion in these mutants, or it may just be that stocks of these mutants tend to contain a higher than usual proportion of defective viruses. If virus is collected from cloned transformed cells the proportion of defective particles is probably reduced (Vogt, personal communication). Virus obtained in this way may thus enable more definitive complementation tests to be performed on these unclassified viruses. The class T mutants all replicate competently at the nonpermissive temperature, and thus their ts lesions involve only transforming functions (Wyke, 1973). Although they form a homogeneous group, certain differences have been detected (Wyke and Linial, 1973), and these show a partial correlation with the complementation groups defined here. Thus, the plaque-supporting mut.ants ts 25 and Is 28 are both in group I, though

COMPLEMENTATION

the other 3 members of this group do not support plaques. Ts 23 and ts 35, comprising group II, are linked by their greater than usual virion heat stability. In addition, the 2 C class mutants tested, ts 335 and ts 338, show patterns of complementation which correspond to their distinctive physiological behavior, thus confirming their difference both from one another and the T class mutants. The complexity and organization of the RSV genome is uncertain, and Vogt (1973) has presented arguments favouring 2 models. The genome may bo polyploid, comprising 3 to 4 identical RNA molecules of about 3 X lo6 molecular weight. On the other hand, it may be a unique sequence of about 10’ MW, which is split into several segments, only one of which is concerned with maintenance of in vitro transformation. The existence of at least 4 complementation groups concerned with transformation alone would seem to favour the larger genome implied in the second model. However, although these groups show strong reciprocal complcmentation, it would be premature to assume t,hat they involve lesions in different proteins. Intragenic complemnntation is possible, as a protein may contain more than one active site. To investigate these possibilit’ics, attempts are being made to study recombination bet,wcen tjhcse mutants, and also t.o achieve a further physiological differentiation of the class T mutant’s. The failure to observe complementation bct,wecn the PR-RSV mutants studied here and t,he B-77 and Schmidt-Ruppin RSV mutants was disappointing. If this is because analogous proteins in these different strains cannot adequately complement one another, then it will be difficult to compare the 4 complcmentation groups defined here with the 2 groups described by Kawai et al. (1972) for Schmidt-Ruppin strain RSV. This failure also cast doubt on previous observations that ts mutants were not complemented by avian leukosis viruses (E’riis et al., 1971; Wylie and Linial, 1973), since t,hc leukosis viruses were oft.en of different strains. It would t’hus be interesting to SCPwhether ts mut’ant’s could be complemented by various transformation-defect,ivc mut#ants of their own strain.

OF AS\,’ Is MUTANTS

3.5

ACKNOWLEDGMENTS I am indebted t,o Drs. It. 1~. Friis, Maxine Linial, W. S. Masoll, I’. K. Vogt,, and It. A. Weiss for helpful discussions during t,he course of this work. Dr. Vogt also provided valuable advice during t,he preparat,ion of this manuscript. I should also like to thank Sophie Yap for conscientious technical assistance and the Horserace Betting Levy Board, London, for the award of a travel grxlit.

.J. 1’. (1972). Temperature-dependent transformation of cells infected with a mutant of Bryan ROIIS sarcoma virus. .J. F’iroZ. IO,

BADWR,

267-276. DLWF, 1~. (:., and VOGT, I’. K. (1969). Character-

ist,ics of t,wo new avian t,umor virus subgroups. vil-olog~/ 40, 102%1029. FRIIS, Il. Ii., TOYOSHIMA, R., and VOGT, P. K. (1971). Conditional lethal mutants of avian sarcoma. viruses. I. Physiology of Is 75 and 2s 149. I'iro2ogy

43, 3755389.

T. (1971). A plaque assay for avian RN,4 tumor viruses. T’irolog?/ 50, 567-57s. KAWAI, S., :md HANAFGSA, H. (197%). Plaque assay for some strains of avian leukosis virus. GRAF,

Vil-OlOg~/ 48, la- 135. KAWAI. s., :tud HANAFUSA,

recombination

H. (197%). Genetic with avian tumor virus. Virol-

ugy 49, 37-14. s., PVIB.TROKA, c. E., aIld HANAFUSA, H. (1972). Complemcrrtation of functions required for cell transformation by double infection with l{SV mutants. Virology 49, 30“-304. LINIAI,, RI., and Iblaso~, W. 8. (19i3). Characterizat ion of two condit,ional early mutants of llous sarcoma virus. Virology, in press. RU~JIN, H. (1960). .4 virus in chick embryos which induces resista~~cc i/l. oitro to infection with 1~011ssarcoma virus. I’roc. Nnl. Bcud. Sci. T’.S.

KAU-AI,

46, 1105..1119.

F. T., and RUBIN, H. (1966). The mechanism of interference between an avian leukosis virus and Ror~s sarcoma virus. l’irology 29, 628-611. ToYosHIMI, K., and VOOT, P. K. (1969a). Enhancement and inhibition of avian sarcoma viruses by polycations and polyanions. i’irology 38, 41il-426. TOY~SHIMA, K., and VOGT, P. Ii. (1969b). Temperat ure set&tive mut,ants of an avian sarcoma virus. I~irology 39, 930.-931. VOGT, I’. K. (1969). Focus assay of Rous sarcoma virus. III, “Fundamental Techniques in Virology” (K. Habel a.nd N. P. Salzman, eds.), pp. 198-211. Academic Press, New York.

STWK,

36 VOGT, P. K. (1971a). Spontaneous segregation

WYKE

of nontransforming viruses from cloned sarcoma viruses. Virology 46, 939-945. VOGT, P. K. (1971b). Genetically stable reassortment of markers during mixed infect,ion with avian tumor viruses. Virology 46, 947-952. VOGT, P. K. (1973). The genome of avian RNA tumor viruses: a discussion of four models. “Possible Episomes in Eukaryotes” (L. G. Silvestri, ed.), Proceeding 4th Lepet,it Colloquium North-Holland Publ., Amsterdam. VOQT, P. K., and ISHIZAKI, Ii. (1965). Reciprocal patterns of genetic resistance to avian tumor

viruses in two lines of chickens. Virology 26, 664-672. WEISS, R. A., MASON, W. S., and VOGT, P. K. (1973). Genetic recombination between endogenous and exogenous avian RNA tumor viruses. Virology, in press. WYKE, J. A. (1973). The selective isolation of temperature-sensitive mutants of Rous sarcoma virus. Virology, in press. WYKE, J. A., and LINIAL, M. (1973). Temperature-sensitive avian sarcoma viruses: a physiological comparison of twenty mutants. VifoZogy, in press.