Tumor antigens induced by nontransforming mutants of polyoma virus

Cell, Vol. 15.485-496,

October

1976,

Copyright

8 1978 by MIT

Tumor Antigens Induced by Nontransforming Mutants of Polyoma Virus Jonathan Silver,*t Brian Schaffhausent Thomas Benjamint

and

Department of Hematology Beth Israel Hospital Boston, Massachusetts 02115 T Department of Pathology Harvard Medical School Boston, Massachusetts 02115 l

Summary We have studied the tumor (T) antigens induced by wild-type polyoma virus and several nontransforming mutants using lmmunoprecipitation with antisera from animals bearing polyoma-induced tumors followed by sodium dodecylsulfate (SDS)polyacrylamide gel electrophoresis. In a variety of mouse cells, wild-type virus induces a major T antigen species with apparent molecular weight of 100,000 daltons, and four minor T antigen species with apparent molecular weights of 63,000, 56,000, 36,000 and 22,000 daltons. Hr-t mutants, which have an absolute defect in transformation, induce a normal 100,000 dalton T antigen but are altered in the minor T antigen species. Hr-t deletion mutants Induce none of the minor T antigen species seen in wild-type virus. In their place, these mutants induce T antigen species with molecular weights in the range of 6,000-9,000 daltons. The size of the very small T antigen products does not correlate in any simple way with the size or location of the deletions in the viral DNA. Point hr-t mutants induce two of the four minor T antigen species; they make apparently normal amounts of the 56,000 dalton product and reduced amounts of the 22,000 dalton product, but none of the 63,000 or 36,000 dalton species. Ts-a mutants, which have a temperaturesensitive defect In the ability to induce stable transformation, and which complement hr-t mutants, induce T antigens with the same mobility as wild-type; however, the 100,000 daiton T antigen of ts-a mutants is thermolabile compared to wild-type. A double mutant virus carrying both a ts-a mutation and a deletion hr-t mutation induces a thermolabile 100,OO dalton product and none of the minor T antigen species. Ceil fractionation studles with productively infected cells have been carried out to localize the T antigen species. Introduction Cells transformed or infected by papovaviruses acquire new antigens recognized by antisera raised in animals bearing virus-induced tumors. These antigens, called tumor (T) antigens, were first rec-

ognized by complement fixation of whole cell extracts (Black et al., 1963; Habel, 1965) and by immunofluorescence of nuclei of infected or transformed cells (Pope and Rowe, 1964). In an effort to identify viral proteins involved in transformation, we have compared T antigen species induced by wild-type polyoma virus and several nontransforming viral mutants. Two classes of transformation defective mutants have been identified for polyoma virus, defined by the ts-a and hr-t complementation groups (Eckhart, 1977; Fluck, Staneloni and Benjamin, 1977). Hr-t mutants have an altered host range for lytic growth (Benjamin, 1970; Goldman and Benjamin, 1975; Staneloni, Fluck and Benjamin, 1977) and are defective in abortive and stable transformation (Benjamin, 1970; Benjamin and Norkin, 1972; Staneloni. et al., 1977). They induce normal T antigen immunofluorescence (Staneloni et al., 1977). Marker rescue experiments show that hr-t mutations map in the proximal part of the early region of the viral DNA (Feunteun et al., 1976). Ts-a mutants have a temperature-sensitive defect in viral DNA replication (Francke and Eckhart, 1973) and in the induction of stable transformation (Fried, 1965; Eckhart, 1969; DiMayorca et al., 1969). They are capable of abortive transformation at the high temperature (Stoker and Dulbecco, 1969), however, and cells transformed at the low temperature may remain transformed when shifted to the high temperature (Fried, 1965; Eckhart, 1969; DiMayorca et al., 1969; Vogt, 1970; Folk, 1973; Seif and Cuzin, 1977; M. Fluck and T. Benjamin, manuscript in preparation). At least some ts-a mutants are defective in T antigen immunofluorescence at the high temperature (Oxman, Takemoto and Eckhart, 1972), and T antigen activity by complement fixation ;s thermolabile in extracts of ts-a-transformed cells (Paulin and Cuzin, 1975). Marker rescue experiments show that ts-a mutations map in the distal part of the early region (Miller and Fried, 1976; Feunteun et al., 1976). Recent experiments have shown that anti-polyoma tumor sera recognize a series of polypeptides in lytically infected and transformed cells (Paulin, Perreau and Cuzin, 1974; Paulin, Gaudray and Cuzin, 1975; Ito, Spurr and Dulbecco, 1977a). We have identified a major T antigen species in lytically infected cells which has an apparent molecular weight of 100,000 daltons by SDS-polyacrylamide gel electrophoresis and 81,000 daltons by guanidine Sepharose chromatography (Schaffhausen, Silver and Benjamin, 1978). Although the latter molecular weight estimate is likely to be more accurate, in this paper we will refer to the large T antigen as the 100,000 dalton species since all other molecular weight estimates are based on

Cell 466

mobility in SDS-polyacrylamide gels. Additional species with apparent molecular weights of 63,000, 56,000, 36,000 and 22,000 daltons were seen in wild-type-infected cells, while only the 100,000 dalton species was seen in cells infected by the hr-t mutant NG-18 (Schaffhausen et al., 1978). Ts-a mutants have been reported to induce a thermolabile 100,000 dalton species (Ito et al., 1977a). In this report, we extend the observations concerning the T antigen species induced by nontransforming mutants. Nine independently isolated hr-t mutants, including both point and deletion mutations, and two ts-a mutants were analyzed for their patterns of T antigens in lytic infection. Preliminary experiments concerning the intracellular localization of the different T antigen species’are also presented.

Results T Antigen Species Induced by Wild-Type Virus Five mouse cell lines were infected with wild type polyoma, labeled with 35S-methionine, lysed with NP-40 and immune-precipitated using Staphylococcus aureus protein A, and the immune precipitates were analyzed by SDS-polyacrylamide gel electrophoresis (see Experimental Procedures). The results shown in Figure 1 indicate that the pattern of T antigen species is essentially the same in all cell lines tested. For each cell line, the major virus-specific band has an apparent molecular weight of 100,000 daltons. Smaller amounts of T antigen species with molecular weights of 63,000, 56,000, 36,000 and 22,000 daltons are also seen. These proteins are not immune-precipitated from mock-infected or infected cells when either pre-immune serum or serum against SV40 T antigens is used as a control. The relative amounts of the smaller T antigen species vary somewhat from experiment to experiment. In some experiments, the 36,000 dalton band is much less intense than the other virus-specific bands. Some variation is also seen in the separation of the 63,000 and 56,000 dalton species on different gels. In some gels, the 36,000 dalton product is seen to migrate just ahead of a cellular protein. The same five virus-specific products are precipitated with three different preparations of anti-tumor serum, two from rat and one from hamster. Some sera precipitate relatively less of the 63,000 dalton product compared to the other bands. Some sera also precipitate large amounts of a nonvirusspecific product with an apparent molecular weight of - 40,000 daltons. This product is probably actin, since a band with the same mobility is precipitated with anti-actin serum plus Staphylococcus aureus

protein A (data not shown). Since anti-actin serum does not bring down other products from infected cells, it is improbable that any of the T antigen species are cellular proteins which precipitate by virtue of interaction with actin, or that actin is precipitated by virtue of interaction with T antigen species. The same five T antigen products are seen with several wild-type stocks. The pattern of T antigens is the same whether or not the stocks contain a significant proportion of defective particles as determined by restriction endonuclease mapping. Thus it is improbable that any of the minor T antigen species are translation products from defective viruses. Several experiments were carried out to test the possibility that some of the T antigen species arise by proteolytic cleavage. The same five T antigen products are present after pulses as short as 10 min. These bands are seen at least as early as 18 hr after infection at 37X, before there is detectable cytopathic effect. Protease inhibitors did not alter the pattern of T antigen products, either when added to the extraction buffer (phenyl-methylsulfonylfluoride, 200 pg/ml) or when present in the culture fluids 20 min before and during a 1 hr labeling period (leupeptin, 3.8 pg/ml; chymostatin, 3.4 pg/ml; antipain, 4.1 pg/ml; pepstatin, 2 x lo-’ M; elastinal, 5 x 1O-g M). Canavanine (3.2 mM), which has been shown to block proteolytic processing of the polio virus precursor protein (Jacobson, Asso and Baltimore, 1970), also failed to alter the pattern of T antigens. Although these experiments offer no evidence that the minor T antigen species arise by proteolytic processing, rapid cleavage of a nascent polypeptide precursor cannot be ruled out.

T Antigen Species Induced by Hr-t Mutants NG-18 is an hr-t mutant with a deletion of approximately 3.7% of the genome in the proximal part of the early region in Hpa II fragment 4. Two other hrt mutants, NG-59 and 3A-1, were found to have no deletion in this region of the genome (Staneloni et al., 1977). Since all three of the mutations map in Hpa II fragment 4 by marker rescue (Feunteun et al., 1976), NG-59 and 3A-1 most probably have point mutations in Hpa II fragment 4, although very small deletions and/or substitutions cannot be ruled out. The DNAs of several additional hr-t mutants have been analyzed using standard restriction enzyme mapping techniques (J. Hattori, G. Carmicheal, J. Silver and T. Benjamin, unpublished results). As shown in Figure 2, seven of the mutants have deletions in the proximal half of Hpa II fragment 4. These deletions vary in size from 2.4-4.7% of the genome. One of the mutants, SD-

T Antigens 407

Figure

of Nontransforming

1. Wild-Type

Polyoma

Mutants

T Antigens

of Polyoma

in Different

Mouse

Cell Lines

Confluent cells were infected with wild-type polyoma at a multiplicity of 5-10 plaque forming units (pfu) per cell and labeled with Yjmethionine (20 &i/ml) from 17-27 hr after infection. Extracts from - 5 x IO’ infected cells were immune-precipitated with (a) anti-T ascites or (b) control serum. Control extracts from uninfected cells were immune-precipitated with (c) anti-T ascites. (1) BALB/c 3T3, (2) A31, (3) 3T3d, (4) ME, (5) BMK, (6) NIH-3T3. Molecular weight estimates in thousands of daltons are shown at left.

15, is also altered in several restriction enzyme sites in the distal half of the Hpa II fragment 4. The host range selection procedure for isolation of hr-t mutants therefore leads with high probability to the selection of mutants with alterations in Hpa II fragment 4. The T antigen products induced by these hr-t mutants are shown in Figure 3. All the hr-t mutants induce a 100,000 dalton T antigen. The “point” mutants NG-59 and 3A-1 also induce apparently normal amounts of the 56,000 dalton product and reduced amounts of the 22,000 dalton product. Another “point” mutant, HA33, also shows this pattern (not shown). The 56,000 and 22,000 dalton bands are not seen with the deletion mutants. None of the hr-t mutants, points or deletions, make detectable amounts of the 63,000 or 36,000 dalton species. Several new virus-specific bands appear in the deletion mutants. All the deletion mutants (including NG-16, not shown) induce small

amounts of T antigen species in the molecular weight range of 6,000 to 9,000 daltons. In addition, SD-15 induces a - 50,000 dalton product. It should be noted that in the lower molecular weight region of the gel (< - 12,000 daltons), the curve of logarithm of molecular weight of standard proteins versus their mobility becomes much steeper. We have calibrated this region of the gels with myoglobin (17,200), lysozyme (14,400), cytochrome c (11,600), parathyroid hormone (9,500), pancreatic trypsin inhibitor (6,200) and insulin (3,100 and 2,700 daltons). The sizes of the very small T antigen products do not correlate in any simple way with the size or position of the deletions in DNA. For example, the same sized product is induced by the largest deletion (82, lane 13), the smallest deletion (11-5,lane 7) and an intermediate sized deletion (3A-4, lane 11). These deletions begin at different points and end at different points within Hpa II fragment 4. These

cell 488

5’-

Figure

*

Early mRNA

2. Size and Location

of Deletion

in Hr-t Deletion

Mutants

Numbered segments refer to Hpa II restriction fragments. Map coordinates of the Hpa II 5-4 junction and 4-8 junction are shown (Fried and Griffin, 1977). (ori) Origin of replication. The mapping of early RNA is from Kamen et al. (1974). Numbers in brackets are estimated sizes of the deletions as percentages of total genome length. Dotted lines indicate regions within which deletions lie. Solid lines indicate more precise locations of deletions, where this is known. Mutant SD-15 has additional altered restriction enzyme sites in the distal half of Hpa II fragment 4 (see text).

Figure

3. T Antigens

Induced

observations argue against the possibility that the 6,000-9,000 dalton species arise as primary translation products from premature chain termination of out-of-phase deletions. In such a situation, the appearance of identical sized’ products in these three mutants would be unexpected. Some mutants, such as SD-15 (lane 10) and 68-5 (lane 12), induce more than one very small T antigen product. In very long exposures, trace amounts of similarly sized products may be seen in extracts from wildtype-infected cells. Taken together, these observations suggest that the very small T antigens are not primary translation products, but probably represent intermediates in the degradation of larger T antigen species. The patterns of T antigens induced by hr-t mutants are unaffected by the degree of permissivity of the host cell for growth of these mutants. Thus the same results are obtained in baby mouse kidney (BMK) cells, which are permissive for

by Hr-t Mutants

Confluent NIH-3T3 cells were infected with wild-type virus or hr-t mutant at a multiplicity of I-10 pfu per cell and labeled for 45 min with Y9-methionine (100 &i/ml) beginning at 22 hr after infection. Controls: extracts from uninfected cells were precipitated with (1) control serum or (2) anti-tumor ascites; extracts from wild-type-infected cells were precipitated with (3) control serum or (4) anti-T ascites. Extracts from hr-t mutant-infected cells were precipitated with anti-tumor ascites. Point mutants: (5) 3A-1, (6) NG-59. Deletion mutants, in order of increasing size of deletion: (7) 11-5, (8) A-8, (9) NG-23, (10) SD-15, (11) 3A-4, (12) 66-5 and (13) 82. Arrow indicates position of - 40,000 dalton band thought to be actin (see text).

T Antigens 489

of Nontransforming

Mutants

of Polyoma

growth of hr-t mutants (Goldman and Benjamin, 1975), and in nonpermissive NIH-3T3 cells. Permissivity for hr-t mutants is therefore not due to the induction of the normal pattern of minor T antigen species seen in wild-type infections. In double infections with wild-type virus and hr-t mutants, the hr-t mutants have been observed to exert a partial “dominant lethal” effect, evidenced by a reduction in virus yield in lytic infections or a reduction in the frequency of transformation in nonproductive infections (Fluck et al., 1977). When BMK cells are doubly infected with wild-type virus and either NG-18 or NG-59, there is a moderate but reproducible decrease in the amount of the minor T antigen species in which the mutant is deficient, compared to cells infected at the same multiplicity with wild-type virus alone. These results are shown in Figure 4. The same results were obtained in NIH3T3 cells. This suggests that the “dominant lethal” effect could be due to partial interference with the production of the minor T antigen products seen in wild-type infections. 1 Antigen Species Induced by Ts-a Mutants We examined the effect of different ts-a mutations on the pattern of T antigens in pulse-chase experiments. NIH-3T3 cells were grown at 32°C for 24 hr after infection with wild-type virus or ts-25D, a ts-a class mutant. After labeling for 10 hr at 32°C with 35S-methionine, the cells were harvested or washed and chased for an additional 10 hr at 39°C in nonradioactive medium containing ten times the normal concentration of methionine. Under these conditions, the 100,000 dalton T antigen of ts-25Dinfected cells turns over more rapidly than that of wild-type infected cells, as shown in Figure 5. In contrast, the 63,000 and 36,000 dalton products appear to be stable or even to increase slightly during the chase. While these results might suggest that the 100,000 dalton product is a precursor of the 63,000 and 36,000 dalton species, stability or enhancement of the 63,000 and 36,000 dalton products are not consistently observed in other cell lines. The 56,000 and 22,000 dalton products are quite labile under these conditions with both wildtype and ts-25D. In shorter chases, no evidence has been obtained for enhanced lability of these products in ts-a or hr-t point mutants compared to wild-type infections. The same results have been obtained with ts-616, another ts-a mutant. Similar pulse-chase experiments were carried out with NG-18, ts-25D and a double mutant carrying the NG-18 deletion and the ts-25D mutation (Feunteun et al., 1976). Infected cells were grown for 40 hr at 32°C and labeled for 1 hr at 32°C with 35S-methionine. The cells were then harvested or chased for 4 hr at 32 or 39°C. The results are shown

Figure 4. T Antigens in Cells Doubly Infected with Wild-Type Virus and Hr-t Mutants BMK cells were infected with wild-type virus, NG-19, NG-59 or wild-type virus plus NG-18 or NG-59. Multiplicity of infection was - 5 pfu per cell for each virus in both single and double infections (total multiplicity - 10 pfu per cell in double infections). Cells were labeled with +S-methionine (20 $Xml) from 12-24 hr after infection. Immune precipitates with anti-T ascites from cells infected with: (1) wild-type virus alone, (2) wild-type virus plus NG-19, (3) NG-19 alone, (4) wild-type virus alone, (5) wild-type virus plus NG-59. (6) NG-59 alone.

in Figure 6. The 100,000 dalton T antigen of NG-18 is stable at 32 and 39X, while the 100,000 dalton product of ts-25D is stable at 32°C but labile at 39°C. None of the minor T antigen species are seen in NG-18 infected cells, even after the high temperature chase. The double mutant shows the defects of both parental mutants: that is, absence of the minor T antigen species and thermolability of the 100,000 dalton product. Disappearance of the 100,000 dalton band of the double mutant during the chase is not accompanied by appearance of 63,000 and 36,000 dalton products. In this experiment, little 36,000 dalton product is

Cell 490

Figure 6. T Antigens Both an Hr-t Deletion

Induced by a Double and a Ts-a Mutation

Mutant

Containing

Confluent NW3T3 cells were infected with NG-16, ts25D or a double mutant carrying the NG-16 deletion and the ts-25D mutation. Cells were grown at 32°C for 46 hr. pulsed for 1 hr at 32°C with %-methionine (100 &i/ml) and then harvested or chased for 4 hr at 32 or 3QC. Extracts were precipitated with anti-T ascites. Cells harvested before the chase: (1) NG-16, (2) double mutant, (3) ts-25D. Cells harvested after the chase at 32°C: (4) NG16, (5) double mutant, (6) ts-25D. Cells harvested after the chase at 39°C: (7) NG-16, (6) double mutant, (9) ts-25D. Panels 1-3 were exposed for 1 day; panels 4-Q were exposed for 3 days. Arrow indicates position of - 46,000 dalton band thought to be VP-l (see text).

Figure 5. Thermolability Ts-a Mutant

of T Antigens

in Cells

Infected

with

a

Subconfluent NIH-3T3 cells were infected with wild-type polyoma or ts-25D at a multiplicity of 5 pfu per cell. After 24 hr at 32”C, cells were labeled with %-methionine (56 &i/ml) for IO hr at 32°C and then (a) harvested or (b) chased for an additional 10 hr at 39°C. Extracts were precipitated with anti-T ascites. (1) Mockinfected cells, (2) wild-type-infected cells, (3) ts-25D-infected cells.

seen with ts-25D after the high temperature chase. This could be related to the fact that cells were harvested late in infection when there was extensive cytopathic effect and possibly increased proteolysis. An additional band with apparent molecular weight of - 48,000 daftons is also seen in the ts-a- and NG-1Sinfected cells. This probably represents VP1 , which occasionally appears in immunoprecipitates from cells harvested late in infection even when pre-immune serum is used.

Intracellular Several different

Localization

experiments T antigen

of T Antigen Species

were performed to localize species in lytically infected

cells. In one approach, wild-type-infected cells were lysed in petri dishes with NP-40 in a buffer containing sucrose and divalent cations (TSCM buffer, see legend to Figure 7). This buffer is reported not to extract most proteins from rat liver nuclei (Kellermayer et al., 1974) and not to extract SV40 T antigen from nuclei of SV40-infected and transformed cells (Kellermayer et al., 1976). As shown in Figure 7, TSCM buffer solubilized the minor T antigen species of polyoma but only small amounts of the 100,000 dalton product. Nuclei, which remained adherent to the petri dishes after extraction, were reextracted with a buffer containing 0.15 M KCI instead of sucrose (TKCM buffer). This modified buffer is reported to soubilize many rat liver nuclear proteins (Kellermayer et al., 1974) as well as SV40 T antigen (Kellermayer et al., 1976). As shown in Figure 7, TKCM buffer solubilizes the polyoma 100,000 dalton product. This suggests that the large T antigen is nuclear while the smaller T antigen products may be cytoplasmic, membrane-associated, or readily extractable from nuclei in low salt buffer. In another approach, wild-type-infected cells were harvested 24 hr after infection and broken by shearing in a Dounce homogenizer. Nuclei were spun out at low speed and membranes were separated from cytosol by higher speed centrifugation. In this crude factionation, the nuclei are visibly contaminated by adherent cytoplasmic tags. The

T Antigens

of Nontransforming

Mutants

of Polyoma

491

Figure

6. Intracellular

Localization

of T Antigen

Species

Subconfluent NIH cells were infected with wild-type polyoma at a multiplicity of 10 pfu per cell, labeled with YGmethionine (50 r&i/ml) from 22-24 hr after infection and harvested by gentle pipetting in phosphate-buffered saline containing 0.5 mM EDTA. Cells were washed and suspended (- 6 x 1V cells/ml) in hypotonic buffer [lo mM Tris-HCI (pH 7.4), 10 mM NaCl, 1.5 mM MgCI,]. After swelling for 10 min at 4”C, the cells were broken with a Dounce homogenizer until - 95% of cells were disrupted as judged by phase microscopy. Nuclei were removed by centrifugation at 2900 x g for 2 min. The supernatant was centrifuged at 10,000 x g for 10 min to pellet membranes. The low speed pellet (“nuclear fraction”) and high speed pellet (“membrane fraction”) were extracted in lysing buffer (see Experimental Procedures). The high speed supernatant (“cytosol fraction”) was modified by the addition of salts, NP-40 and glycerol to match the concentrations in the lysing buffer. Extracts were immunoprecipitated with (a) preimmune serum followed by (b) anti-T ascites. (1) Whole cells, (2) cytosol fraction, (3) membrane fraction, (4) nuclear fraction.

Figure

7. Differential

Extraction

of T Antigen

Species

NIH-3T3 cells infected with wild-type polyoma were labeled from 21-25 hr after infection with Yr-methionine (20 &i/ml). Cells were washed with phosphate-buffered saline and extracted for 20 min at 4°C with 1 ml per 90 mm petri dish of TSCM buffer [IO mM Tris-HCI (pH 7.2), 250 mM sucrose, 3.7 mM CaCI,, 12 mM MgCI,, 1% NP-401. The supernatant was removed and the nuclei were reextracted for 20 min at 4°C with 1 ml of TKCM buffer 110 mM Tris-HCI (pH 7.2). 150 mM KCI, 3.7 mM CaCI,, 12 mM MgCI,, 1% NP-401. Extracts were immunoprecipitated with control serum followed by anti-T ascites. TSCM extract: (1) pre-immune serum, (2) anti-T ascites. TKCM extract: (3) anti-T ascites, (4) pre-immune serum.

precipitated. The results are shown in Figure 8. The nuclear fraction is enriched for the 100,000 dalton product but also contains some 56,000 dalton product. The membrane fraction contains mainly the 56,000 dalton product, with small amounts of the 100,000 and 63,000 dalton species. The cytosol contains all the T antigen species, but is relatively depleted in 100,000 and 56,000 dalton products. These results suggest that the 56,000 dalton product is associated with membranes, while the other minor T antigen species are cytoplasmic or released from nuclei during the cell fractionation. The 56,000 dalton T antigen is also found in the membrane fraction of cells infected with the point hr-t mutant NG-59, but not with the deletion hr-t mutant NG-18.

Discussion cytosol fraction may also contain material solubilized from nuclei and the crude membrane preparation may be contaminated with nuclear debris. Each cellular fraction was extracted and immuno-

Wild-type polyoma virus induces at least five T antigen species in lytically infected cells. These have apparent molecular weights of 100,000,

Cell 492

63,000, 56,000, 36,000 and 22,000 daltons on SDSpolyacrylamide gels. Since the coding capacity of the early region of the virus is only about 100,000 daltons in a single reading frame, these polypeptides cannot all be primary viral gene products unless the genome is read in multiple frames or there is extensive RNA splicing to generate different polypeptides from the same coding sequences. It is therefore important to determine which of the T antigen species are virally encoded, which are primary gene products and where on the genome their coding sequences lie. SV40, a virus structurally and biologically similar to polyoma (Fried and Griffin, 1977), is reported to induce two T antigen species in lytic infection: a large T antigen with molecular weight of - 90,000 daltons (Carroll and Smith, 1976; Ahmed-Zadeh et al., 1976; Rundell et al., 1977) and a small T antigen with molecular weight of - 17,000 daltons (Prives et al., 1977). A third T antigen with molecular weight of - 60,000 daltons has been observed in some SV40-transformed cells (Lichaa and Niesor, 1977; Griffin, Light and Livingston, 1978). It has been proposed that the coding sequences for the large and small SV40 T antigens overlap in an unusual way: the small T antigen is encoded by the sequence proximal to and including the 0.59-0.54 region, while the large T antigen is encoded by noncontiguous sequences lying proximal to 0.59 and distal to 0.54 (Berk and Sharp, 1978; Crawford et al., 1978; Fiers et al., 1978; Reddy et al., 1978). Whether the middle sized T antigen is virally encoded has not been determined. Table 1 summarizes the T antigen species induced by different point and deletion mutants of polyoma virus. Ts-a mutants induce a thermolabile 100,000 dalton product, indicating that this major T antigen is a product of the ts-a gene. The assignment of the 100,000 dalton product to the ts-a gene is also consistent with the production of this protein by all hr-t mutants and the ability of these mutants to complement the growth and transforTable

1. T Antigen

Species

Molecular Weight (Daltons)

of Wild-Type

Polyoma

Virus,

mation defects of ts-a mutants. In SV40 the large T antigen is thought to be the product of the analogous ts-A gene, since it can be synthesized in vitro from viral-specific RNA (Prives et al., 1977) and since the partially purified product from ts-A-infected cells is thermolabile compared to the wildtype product in complement fixation assays and in DNA binding (Tenen et al., 1977). Since hr-t mutants with deletions in the proximal part of the early region induce a normal sized 100,000 dalton T antigen product, the hr-t region of polyoma must be excluded from the coding region for the large T antigen (Schaffhausen et al., 1978). This parallels the situation found in SV40, in which similarly placed deletions do not alter the size of the large SV40 T antigen (Crawford et al., 1978). The amounts of the 63,000 and 36,000 dalton products appear to increase during a high temperature chase, particularly in ts-a mutant-infected cells in which the 100,000 dalton T antigen disappears. This result raises the possibility that the 63,000 and 36,000 dalton species are not primary gene products but rather derivatives from the 100,000 dalton T antigen. Hr-t mutants fail to induce the 63,000 and 36,000 dalton species. An hrt&-a double mutant which induces a thermolabile 100,000 dalton product also fails to induce these species. Thus if the 63,000 and 36,000 dalton products do derive from the 100,000 dalton T antigen, a functional hr-t gene may be required for this conversion. Other evidence, however, fails to support a precursor-product relationship. The 63,000 and 36,000 dalton products are present after pulses as short as 10 min, and protease inhibitors do not block their appearance. Furthermore, in mixing experiments, extracts from wildtype-infected cells do not generate 63,000 and 36,000 dalton products from the 100,000 dalton T antigen of NG-18-infected cells (Schaffhausen et al., 1978). If the 63,000 and 36,000 dalton species do not

Hr-t and Ts-a Mutants

Wild-Type

Hr-t deletion Mutantsa

Hr-t Point Mutants

100,M)o

+

+

+

63,WQ

+

56,ooO

+

36,ooO

+

22,c#6

+

B These mutants induce -5O.ooO dalton product. b Thermolabile. c Reduced amounts.

Tea Mutants

Double Mutant (NG-lB/Ts25D)

+b

+b -

+ +

+

+= additional

T antigen

species

with

molecular

weights

-6QoO-QCNM

+

-

+

-

daltons.

One

mutant,

SD-15

also

induces

a

T Antigens 493

of Nontransforming

Mutants

of Polyoma

derive from post-translational processing but are primary products, their absence in the hr-t mutants could be explained by at least three different mechanisms. First, they are encoded by the hr-t region, and the altered products of hr-t mutants are either rapidly degraded or not recognized by the anti-T sera; second, they are the products of spliced mRNAs which are not produced by hr-t mutants; or third, they are cellular products not induced by hrt mutants. The last possibility is improbable because preliminary results from complete and partial proteolytic digestion indicate that the 63,000 and 36,000 dalton species are related to the 100,000 dalton T antigen (unpublished observations). The possibility that a splicing defect may account for the absence of these species in hr-t mutants is attractive for several reasons. A mutation interfering with the production of correctly spliced mRNAs could lead to the absence of several virally encoded species. Furthermore, one of the SV40 early mRNAs is spliced within the region corresponding to the hr-t mutations of polyoma (Berk and Sharp, 1978). The absence of the 56,000 and 22,000 dalton species in hr-t deletion mutant-infected cells could be explained by the same mechanisms proposed for the absence of the 63,000 and 36,000 dalton species. Normal amounts of the 56,000 and reduced amounts of the 22,000 dalton products, however, are induced by point hr-t mutants. The simplest explanation for this observation is that these products are encoded by the hr-t region. In this case, the 56,000 and 22,000 dalton products induced by hr-t point mutants would be expected to be abnormal due to an amino acid substitution. The quantitative defect in the 22,000 dalton species of point hr-t mutants might then be explained by decreased affinity for antibody of the altered 22,000 dalton product or reduced amount of appropriately spliced mRNA. This possibility is being tested by comparison of the 56,000 and 22,000 dalton products of wild-type virus and hr-t point mutants, using isoelectric focusing and tryptic peptide analysis. Despite the differences in T antigen species induced by point and deletion hr-t mutants, no biological differences between these mutants have so far been observed. In place of the minor T antigen species, hr-t deletion mutants induce new products with molecular weights in the range of 6,000 to 9,000 daltons. The small size of these proteins, as well as the failure of their size to correlate with the position or size of the deletions in viral DNA, suggests that they are breakdown products rather than primary translation products of genes bearing deletions. SV40 mutants with deletions in the 0.59-0.54 region also fail to induce the - 17,000 dalton T

antigen (Crawford et al., 1978), and in some cases induce smaller T antigen species (Feunteun et al., 1978; Sleigh et al., 1978). One hr-t mutant, SD-15, induces a 50,000 dalton T antigen product. This size is about what one would expect for a deleted 56,000 dalton product, assuming that the SD-15 deletion occurred in sequences which coded for the 56,000 dalton product and did not alter the reading frame. The absence of a T-antigen species of predictable size in other hr-t mutants may be due to frameshifts leading to premature chain termination. It is also possible that the appearance of the - 50,000 dalton T antigen product of SD-15 is related to the location of the deletion or to the additional sequence changes in the distal part of Hpa II fragment 4. Cell fractionation experiments suggest that the 100,000 dalton T antigen is nuclear, a finding consistent with the predominantly nuclear fluorescence of lytically infected and transformed cells (Pope and Rowe, 1964) and the ability of T antigen to bind DNA (Reed et al., 1975; Jesse11 et al., 1976). The other T antigen species, with the exception of the 56,000 dalton product, were found predominantly in the cytoplasm. These results were obtained in productively infected mouse cells and are therefore subject to artifacts resulting from nuclear membrane breakdown and other degradative processes occurring in lytic infection. More definitive experiments on intracellular localization of T antigen species will require an examination of transformed cells. The 56,000 dalton product was found within the membrane fraction. Ito, Brocklehurst and Dulbecco (1977b) also found a membrane-associated polyoma T antigen of approximately this molecular weight using a different cell fractionation procedure. Similarly sized membrane-associated SV40 T antigens have been identified in cells infected with adenovirus-SV40 hybrids that contain only the distal half of the early region of SV40 (Deppert and Walter, 1976). The same adenovirus-SV40 hybrids are capable of inducing SV40 tumor-specific transplantation antigen (TSTA) (Lewis and Rowe, 1973), indicating that the distal half of the SV40 early region encodes protein(s) with determinants of the SV40 TSTA. Although there is no direct evidence, it seems probable that the 56,000 dalton membraneassociated polyoma T antigen contains the TSTA recognized by effector cells mediating polyomaspecific transplantation immunity. If the organization of the early region of polyoma were the same as in SV40, then part of the coding sequences for the 56,000 dalton product would come from the distal half of the early region. Since the evidence discussed above suggests that the 56,000 dalton species is an hr-t gene product, the most plausible

Cell 494

hypothesis for the molecular origin of this T antigen is that it derives from both the hr-t region and the distal half of the early region. Mutations in the hr-t region of polyoma have complicated effects upon the patterns of the minor T antigen species. From the data presented here, one cannot determine which, if any, of the minor T antigen species are responsible for the biological effects ascribed to the hr-t gene. Further study of the hr-t mutants should prove useful in understanding the detailed organization and mode of expression of genes in the early region of polyoma, as well as help in understanding the mechanism of transformation. ExperImental

Procedures

Cells Primary cultures of baby mouse kidney (BMK) cells were prepared as described (Winocour, 1963). Mouse embryo (ME) cells were the second passage of primary mouse embryo cultures from Swiss mice (Charles River). 3T6 and 3T3 are lines established from NIH Swiss mice (Todaro and Green, 1963); 3T3d is a subclone of 3T3. NIH-3T3 is a line established from inbred Swiss mice (Jainchill, Aaronson and Todaro, 1969). BALB 3T3 is a line established from BALB/c mice (Aaronson and Todaro, 1966); A31 is a subclone of this line provided by G. Todaro.

Viruses Ts-a mutants included ts-25D (referred to as HA-25, DiMayorca et al., 1969) and ts-616 (Echkart, Dulbecco and Burger, 1971). Hr-t mutants included NG-16, NG-23, NG59 (Benjamin, 1970) and A6, 3A1, 3A4, 82, 68-5, II-5 and SD-15 (Staneloni et al., 1977). A double mutant constructed to contain the NG-16 and ts-25D mutations (Feuntaun et al., 1976) was used. Wild-type viruses were derived by marker rescue from hr-t and ts-a mutations (Feunteun et al., 1976).

Antlsera Anti-polyoma tumor sera (anti-T sera) came from Brown Norwegian (BN) rats (Microbiological Associates) bearing a syngeneic polyoma-induced tumor. The tumor was derived from PyB4, a polyoma-transformed BN fibroblast (Goldman and Benjamin, 1975). PyB4A is a cell line isolated from a mixed ascites-solid tumor induced by PyB4. When PyB4A is injected intraperitoneally into BN rats, it routinely induces a mixed ascites-solid tumor. The ascites has a titer of P 1:200 by nuclear immunofluorescence on polyma-infected NIH-3T3 cells, and gives the same results upon immunoprecipitation as anti-T serum. Additional anti-polyoma tumor sera were gifts from Y. Ito (derived from inbred rat) and K. Takemoto (derived from hamster).

Lsbellng

Condltlons

Cells were labeled with S6S-methionine (400-600 Ci/mM, New England Nuclear) in methionine-free Dulbecco’s modified Eagle’s medium (pulses >l hour) or Hanks’ balanced salt solution (pulses ~1 hour). In pulse-chase experiments, cells were washed with phosphate-buffered saline (PBS) and chased with Dulbecco’s modified Eagles medium containing 10 or 20 times the normal concentration of methionine.

1 Antlgen

Extraction

Cells were washed twice with cold PBS and rinsed with isotonic buffer containing 20 mM Tris-HCI (pH 9). To each 90 mm petri dish was added 0.6-l .O ml of lysing buffer [20 mM Tris-HCI (pH 9), 137 mM NaCI, 1 mM Car&, 1 mM MgCI,, 10% (v/v) glycerol, 1% (v/v) NP-40 detergent]. Cells were incubated for 20 min at 4°C.

The extract was clarified Microcentrifuge.

by spinning

for 10 min in an Eppendorf

Immunopreclpltstlon To each 1 ml of extract was added 10 A of anti-T serum or ascites, or 10 A of pre-immune serum, plus 40 A of a settled suspension of Staphylococcus aureus protein A coupled to Sepharose CL-4B beads (Pharmacia). The mixture was vortexed gently for 30 min in the cold. The Sepharose beads were then washed 3-4 times with washing buffer [lo0 mM Tris-HCI (pH 9), 500 mM LiCI] and eluted by heating to 90°C for 5 min in 39-50 A of sample buffer [lo mM Tris-HCI (pH 6.6) 5% 2-mercaptoethanol. 5% sodium dodecylsulfate, 0.003% bromophenol blue].

Gel Electrophoresls Immune precipitates were analyzed on discontinuous SDS-polyacrylamide gels (12.5% acrylamide) as described by Laemmli (1970). Molecular weight standards included 6-galactosidase (116,ooO). phosphorylase A (93,ooO), bovine serum albumin (69,000), gamma globulin heavy chain (53,000), ovalbumin (43,ooO), chymotrypsinogen (26.090). myoglobin (17,200), lysozyme (14,000), cytochrome c (11,600), parathyroid hormone (9,500), pancreatic trypsin inhibitor (6,200) and insulin (3.100 and 2,700 daltons). Gels were stained with 0.1% Coomassie brilliant blue in 50% methanol-7.5% acetic acid, destained in 5% methanol-10% acetic acid, prepared for fluorography as described by Bonner and Lasky (1974) and fluorographed at -70°C with Kodak XR-5 film.

Acknowledgments We thank Ann Chen and lngrid Lane for their expert technical assistance, Patricia Weliska for her assistance in typing the manuscript, and Drs. P. Libby and A. Goldberg for their gift of protease inhibitors. J. S. was the recipient of a National Cancer Institute Fellowship. This work has been supported by a grant from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received

June

27,1976;

revised

August

2,1976

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