A mutation increasing the size of the polyoma virion proteins, VP2 and VP3

VIROLOGY

109, 35-46

A Mutation WALTER “Tumor and

(1981)

Increasing

the Size of the Polyoma

Virion Proteins,

VP2 and VP3

ECKHART,” SUZANNE DELBRUCK,” PRESCOTT DEININGER,+ THEODORE FRIEDMANN,t AND TONY HUNTER”” Virology Laboratory, The Salk Institute, Post Of$ce Box 85800, San Diego, +Depatiment of Pediatrics, School of Medicine. University of Caljfornia, La Jolla, California !?ZOSS ilccepted

Sepfwn

her

California Sari Diego.

!?2138

I 1% 1980.

The ts48 mutant of polyoma virus carries a temperature-sensitive mutation (ts A) which affects the polyoma large T antigen, blocking viral DNA synthesis at the nonpermissive temperature. In addition, there are at least two other changes in the ts48 genome which affect virus-coded proteins. There is a single base change which abolishes an ZZpaII restriction enzyme cleavage site in the portion of the early region coding simultaneously for the polyoma medium and large T antigens. There is also a single base change in the late region of the genome which results in the synthesis of virion proteins, VP2 and VP3, larger than the correspondingwild type proteins. The change in the late region abolishes the normal termination &don for VP2 and VP3.

The corresponding virion proteins of SV40 have a similar arrangement of coding sequences on the SV40 genome, and have molecular weights of 39,700, 38,500, and 27,000, respectively (Fiers et al., 1978; Reddy et al., 1978). Little is known about the process of virion assembly, or about the role of the three proteins in the process. Temperature-sensitive mutations in VP1 result in defective assembly (Friedmann and Eckhart, 1974). Temperature-sensitive mutations in VP2 andior VP3 result in failure to express early functions (Cogen and Eckhart, 1977; Robb and Martin, 1972). Apparently VP2 is not absolutely required for assembly of SV40 virions because mutants lacking VP2 can still produce infectious virions (Cole et al., 1977). We describe here some unusual features of the genome of a polyoma temperaturesensitive mutant, ts48. The temperaturesensitive phenotype results from a tsA mutation in the early region of the genome, affecting the polyoma large T antigen, blocking viral DNA synthesis at the nonpermissive temperature (Francke and Eckhart, 1973; Hutchinson et al., 1978). The ts48 mutant contains at least two other changes in its genome: a single base change in the early region which abolishes an UnaII re-

INTRODUCTION

The polyoma virus genome encodes three virion proteins, VPl, VP2, and VP3. The arrangement of the coding regions for the three proteins has been deduced from a combination of genetic mapping, peptide analysis, mRNA characterization, and DNA nucleotide sequence analysis (reviews, Deininger et al., 1979; Soeda et al., 1980). The coding regions for the three proteins are in the late region of the polyoma genome, between 70 and 25.5 map units, counterclockwise (Griffin et al., 1974). The VP1 protein is encoded between 47.7 and 25.9 map units; VP2 and VP3 are encoded between 65.2 and 47.1 and 58.7 and 47.1 map units, counterclockwise, respectively, sharing common C terminal sequences (Gibson, 1974; Fey and Hirt, 1975; Hewick et al., 1975). The arrangement of the coding regions on the polyoma genome is shown in Fig. 1. The proteins are translated from mRNAs having leader sequences derived from the region around 65 map units (Flavell et al., 1979; Kamen et a.L., 1980). The molecular weights of the three proteins, predicted from the DNA nucleotide sequence, are: VPl, 42,834; VP2, 35,007; and VP3, 22,979 (Deininger et al., 1979). 35

36

ECKHART

ET AL

Sau3a WT

Hpa II WT

ls48

Is48

56-

6-

78-

HpaIl Sau3a

a ’ 5 ,

4

5 1

3

1

1

6

2

6

4

2 3

5

-VP2-VP3-

VPl-

FIG. 1. Comparison of restriction endonuclease fragments of ts48 and wild-type DNAs. ZY’P-labeled DNA was isolated from approximately lo7 cells as described in Materials and Methods. Of each preparation li60th was digested with 2 units of either HpaII or Sau 3a for 2.5 hr at 37”. The digests were resolved by electrophoresis on a 5% acrylamide gel as described in Materials and Methods. The gel was dried and exposed against NS5T X-ray film for 4 hr. The diagram below is a representation of the position on the polyoma genome, linearized at the EcoRI site, of the fragments generated by HpaII (upper scale) and Sau 3a digestion (bottom scale). The late region of the virus is also indicated, together with the coding regions for VPl, VP2, and VP3. The labeled bands appearing in submolar amounts differ from one preparation to another. We attribute this to contaminating cellular DNAs.

enzyme cleavage site between HpaII fragments 7 and 8, and a change in the late region resulting in the synthesis of VP2 and VP3 proteins larger than the norma1 wild type proteins. The alteration in the late region abolishes the normal termination codon for VP2 and VP3.

striction

MATERIALS

AND

METHODS

Cell culture a?bd virus infection. Cells were routinely grown on plastic tissue culture dishes in Dulbecco’s modified Eagle’s medium (DME) supplemented with 5-100/o calf serum. Polyoma virus stocks were

SIZE OF POLYOMA

grown in 3T6 cells. The large plaque wildtype strain, WS, was grown from a plaque isolated from the LP strain (Eckhart, 1969; Friedmann et al., 1978). The isolation and properties of the temperature-sensitive mutant, ts48, have been described previously (Eckhart, 1969, 19’74; Miller and Fried, 1976). Virus stocks were tested for the presence of defective genomes by digestion with the HpaII restriction enzyme. Virus infections were carried out by adding virus suspended in Tris-buffered saline (TBS) to cultures for 1 hr at 37”. After adsorption, fresh medium was added and incubation was continued at 32”. Viru.s labeling and purilfication. 3T6 cells infected with either wild-type virus or the ts48 mutant at 32” were radiolabeled 24-36 hr postinfection by removing the media, rinsing two times with TBS, and adding 5 ml per 9 cm plate DME made 10% in amino acids, 2% dialyzed calf serum, and containing 2.5 @Zi/ml of 15 ‘“C-labeled amino acids (NEN, NEC145 L-amino acid mix [‘4C(U)J). Radiolabeled virions were isolated from infected cell lysates by equilibrium centrifugation in cesium chloride. Details of this procedure are described by W. Gibson (1974). Radiolabeled infected cultures were fractionated into cytoplasm and nuclei using 0.5% NP40 in 0.05 M Tris (pH 7.0). One hundred microliters of solubilizing buffer [50 mM Tris (pH 7.0)], 2.8 M P-mercaptoethanol, 5% glycerol, 2% SDS, and 0.005% bromophenol blue were added to nuclear fractions and to pelleted virions and boiled for 10 min. Electrophoresis. Solubilized proteins of purified virions and in vitro translation products were analyzed by SDS-polyacrylamide slab electrophoresis as described previously (Gibson, 1974). The gels contained acrylamide cross-linked with N,N’-methylene-bis-acrylamide (Bis) at a ratio of 28.8: 0.735. The gels were stained and prepared for fluorography as described by Bonner and Laskey (1974). Analysis of “2P-labeled DNA restriction fragments was done by separation on 5% acrylamide 0.25% Bis acrylamide slab gels made 25% with glycerol in Tris borate buffer (0.089 A4 Trizma base, 0.089 M boric acid, lo-” M EDTA, pH 8.2).

VIRION

PROTEINS

37

Peptide analysis. Solubilized 14C-labeled virion or nuclear fractions were electrophoresed through 2 mm 12% acrylamide gels. The gels were dried; VPl, VP2, and VP3 were cut out and subjected to tryptic digestion, and peptide analysis as described elsewhere (Gibson, 1974; Hutchinson et a,Z., 1978) with one modification. After the plates were dried, they were dipped in melted 2methylnaphthalene (Aldrich) containing 0.4% diphenyloxazole and exposed on preflashed Kodak XR-5 film (Bonner, 1978). Isolation of “2P-radiolabeled DNA. Infected 3T6 cells were labeled with 32P from 42 to 66 hr postinfection by removing media, rinsing two times with DME without phosphate, and adding 2 ml/plate DME without phosphate containing 100 pCi/ml of 32P (ICN, carrier free). Viral DNA was extracted by the method of Hirt (Hirt, 1967) and purified as described by Wells (Wells, et al., 1979). DNA nucleotide sequence analysis. The DNA sequence of the polyoma wild type genome and its restriction enzyme recognition sequences have been reported previously (Deininger et al., 1980). DNA fragments were labeled at their 3’ ends by incubation with reverse transcriptase in the presence of a 32P-labeled deoxynucleoside triphosphate as described previously (Friedmann et al., 1978). To determine the sequence in the vicinity of the HpaII 8/7 junction, we radiolabeled MboI fragment 7 from ts48 DNA, redigested with AluI and separated the two end-labeled fragments by polyacrylamide gel electrophoresis. To isolate a fragment to determine the sequence in the vicinity of the VP2iVP3 terminator, we radiolabeled Hind111 cleaved ts48 DNA and redigested with HpaII before isolation of the 494 base pair fragment spanning this region. DNA sequence analysis was carried out by the method of Maxam and Gilbert (1977) with modifications as described previously (Friedmann and Brown, 1978). Isolation of polyoma

mRNA and synthe-

3T6 cells were infected at 32” with wild-type polyoma at a multiplicity of 2.5 PFU/cell. Total cytoplasmic RNA was isolated at 48 hr postinfection and polyomaspecific RNA selected as described elsewhere (Hunter and Gibson, 1978). DNA was

sis of cRNA.

harvested from a parallel infection at 70 hr postinfection. Polyoma form I DNA was isolated and cRNA \vas prepared with E. co/i RNA polymerase (Miles Laboratories) as described (Cogen, 1978). Zw vitro translntioic. Polyoma-specific RNA and cRNA were translated in the mRNA-dependent reticulocyte lysate (Pelham and Jackson, 1976) in the presence of l”“S]methionine (AmershamiSearle, XJ204; specific activity >300 Ci/mmol) at a final concentration of 500 $.X/ml as described (Hunter et nl., 1978). For preparation of [‘l~S]formyl-methionine-labeled proteins polyoma-specific RNA was translated in the mRNA-dependent reticulocyte lysate in a final volume of 75 ~1 containing 1.2 x 10’ cpm ]:‘“S]f-met-tRNA;“‘l (Hunter and Gibson, 1978). Alzalysis bg partial proteolysis. [““S]Formyl-methionine-labeled irr vitro translation products Lvere partially digested with Staphylococucs (x1cre%Ls V8 protease and analyzed using the methods for proteins in gel slices as described by Cleveland pt ctl. (1977) with the following changes. Solubilized i?? ?:itro translation products were run on a l-mm slab gel containing 12% acrylamide as described above. The gels were then soaked for 2 hr at 4” in H,O with Amberlite mixed bed resin. The gel was then dried under vacuum over a boiling water bath and exposed against NS5T film. Using the autoradiograph as a guide, the VPl, VP2. and VP3 bands were cut, the paper scraped off, and the gel slices rehydrated. The slices were then loaded on a l-mm slab gel containing 16% acrylamide with a 3-cm stacking gel. The embedded gel slices were overlaid with 0, 10, and 50 ng protease and electrophoresis was performed. RESULTS The ts48 Mutant DNA Clecrvage Site.

Lacks an HpaII

We analyzed the DNA of wild-type polyoma and the ts48 mutant by digestion with the Hpa II restriction enzyme. The patterns of the HpaII digestion products are shown in Fig. 1. The ts48 mutant lacks the fragments 7 and 8 present in the wild-type digest. (Fragments 7 and 8 are 275 and 112

base pairs, respectively. The fused fragment comigrates with fragment 5, 390 base pairs.) We analyzed the nucleotide sequence of the ts48 mutant DNA in the region of the ,junction between HpaII fragments 7 and 8. The sequence in this region \vas identical to that of wild-type polyoma DNA except in one position, within the recognition site for the Hprr II enzyme. The wild-type sequence is -5 ACCGGA 3’: the fs48 mutant sequence is 5’ ATCGGA 3’. We conclude that the normal HpaII fragments 7 and 8 are missing in the ts48 mutant digest because the wildtype HpaII recognition site has been abolished in that region due to a c‘ to T transition at nucleotide 1228.

While studying the T antigens ofpolgoma temperature-sensitive mutants we noticed proteins which Lvere present in the lgsates of ts48-infected cells but not in wild-type infected cells late in infection (Hutchinson it ol., 1978). To see whether these proteins came from fs48 virions \ve compared the radiolabeled proteins of wild-type and ts48 virions. The comparison is shown in Fig. 2. The proteins corresponding to the normal wild-type VP2 and VP3 are missing in fs48. and are replaced by t\vo neu proteins, each approximately 2500 daltons larger than the corresponding wild-type proteins. To verify that the apparent size differences resulted from a change in the coding sequences for the fs48 proteins, rather than from posttranslational processing, \ve isolated polyoma-specific cytoplasmic RNA from cells infected by wild-type polyoma or ts48, and translated the RNA it{ ~‘jtro. The results are shown in Fig. 3. The irt vitro products show the same apparent size differences as the proteins isolated from virions. This suggests that the alterations in the fs48 proteins result from changes in the sequences of the mRNAs coding for the proteins rather than from posttranslational processing, although it cannot be ruled out that some processing occurs in the in rlit,~~ system. [Figure 3 also shows the product:: of translation of complementary RNA (cRNA). These results are discussedbeloiv.]

SIZE

WT

OF POLYOMA

ts48

VIRION

39

PROTEINS

We considered a number of possible esplanations for the apparent increase in size of the ts48 VP2 and VP3. The first was that the increase resulted from an insertion of new DNA sequences in the coding regions for VP2 and VP3 in the ts48 mutant. As can be seen in Fig. 1, the HpnII fragment 3 of ts48, spanning the region between 54 and

VP1 a

1

% vp2t

-

E 5

.qi* -An WT

ts48

& ” ta?

E aa WT

ts48

WT

ts48

---

VPI-

FIG. 2. Mobility

differences between the virion proteins VP2 and VP3 of ts48 and wild-type virions. [‘?ClAmino acidlabeled ts48 and vvild-type virions were isolated as described in Materials and Methods from approximately 4 x 1O’cells. The virion pellet was soluhilized in 30 ~1 and IO ~1 was subjected to electrophoresis on a 12% polgacrylamide gel. The gel \vas dried and autoradiographrtl for 6 days against NS5T film.

We analyzed the tryptic peptides of the wild-type and ts48 VP2 and VP3 proteins by two-dimensional electrophoresis and chromatography to see whether the apparent size changes were reflected in the presence of altered peptides. The comparison is shown in Fig. 4. As reported previously, VP2 and VP3 share many tryptic peptides (Gibson, 1974). A comparison of the ts48 and wild-type peptides shows that at least one wild-type peptide (designated i) common to VP2 and VP3 is missing in ts48, and at least four new peptides (designated 1, 2, 3, and 4) appear in the ts48 proteins. (Peptides 3 and 4 are not seen clearly in the VP2 map.) This confirms that the apparent size change of the fs48 protein is accompanied by alterations in the amino acid sequence of the proteins.

VP2[

VP3[

FIG. 3. Comparison of polyoma proteins from purified virions and in z?tro translation products of polyomaspecific RNA and complementary RNA. (“ClAmino acid-labeled purified virions were analyzed as described in Fig. 2. Polyoma-specific cytoplasmic RNA, isolated from cells infected with either ts48 or wild-type virus, was translated in the mRNA-dependent reticulocyte lysate in the presence of [%]methionine in a final volume of 10 ~1 as described in Materials and Methods. The cRNA was prepared from the viral DNA isolated from parallel infections as described in Materials and Methods and translated in z&o as described above. Two microliters of each translation reaction were resolved on a 12% polyacrylamide gel which was subjected to fluorography for 3 days. The background of bands in the overexposed mRNA tracks consists mainly of premature termination products of the viral proteins.

40

ECKHART

ET AL.

tda vpl

WT VP1

U-

‘X

l

tsda vp3

WT VP3

FIG. 4. Tryptic peptide maps of l’%]amino acid-labeled polyoma proteins VPI, VP2, and were infected with either&48 or wild-type virus and labeled with [YJamino acids. Purified nuclear fractions were prepared and subjected to electrophoresis as described in Materials ods. The wild-type preparation is from a nuclear extract; the map is identical to a map Lvild-type virions. The ts48 preparation is from purified virions. Individual protein bands of

VP3 Cells virions, or and Methof purified VPl. VP2,

SIZE

OF

POLYOMA

70.7 map units, clockwise, is, in fact, larger than the corresponding wild-type fragment, and includes coding regions for amino acids common to VP2 and VP3. Nevertheless, the increase in size of the HpaII fragment 3 of ts48 does not result from additional DNA in the coding regions for VP2 and VP3. This conclusion is based on the fragments produced by digestion of wild-type and ts48 mutant DNAs by the enzyme Sau 3a (Fig. 1). This enzyme has cutting sites within HpaII fragment 3. If the additional DNA sequences in the ts48 mutant occurred in the coding regions common to VP2 and VP3, it would be expected that either the Sau 3a fragment 2 or 6 of ts48 would be larger than the corresponding wild-type fragment. The digestion patterns in Fig. 2 show that this is not the case. Instead, Suu 3a fragment 1 of ts48 is increased in size relative to wild type, showing that the additional sequences in the ts48 DNA occur outside the coding region common to VP2 and VP3 in wild-type polyoma DNA. The additional sequences probably occur in the vicinity of the HpnII 315 fragment junction, where sequence differences among polyoma wild-type strains have been noted previously (Fried et al., 1975). The ts48 mutant was isolated from a mutagenized polyoma large plaque population which contained a mixture of wild-type genomes having HpaII fragments 3 and 5 of differing sizes. We considered the possibility that the ts48 DNA might be altered so that new mRNA leader sequences were produced and that these new leader sequences might be translated, adding to the N-termini of the VP2 and VP3 proteins of ts48. (This would be consistent with the location of the additional DNA sequences described above.) We did two experiments to test this possibility. The first was to translate cRNA in vitro. If the size change in the ts48 proteins were caused

VIRION

PROTEINS

41

by new translated leader sequences, the size change should not appear in proteins produced by in vitro translation of cRNA, since the cRNA is an unprocessed transcript of the viral DNA. Figure 3 shows that the translation of cRNA from wild-type or fs48 results in the production of proteins having the characteristic mobilities of the VP2 and VP3 proteins of each virus. This result does not support the idea that the ts48 mutant contains new leader sequences which are translated to yield additional amino acids at the N-termini of VP2 and VP3. Further evidence on this point came from the analysis by partial proteolytic digestion of VP2 and VP3 proteins radiolabeled specifically in their N-terminal methionine residues. Separation of the partial digestion products by size allows a comparison of the length of polypeptide chains between the labeled N-terminal residue and the sites of cleavage in the VP2 and VP3 proteins of wild-type polyoma and fs48 (Hunter, 1979). If the change in the amino acid sequencr’ of the ts48 protein ivere at the N-terminal end, the labeled fragments of the fs48 proteins should be larger than the corresponding fragments of wild-type proteins. Figure 5 shows that this is not the case: the partial digestion products of the wild-type and ts48 proteins are the same size, showing that the change in amino acid sequence occurs in the C-terminal portion of the ts48 proteins. From the size of the largest fragment produced we can deduce that this change must be at least 24K from the N-terminus of VP2. A Terminatiorl Codon Is Abolish,ed irr fh,e ts48 Mutant

We analyzed the nucleotide sequence of DNA in the coding region of the C-terminal end of VP2 and VP3. Figure 6 shows the relevant portion of the sequencing

ts48 mutant

and VP3 for each virus were eluted and digested with TPCK-treated trypsin as described in Materials and Methods. The digests were resolved by two-dimensional separation on cellulose thin-layer plates, by electrophoresis at pH 4.7 from left to right toward the cathode, followed by chromatographic development from bottom to top. The plates were dipped in 2-methylnaphthalene containing 0.4% diphenyloxazole as described by Bonner and Stedman (1978). The amounts of radioactivity applied to each plate and exposure times are: ts48 VPl, 15,000 cpm for 4 weeks; ts48 VP2, 2000 cpm for 8 weeks; ts48 VP3,4000 cpm for 8 weeks; Wt VPl, 30,000 cpm for 4 days; Wt VP& 11,000 cpm for 8 weeks; Wt VP3, 12,000 cpm for 8 weeks.

4%

fs48 0

VP1 10

50

WT 0

ECKHART

ET AL.

VP2

fr48

1050

0

v

x

lb’ +

X

id X

d’

X

VP2 10

50

Is48 VP3

WT VP3 0

10

50

0

10

50

WT VP2 +c ce

lr48

VP2

WT VP3 ls48VP3

FIG. 5. Analysis by partial proteolysis of [““S]formyl-methionine-labeled it/ rlifm translation products of ts48 and wild-type polyoma-specific RNA. Polyoma-specific ts48 and wild-type cytoplasmic RNAs \rere isolated and translated ii/ vitro in the presence of [,‘%]f-met-tRNky”’ as described in Materials and Methods. Two microliters of the translation product was subjected to electrophoresis on a 129 polyacrylamide gel after which the individual, radiolabeled bands corresponding to VPl, VP2, and VP3 fol each virus were excised from the dried gel. The gel slices were reelectrophoresed on a 16% polgacrylamide gel in the presence of 0, 10, and 50 ng of Sfnphy/oco~n~.s nwews V8 protease as described by Cleveland (19’77). The molecular weights in daltons of the fragments generated were determined to be: Wt a, fs48 a’, %K; Wt b, ts4X b’. 17K: JVt c. ts48 c’. 13K; Wt d. ts48 d’. 7K.

gels, and Fig. 7 the partial sequences for the wild-type and ts48 DNAs, and the amino acid sequences that would be encoded in them. The ts48 mutant shows a single base change (T to C) at nucleotide 4070, changing a TAA termination codon to a CAA codon for glutamine. This change, which is in the expected termination codon for VP2 and VP3, would result in the extension of the VP2 and VP3 proteins by an additional 21 amino acids at their common C-termini. DISCUSSION

We have analyzed changes in the genome of the ts48 mutant of polyoma virus which affect virus-coded proteins. The mutation

causing the temperature-sensitive phenotype has been mapped by marker rescue in the early region of the genome, roughly between 14 and 26 map units (Miller and Fried, 1976). Preliminary sequence analysis in this region indicates a G to T transversion at nucleotide 2341 (P. Deininger, unpublished). We can tentatively conclude that the resultant serine to isoleucine change in the sequence of the large T antigen is responsible for the temperature-sensitive phenotype of ts48. A second change in the early region abolishes the wild-type Hpa II cleavage site at 93.5 map units, by changing the DNA sequence at the recognition site from 5’ CCGG 3’ to 5’ TCGG 3’. This site is in the region of the polyoma genome coding simultaneously

SIZE OF POLYOMA WT

G

A

VIRION

ts48

1

C

C+T

43

PROTEINS

G

A

C

c +1

FIG. 6. Nucleotide sequences ofts48 and wild-type DNA at the termination of VP2 and VP3. Nucleotide sequencing was carried out as described in Materials and Methods. The region between nucleotides 3943 and 4105 was displayed on the sequencing gels. The region including the termination codon (nucleotides 4068-4070) is shown.

for the medium and large T antigens (Hunter et nl., 1979). The change in the DNA sequence would be expected to cause a change from threonine in the wild-type large T antigen to isoleucine in the mutant, and a change from proline in the wild-type medium T antigen to serine in the mutant (Hunter et al., 1979). The ts48 mutant has not previously been observed to be defective in DNA replication or cell transformation at the permissive temperature (Eckhart, 1975). However, the proline to serine change in the medium T antigen might be expected to affect the configuration of the protein, and the change is in the same region of the genome deleted in the polyoma mutant, d123, which is defective for transformation (Griffin and Maddock, 1979). Therefore, we are reexamining transformation by the ts48 mutant. The increase in size of the ts48 virion proteins, VP2 and VP3, apparently results from a base change which abolishes the normal termination codon for the two proteins. Although the general arrangement of the virion protein coding regions is well established from genetic mapping, DNA sequencing, mRNA characterization, and peptide analysis, the previous analysis of the proteins has not been done in sufficient detail so that one could be confident of the correct assignment of the termination codon.

Therefore, we have sought to rule out alternative explanations of the size increase of VP2 and VP3, particularly the presence of additional DNA sequences in the coding region and the possibility of new leader sequences which might be translated in the case of the mutant. None of these alternatives seems feasible, so we conclude that the base change we have identified by nucleotide sequencing does, in fact, affect the normal termination codon for VP2 and VP3. The tryptic peptide analyses shown in Fig. 5 demonstrate that at least four additional peptides are present in the k-48 VP3 protein, compared to wild-type. This is consistent with deductions from the DNA sequence, shown in Fig. 6: if the normal termination codon were abolished, seven new peptides would be created (assuming that termination takes place at the next termination codon in this reading frame). Of the tryptic peptides predicted from the additional sequence, five are basic and two are neutral. The four identified new peptides (l-4) in ts48 VP2 and VP3 are all basic. Since the C-terminal sequence of wild-type VP2 and VP3 is Lys-Glu-Lys-Arg-ArgLeu, the C-terminal tryptic peptide should be simply leucine. However, cleavage at Lys-Arg-Arg may be incomplete and generate a series of partial digestion products. We would expect free leucine and any

44

ECKHARTETAL other partial digestion products containing the C-terminal leucine to be absent from the tryptic digest of ts48 VP2 and VP3. Peptide i (Fig. 4) is absent from the tryptic digestion products of ts48 VP2 and VP3. Although peptide i does not comigrate with free leutine, it is basic and could represent a partially cleaved C-terminal peptide. Alternatively there may be a second change in another region of the coding sequence for VP2 and VP3 in the ts48 genome which results in a peptide change compared to the wild-type. Abolishing the normal termination codon for VP2 and VP3 would cause the addition of 21 amino acids to the C-termini of the proteins in ts48 (again assuming the next available termination codon is used). The apparent increase in molecular weight of the proteins is about 2500 which is in good agreement with the 2533 predicted from the DNA sequence. Due to the redundancy of the genetic code, the mutation abolishing the VP2lVP3 termination codon does not alter the sequence of VP1 (see Fig. 6). There are no observable differences between the tryptic peptide maps of ivild-type and fs48 VPls (Fig. 5). It is interesting that virion assembly appears to proceed normally in the ts48 mutant, using a wild-type size VPl, plus VP2 and VP3 of 37,500 and 26,000 daltons, respectively, rather than 35,000 and 23,000 daltons, as in the wild-type. Apparently the assembly mechanism is not exquisitely sensitive to changes in the size of VP2 and VP3 by extension at the C-terminus. Indeed the role of VP2 and VP3 in virion assembly is not well defined. There is at least one mutation in VP2 and VP3 which affects virus production late in the replication cycle, but it is not clear whether the only defect is one in virus assembly at the restrictive temperature (Gibson et nl., 1977). In the caseof SV40 VP2 appears to be dispensable for virus assembly because there are viable deletion mutants which possessno detectable VP2 in their virions (Cole et al., 1977). In addition, there are viable deletion mutants which have shortened VP2 and VP3 proteins in their virions (Cole et al., 1977). Furthermore, the overlap between the C-terminus of VP2 and VP3 and the N-terminus of VP1

SIZE OF POLYOMA

is considerably greater for SV40 than polyoma, being 120 nucleotides instead of 27. This overlap is increased to 93 nucleotides in ts48, but while there is extensive homology between SV40 and polyoma in other parts of the VP2lVP3 gene, there appears to be little homology in the overlap region beyond the normal termination site for VP2i VP3 (Deininger et al ., 1979). This is a further indication that the C-terminal regions of VP2 and VP3 probably do not play a crucial role in virus assembly. Multiple changes in the genomes of mutants isolated after potent mutagenesis are not unexpected. We have observed in a survey of alterations in restriction enzyme cleavage patterns among a collection of polyoma temperature-sensitive mutants that these genomes may contain several base changes in addition to the ones resulting in a temperature-sensitive phenotype (W. Eckhart, unpublished observations). The changes in the ts48 genome described here emphasize the importance of considering “silent” mutations in assessing the properties of mutants. A(‘KNOWLEDGMENTS This investigation was supported by research grant Nos. CA 13884, CA 14195. CA 17096. and CA 24288 a\vardrd by the National Cancer Institute, and by grant No. 78 SDS from the Cancer Coordinating Committee, Universitv of California. P. Deininger IT-as supported hy Training Grant No. GM 07199. awarded by the National Institute of General Medical Sciences. We thank Bill (‘ogen for generously providing polyoma complementary RNA. REFERENCES BOXNEK. LV., and LASKEY, R. (1974). A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Kiocherr~. 46,83-88. BONNER, W. M.. and QTEDMAN, .J. D. (1978). Efficient fluorography of :‘H and “C on thin layers. Ann/. RKwhrrrr. 89 247-256. CLEVELAND. 6. W;.. STUART. G. F., KIRSCHNER, M. \V.. and LAEMMLI, c. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Riochrw. 252, 1102-1106. (‘oGEN, B. (1978). Virus specific early RNA in 3T6 cells infected by a ts.4 mutant of polyoma virus. I’iro/og,q 8;i I-‘WL230.(‘OGEN, B.. and ECKHART. R’. (1977). BALBIXT:~ cells

VIRION

45

PROTEINS

infected by the ts3 mutant of polyoma virus fail to accumulate virus-specific early RNA at the nonpermissive temperature. J. Viral. 24, 701-703. COLE, C. N., LANDERS, T.. GOFF, S. P., MANTEUILBRUTLAG, S., and BERG, P. (1977). Physical and genetic characterization of deletion mutants of simian virus 40 constructed in vitro. d. Viral. 24, 277-294. DEININGER. P., ESTY, A., LAPORTE, P., and FRIEDMANN, T. (1979). Nucleotide sequence and genetic organization of the polyoma late region: features common to the polyoma early region and SV40. c’cll 18, 771-779.

DEININGER, P.. ESTY. A., LAPORTE, P., Hsu, H.. and FRIEDMAXN. T. (1980). The nucleotide sequence and restriction enzyme sites of the polyoma genome. Nu,cZ. Acids. Res. 8, 855-860. ECKHART, W. (1969). Complementation and transformation by temperature-sensitive mutants of polyoma virus. Virology 38, 120- 125. ECKHART, W. (1975). Properties of temperature-sensitive mutants of polyomavirus. C’oldSprircg Htrrhor Synrp. &unnt. Bid. 39, 120-125. FEY, G., and HIRT, B. (1975). Fingerprints of polyoma virus proteins and mouse histones. Co/d Spting Herrbar S?/tr/p. Q/rant. Rid. 39, 235-241. FIERS. W., CONTRERAS. R.. HAEGEMAN, G.. ROGIERS, R., VAN DE VOORDE, A., VAN HEUVERSWYK, H., VAN HERREIVEGHE. .J.. VULCKAERT. G., and YSEBAERT, M. (1978). Complete nucleotide sequence of SV40 DNA. A’otzrw (London) 273, 11% 120. FLAVELL, A. J., COWIE, A., LEGON, S., and KAMEX, R. (1979). Multiple 5’ terminal cap structures in late polyoma virus RNA. Ceil 16, 357-371. FRANCKE, B., and ECKHART, W. (1973). Polyoma gene function required for viral DNA synthesis. Viro/og,g 53,

127-

1’.3’ .).

FRIED, M., GRIFFIN, B. E., L~xD, E., and ROBRERSON. D. L. (1975). Polyoma virus--A study of \viltl type. mutant and defective DNXs. Co/d Sprfng

Hnrhor

&/n/p.

f&nnt.

Bid.

39, 45-52

FRIEDMANN, T.. and BROW-r;. I). (1978). Base-specific reactions useful for DNA sequencing: methylene blue-sensitized photo oxidation of guanine and osmium tctraoxide modification of thyminr. Slrc/. Acids.

RPS. 5, 615-622.

FRIEDMANN, T., and ECKHART, W. (1975). Virion proteins of temperature-sensitive mutants of polyoma virus: late mutants. Cold Sp)-ing Hnrhor Sqrt~p. Qm~t.

Rio/.

39,

243-246.

FRIEDMANN. T.. LAPORTE, P.. and ESTY, A. (1978). h’ucleotide sequence studies of polyoma DiXA. J. Bid.

(‘hen/.

233,

6561-6567.

W. (1974). Polyoma virus proteins: a description of the structural proteins of the ririon based on polyacrylamide gel electrophoresis and peptide analysis. Virology 62, 31%336. GIBSON, W., HUNTER, T., COGEN. B., and ECKHART, LV. (1977). Altered virion proteins of a temperatureGIBSON,

46

ECKHART

sensitive mutant of polyoma virus, ts59. Virology 80, 21-41. GRIFFIN, B. E., FRIED, M., and COWIE. A. (1974). Polyoma DNA-a physical map. Proc. Xnt. Aced. Sri. USA 71, 2077-2081. GRIFFIN, B. E.. and MADDOCK. C. (1979). New classes of viable deletion mutants in the early region of polyoma virus. J. Viral. 31, 645-656. HEWICK, R. M., FRIED, M., and WATERFIELD, M. 1). (1975). Nonhistone virion proteins of polyoma: characterization of the particle proteins by tryptic peptide analysis using ion-rschange columns. Virology 66, 408-419. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Hiol. 26, 265-869.

HUNTER. T. (1979). Functional characterization of the early and late mRNA’s of simian virus 40. Virology 95, 511 -.i22.

HUXTE:R, T.. and GIBSON, W. (1978). Characterization of the mRNA’s for the polyoma virus capsid proteins. VPl, VP2 and VP3 .I. Viral. 28, 240-253. HUNTER, T.. HUTCHINSON, M. il.. and ECKHART, W. (1978). Translation of polyoma virus T antigens in vitro. Proc. h’uf. Acrid. Sci. L’S’A 75, 5917-5921. HUNTER, T., HUTCHINSON. M. A., ECKHART, W., FRIED~~ANN, T., ESTY, A., LAPORTE, P.. and DEININGER, P. (1979). Regions of the polyoma genome coding for T antigens. A;wc/. Acids. Kes. 7, 227.5-2288.

HUTCHIXSOK. M. A., HUNTER, T., and ECKHART, LV, (1978). Characterization of T antigens in polyomainfected and transformed cells. Ce// 15, 65-77.

ET AL.

KAMEN, R., FAVALORO, .J., and PARKER, J. (1980). Topography of the three late mRNA’s of polyoma virus which encode the virion proteins. J. Vim/. 33, 637-651. MAXAM, A., and GILBERT, W. (1977). A new method for sequencing DNA. Proc. Nat. Acnd. Sri. uSA 74, 560-564.

MILLER, L., and FRIED, M. (1976). (‘onstruction of the genetic map of the polyoma genome. J. Viral. 1X. 824-842. PELHAM, H. R. B., and JACKSON, R. J. (1976). An efficient mRNA-dependent translation system from reticulocyte lysates. Ezlr. d. Biochem. 67, 247-266. RE:DDY. V., THIMMAPPAYA, B.. DHAR, R., SUBRAMANIAN, K.. ZAIN, B., PAN, J., CELINA. .J.. and WEISSMAN. S. (1978). The genome of SV40. Scicwcv 200 9 494-602. . Ross, .J. il., and MARTIN, R. G. (1972). Genetic analysis of simian virus 40. III. Characterization of a temperature-sensitive mutant blocked at an early stage of productive infection in monkey cells. .J. Viral. 9, 95fi-968.

SOEI)A, E., ARRAND, .J. R., SMOLAR, N., WAISH. .J. E.. and GRIFFIN, B. E. (1980). Coding potential and regulatory signals of the polyoma virus genome. Nnluru

(London)

283, 445-463.

WELLS, R. D., HUTCHIXSO&. M. A., and ECKHART, %‘. (1979). Isolation and characterization of polyoma virus genomes with deletions between the origin of viral DNA replication and the site of initiation of translation in the early region. .I. Viral. 32, .517-622.