Altered DNA binding and replication activities of JC Virus T-antigen mutants

VIROLOGY

183, 239-250

Altered

(199 1)

DNA Binding and Replication

Activities

of JC Virus T-Antigen

Mutants

JOHN E. TAVIS AND RICHARD J. FRISQUE’ Department

of Molecular

and Cell Biology, The Pennsylvania

State University,

University

Park, Pennsylvania

16802

Received January 2, 199 1; accepted April 1, 199 1 Ten mutations were introduced into the JC virus (JCV) T antigen within a region corresponding to the SV40 T-antigen DNA binding domain (SV40 amino acids 131 to 220); nine of these increased homology between the two proteins in sequences critical for SV40 T antigen DNA binding. All mutant JCV T antigens bound to JCV and SV40 origins of DNA replication. Binding efficiency relative to that of wild-type JCV T antigen ranged from 83 to 301% for the JCV binding sites and from 44 to 240% for the SV40 binding sites. Nine mutant proteins promoted viral DNA replication in primary human fetal glial (PHFG) and CV-1 cells. In PHFG cells, promotion of DNA replication ranged from 26 to 220% relative to that of wild-type T antigen; in CV-1 cells it ranged from 14 to 522%. Coding sequences for five mutant proteins were transferred into the hybrid virus Ml (SV40) [Ml (SV40) contains coding sequences from JCV and regulatory sequences from SV40]. Wild-type T antigen promoted replication weakly from the SV40 origin in these hybrid viruses in CV-1 cells (2% that from the JCV origin); replication driven by the mutant proteins ranged from 110 to 412% of that induced by the wild-type protein. Efficient specific DNA binding by a mutant T antigen was not a reliable indicator of that mutant o 1991 Academic PESS, I~C. protein’s ability to promote DNA replication.

The lytic cycle of the human polyomavirus JC virus (XV) in cell culture is prolonged and is limited to primary human fetal glial (PHFG) cells (Padgett et a/., 1971, 1977) and their transformed derivatives, SVG and POJ cells (Major eta/., 1985; Mandl era/., 1987). In viva, JCV resides in the kidneys, but under certain conditions it can replicate in the brain in an uncontrolled manner, leading to the fatal disease progressive multifocal leukoencephalopathy (Padgett et a/., 1976). JCV is 699/o homologous at the nucleotide level to simian virus 40 (SV40), and the genomic organization of these viruses is nearly identical (Fiers et a/., 1978; Frisque et al., 1984). Yet despite this high homology, these viruses are biologically distinct both in viva and in vitro. Relative to SV40, JCV is reduced in its lytic and transforming activities in cell culture, and altered in its pathogenic potential in its natural host and in its tumorigenic capacity in experimental animals (for reviews, see Tooze, 1981; Walker and Frisque, 1986). Large T antigen (T antigen) is the major viral regulatory protein of JCV and SV40; it is a multifunctional phosphoprotein located primarily in the nucleus (Tooze, 1981). SV40 T antigen initiates viral DNA replication by binding to specific sites within the origin of DNA replication and unwinding the DNA to create an open complex for entry of DNA polymerase and primase (Smale and Tjian, 1986; Dean et al., 1987a; Bullock et al., 1989; Mastrangelo et al., 1989; Roberts,

’ To whom correspondence dressed.

and reprint requests

1989; Tsurimoto et al., 1989; reviewed in Stillman, 1989). SV40 T antigen binds to SV40 DNA at three sites in the regulatory region. Binding to site I (BS I) (highest affinity) and site II (BS II) is involved in negative regulation of early transcription (Rio and Tjian, 1983). Binding to BS II is essential for initiation of viral DNA replication, and binding to site III (BS Ill) (lowest affinity) has no known function. JCV T antigen binds to JCV sequences corresponding to BS I and BS II (Lynch and Frisque, 1991). Both SV40 and JCV T antigens are believed to recognize the pentanucleotide (G>T)(A>G) GGC (Tjian, 1978; DeLucia et al., 1983; Jones and Tjian, 1984); this sequence is present in multiple copies in each binding site. JCV BS I and BS II are nearly identical to their SV40 counterparts. SV40 T antigen binds to the DNA through a discrete binding domain, amino acids (aa) 131 to approximately 220 (Clark et al., 1983; Kalderon and Smith, 1984; Margolskee and Nathans, 1984; Cole et a/., 1986; Paucha et al., 1986; Simmons, 1986; Strauss et a/., 1987; Arthur et al., 1988; Simmons et al., 1990a,b). The zinc finger motif of T antigen (aa 302-320; Berg, 1986) is not needed for binding to origin sequences, but when present it affects the binding site preference (Strauss et al., 1987; Arthur et al., 1988; H&s et al., 1990). The onset of JCV DNA replication is delayed relative to that of SV40, and JCV replicates its DNA to lower levels than does SV40 (Lynch and Frisque, 1990, 1991). The JCV T antigen does not interact with viral DNA control sequences in the same manner as does

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TAVIS AND FRlSQUE

240

the SV40 protein; it binds DNA less efficiently and has a higher BS I to BS II binding ratio (Lynch and Frisque, 1991). Also, the SV40 T antigen interacts productively with the JCV origin to drive replication, but the JCV T antigen does not do so with the SV40 origin. In COS cells (CV-1 cells expressing the SV40 T antigen) plasmids containing wild-type SV40 or JCV origins replicate, but in POJ cells (PHFG cells expressing the JCV T antigen) only the JCV origin-containing plasmids replicate detectably (Lynch and Frisque, 1990, 1991). Furthermore, the hybrid virus M 1(SV40) (containing JCV coding sequences and the SV40 regulatory region) does not replicate in PHFG cells, whereas SV40(Ml) (SV40 coding sequences, JCV regulatory region), and the parental viruses JCV and SV40, do replicate in these cells (Chuke et a/., 1986; Lynch and Frisque, 1991). Because both the JCV T antigen and the SV40 replication origin are functional in other combinations, the failure of SV40 origin-containing plasmids to replicate in POJ cells and of M 1(SV40) to replicate in PHFG cells must be due to the interaction between T antigen and the origin sequences. Sequences within the JCV T antigen which restrict productive interactions to the JCV origin have been mapped to a region of the protein which includes the sequence-specific DNA binding domain (Lynch et a/., manuscript in preparation). To analyze these differences between JCV and SV40 DNA replication activity, we performed a mutational analysis of the JCV T-antigen DNA binding domain. We attempted to confirm the identity of the DNA binding region of this protein and to determine the contribution of DNA binding to JCV’s restricted DNA replication. The mutations introduced into the JCV T antigen (Table 1) affected sequences homologous to a region critical to SV40 T antigen DNA binding (SV40, aa 147-l 66; Paucha et a/., 1986); all sites of difference between the two proteins within this region were altered to convert the JCV sequences to those of SV40. MATERIALS Recombinant

AND METHODS

DNAs

Two prototype JCV clones (Mad 1 strain) were employed: pM 1TCRlA contains the complete JCV genome inserted into pBR322 at the unique EcoRl site, and pM lTC(dlBam) is pM 1TCRlA with a deletion in pBR322 from the C/al to Sal1 sites. pMl(B-B) is pM lTC(dlBam) with a deletion in the T antigen coding sequences from nucleotides 4307 to 4242. pM 1oriCAT is a pSVO-CAT derivative containing the JCV promoter-enhancer (nucleotides 5 1 12 to 270). The hybrid virus Ml(SV40) contains the JCV protein coding sequences and the SV40 noncoding regulatory sequences (Chuke et a/., 1986). pSV40Rl B contains the

complete SV40 genome (strain pBR322 at the EcoRl site. Nomenclature and cell lines

of JCV T-antigen

776) inserted

into

mutants

The nomenclature used for the JCV T-antigen mutants is as follows. Individual mutations are indicated by the single letter amino acid code for the mutant residue and the position of that alteration (e.g., L157). Mutant T antigens are indicated by the notation for the mutation followed by “-T” (e.g.? L157-T). Mutant JC viruses or genomes are indicated by “M 1” (strain Mad 1) followed by the mutant T-antigen designation [e.g., Ml(L157-T)]; plasmid clones are indicated by a “p” preceding the viral designation [e.g., pM1 (L157-T)]. M 1(SV40) viruses or genomes carrying T-antigen mutations are indicated by “MS” followed by the mutant T-antigen designation [e.g., MS(L157-T)]. Cloned BHK2 1 cell lines transformed by mutant T antigens are denoted by a “c” followed by the viral designation and a letter to distinguish between multiple cell lines generated by each mutant [e.g., cMl(L157-T)a]. Multiple mutations are indicated by a similar scheme, except that they are designated by the amino acid code for the altered residues only (e.g., SHLCI). Site-directed

mutagenesis

M 1(R168-T) was generated by converting JCV nucleotide 4167 from T to C by the single oligonucleotide site-directed mutagenesis protocol of Zoller and Smith (1982) to create a Sac1 site for use in the subsequent cassette mutagenesis procedures; it also created a lysine to arginine substitution at aa 168 in T antigen. Ml(R131-T) contains a T to C transition at nucleotide 4277 that changes T-antigen aa 13 1 from a lysine to an arginine. This mutant was generated by the method of Zoller and Smith (1982) as modified by Kunkel (1985). M 1(R131 -T) was created because aa 131 is at the amino-terminal end of the DNA binding domain (Rutila et al., 1986; Simmons, 1986; Arthur et al., 1988) and because the alteration makes the JCV T-antigen nuclear localization signal identical to that of the SV40 protein (Kalderon et al., 1984; Lanford and Butel, 1984). Cassette

mutagenesis

Eight additional mutants were constructed by the cassette mutagenesis procedure of Grundstrom and co-workers (1985) as adapted by the laboratory of P. Tegtmeyer at SUNY, Stony Brook. A substrate for cassette mutagenesis was constructed that contained unique Bglll and Sacl sites (JCV nucleotides 3357-4309) flanking the region to be

JCV T-ANTIGEN

altered. The Sac1 site in Bluescript Minus, KS polylinker (BSMKS, Stratagene Cloning Systems) was removed and then the Pstl to BarnHI fragment of pMl(R168-T) (nucleotides 3357-4309) was inserted to create pR168BP-BSMKS(dlSacl). This plasmid was cut with Sac1 and Bg/ll and the large fragment was isolated and dephosphorylated. The oligonucleotides to replace the wild-type sequence were phosphorylated, annealed by combining 2.5 pmol of each (15 oligonucleotides total) in 50 mMTris-HCI, pH 7.5, 10 mM MgCI,, 5 ml\/l D-TT, and 1 mMATP to a final volume of 30 ~1, and heating to 90” for 3 min, and incubated at 37” for 1 hr, and at 4 or 9” for an additional hour. The oligonucleotides and vector were ligated by adding 0.07 pg of the large Bglll and Sac1 fragment of pR168BP-BSMKS(dlSacl) and 1 U T4 DNA ligase to the annealing mixture and incubating for 2 days at either 4 or 9”. Those clones with the correct restriction digestion patterns were sequenced to confirm their identity, and sequences surrounding the mutated site were transferred into pM 1TC(dlBam) by standard cloning procedures. The R168 mutation that created the Sac1 site was reversed by the cassette procedure. Five of the mutations to the JCV T-antigen coding region (L157, Cl 59, R168, SHLCI, and HLC) were cloned into the hybrid virus Ml(SV40) by standard cloning techniques. The constructions were confirmed by restriction analysis and by sequencing the mutated regions.

Generation of transformed BHK-21 cell lines Two independent transformed cell lines that constitutively expressed T antigen were generated for each mutant; BHK-21 (baby hamster kidney) cells were used because they can be transformed readily by JCV. DNA transfection and cloning of the cell lines by limiting dilution were as described earlier (Bollag et al., 1989). Cells were screened for the presence of T antigen by indirect immunofluorescence staining.

lmmunoprecipitation T-antigen content was determined for the JCV BHK21 transformants and for cSVcl3 (SV40 transformed BHK-21 cells) by immunoprecipitation as described previously (Bollag et a/., 1989); T antigen was precipitated with a hamster polyclonal anti-SV40 T serum. The relative amount of T antigen in each sample was determined by excising two slices from each lane of the polyactylamide gel and measuring the radioactivity in each; one slice contained the appropriate T-antigen band, and the other contained no visible band. Background radioactivity (as determined from the control slice) was subtracted from each T-antigen value before

MUTANTS

241

TABLE 1 JCV T-ANTIGENMUTANTS SV40 Mutant JCV Mutant Name

Phenotypea Change

Change

Txn

Repln

D.B.

Ml (R131-T) Ml (S145-T) Ml (H149-T)

b%,Aw Ala,,,Ser Gln,,,His

-

*

Val,,,Leu

T -

Ml Ml Ml Ml

Ser,&ys Val,,,lle b%&g Gln,,,His Val,,,Leu Gln,,,His Val,,,Leu Ser,,,Cys Ala,,,Ser Gln,,,His Val,,,Leu Ser,,,Cys Val,6211e

N.*A. N.A. + N.A. + N.A. N.A.

N.A. * -

Ml (L157-T)

Arg,dys Ser,,,Asn His,,,Tyr His,,,Asn Leu,,,Phe Leu,,,lle Cys,,,Tyr -6

(C159-T) (1162-T) (R168-T) (HL-T)

Ml (HLC-T)

M 1 (SHLCI-T)

b%7Arg

r N.A. -

N.A. N.A. *

a Phenotypes: Txn. transformation; Repln, viral or DNA replication; D.B.. DNA binding. “+” indicates mutant phenotype is greater than wild-type, “:” indicates mutant phenotype is roughly equivalent to wild type, “-” indicates mutant phenotype is less than wild-type, and N.A. indicates information is not available, References are: Paucha eta/. (1986); Peden and Pipas (1985) and Simmons eta/. (1990a, b). b No known SV40 mutant. Construction of the JCV mutant was based on SV40 homology only.

T-antigen content was determined relative to that in the wild type JCV-transformed cell line, CM 1c13.

McKay DNA binding assay Sequence-specific DNA binding activities of the mutant T antigens were determined by the McKay DNA binding assay (McKay, 1981) as adapted by K. Lynch (Lynch and Frisque, 1991) with minor modifications; the nuclear extracts employed were prepared by a protocol based on those of Lynch and Frisque (1991) and Ausubel et al. (1990). Cells were seeded onto four loo-mm tissue culture plates (2.7 X 1O6 per plate) in Dulbecco modified Eagle’s medium (DME) plus 10% bovine calf serum (BCS). Total cell protein was determined 24 hr later from one of the four plates by the Bio-Rad protein microassay with IgG as a standard. Cells were collected from the remaining three plates by rinsing each plate twice with 5 ml cold PBS (150 mM NaCI, 20 mlLl NaH,PO,, pH 7.2), adding 5 ml cold PBS,

242

TAVIS AND FRISQUE TABLE 2 RELATIVEDNA BINDINGACTIVITYOF MUTANT JCV T ANTIGENSTO JCV AND SV40 DNA SEQUENCES’ JCV binding sates

SV40 binding sites

T antigen or control

BS I + II

BS I

BS I + II + III

BS I

ICI-T SV40-T BHK-21 Probe (20 pg) R131-T S145-T H149-T L157-T Cl 59-T 1162-T R168-T SHLCI-T HLC-T HL-T

10ob 980+ 115 0 127f 19 193a 22 19Ok 38 173f 32 200 + 33 137f 39 123If: 20 301 f 43 83+ 18 166+ 32 171 f 17

1OOb 310 k 69’ 0 127 _+ 22 134k20 182 + 19 96+ 14 175 2 34 119*38 87+ 3 222 * 33 58+ 13 166 k 29 113f 16

1oob 464 rf: 51 0 42f 8 151+29 154 f 32 109 * 17 156+ 2 56* 8 a2+ 9 240 + 39 44* 3 84+ 11 116f 14

1OOb 194 zk 15c 0 5Ok 6 1192 11 192 + 18 104k 5 126 k 10 87k 5 124+37 223 zk 21 44+- 7 142 + 12 91k 5

a Binding activity of T antigen is relative to that of JCV-T to DNA fragments carrying either all binding sites or to BS I alone, plus or minus the standard error of measurement (three to five experiments). Values for the probe indicate amounts of radioactivity in the appropriate fragment relative to that bound by JCV-T. b JCV-T bound 0.0025 fmol of the fragment containing combined JCV binding sites, 0.0027 fmol of the fragment with only JCV BS I, 0.0075 fmol of the fragment containing the SV40 combined binding sites, and 0.0063 fmol of the fragment with only SV40 BS I. c SV40 T antiaen binds both BS I and BS II when the sites are separated. Binding to BS II (relative to JCV-T binding to BS I) is 182 + 41 for JCV DNA and 45 f 2 for SV40 DNA.

scraping the cells free, and storing the suspended cells on ice. PBS (3 ml) was added and the plates were scraped again. Cells were collected by centrifugation at 500 g for 5 min at 4”. The pelleted cells were suspended in 4 ml NP-40 lysis buffer (10 m/l/l Tris-HCI pH 7.4, 10 mM NaCI, 2 mNI MgCI,, 0.5% Nonidet P-40 [Sigma Chemical Co.]) containing 100 KIU/ml aprotinin and 1.O mM PMSF, and were incubated on ice for 5 min. Nuclei were collected by centrifuging at 500 g for 5 min at 4’. The nuclei were suspended in 300 ~1 HIP (0.45 1\/1NaCI, 20 mM HEPES, pH 8.5, 1 rnM EDTA, 1% NP-40) containing 100 KIU/ml aprotinin and 1 .O mM PMSF, and the suspension was incubated on ice for 20 min. The nuclear extract was clarified by centrifuging at 15,000 rpm for 60 min at 4’ in a Sotval SS-34 rotor. The supernatant was collected and diluted with HIP based on total cell protein, divided into 60-~1 aliquots, and stored at -75”. Five probes were used in the McKay assays. To detect binding to the combined JCV BS I and II, a HinFIl Avall digest of pMlTCR1A was used, and to the separated JCV BS I and II, a Ddel digest of pMlTC(dlBam) was used. To detect binding to the JCV BS II alone, a HinFl digest of pM1 ori-CAT was employed. To measure binding to the combined SV40 BS I, II, and Ill, an Avall digest of pSV40Rl B was used, and to the separated SV40 BS I and BS II + III, a Dde I digest of pSV40Rl B was used.

HIP and HM (10 mM MES, 10 mM HEPES, 0.1% NP-40) buffers, each containing 100 KIU/ml aprotinin and 1 .O mNI PMSF, were combined to make HM:HIP (2:l HM:HIP), and the pH was adjusted to pH 7.0. Nuclear extracts (60 ~1) were combined with 120 ~1 HM dilution buffer and preabsorbed with 50 ~1 10% Staphy/ococcus aureus in HM:HIP (pH 7.0) for 20 min at 0”. The pH was adjusted to 7.0, salmon sperm DNA(60 ~1, 0.1 pglpl) and 12 ~1 radioactively labeled probe DNA (1 ng/pl) were added, and the mixture was incubated on ice for 1 hr. The final conditions for the binding reactions were 112 mM NaCI, 10 mM HEPES, 5 mM MES, 2.5 mll/l Tris-HCI, 0.5 mM EDTA, 0.3% NP-40, 20 pgl ml salmon sperm DNA, 60 rig/ml probe DNA, pH 7.0. T antigen-DNA complexes were immunoprecipitated by adding 2 ~1 polyclonal hamster anti-JCV T-antigen serum (antibody excess) and incubating on ice for 45 min. The immunocomplexes were collected by adding 30 ~1 10% S. aureus in HM:HIP (pH 7.0, 1 mg/ml BSA) and incubating on ice for 20 min. Bacterial pellets were washed three times at 4” with freshly made wash buffer (20 mM Tris-HCI, pH 7.0, 150 mM NaCI, 2 mM DlT, 1 m/W EDTA, 0.01% BSA, 10 pg/ml calf thymus DNA, 0.5% NP-40). Coprecipitated DNA was released from the immunocomplex by adding 100 ~1 SE (1% SDS, 10 mM EDTA pH 8.0) to the bacterial pellet and heating to 70” for 30 min. The bacteria were removed and the DNA in the

JCV T-ANTIGEN

BS I + II -

FIG. 1. DNA binding activity of T antigens to combined JCV BS I and Il. Binding activity was determined by the McKay coimmunoprecipitation method (McKay, 1981; Lynch and Frisque, 1991) using end-labeled pMlTCRlA digested with Avall and HinFl as a probe. MK denotes the marker lane (20-pg probe DNA from the same labeling reaction as was used in the binding reactions). The fragment bearing JCV BS I and II is indicated. T antigens present in the nuclear extracts used in the binding reactions are indicated above the lanes; no T antigen was present in the control extract from untransformed BHK-21 cells.

supernatant was purified by phenol and chloroform extraction, and ethanol precipitation (1 .O pg salmon sperm carrier DNA was added to each sample). DNA samples were dissolved in varying volumes of electrophoresis loading buffer to compensate for the differing levels of T-antigen in the nuclear extracts. DNA precipitated by equivalent amounts of T-antigen was electrophoresed through 5% polyacrylamide 1X TBE gels and detected by autoradiography. Radioactivity in the appropriate bands was determined by excising and counting the gel slices in a liquid scintillation counter. Background radioactivity (radioactivity present in the control BHK-21 sample lane at the position corresponding to that of T antigen) was subtracted from all samples, and DNA binding activity was determined relative to that of the wild-type JCVT antigen from CM 1cl3 cells. Dpnl DNA replication assay DNA replication of the T-antigen mutants relative to that of wild-type JCV was measured by the Dpnl DNA replication assay as described previously (Lynch and Frisque, 1990). Viral sequences were isolated from plasmids, circularized, and transfected into cells by the

MUTANTS

243

modified DEAE-dextran method (Sompayrac and Danna, 1981). PHFG cells were seeded onto 35 mm tissue culture plates in DME plus 10% fetal bovine serum (FBS) by transferring cells from 1 confluent 1OOmm plate to 10 35-mm plates, and were transfected 2 days later. CV-1 cells (5 x 1O5per 60-mm plate) were plated in DME plus either 10% FBS or BCS and transfected 12 to 16 hr later. At 0,3,7, and 14 days post-transfection low molecular weight DNA was isolated by the method of Hit-t (1967). This DNA was digested with EcoRl and Dpnl; Dpnl distinguishes DNA replicated in bacterial and mammalian cells based upon its ability to cleave only fully methylated DNA. DNA fragments were resolved by electrophoresis through 0.8% agarose 1x TBE gels and were detected by Southern hybridization. I?coRI cut plasmid DNA [either pM lTC(dlBam), pSV40Rl B, or pM 1(SV40); 1.Opg per reaction] was labeled with the Pharmacia random priming kit according to the manufacturer’s instructions. Radioactivity in the hybridized bands was quantitated by excision and liquid scintillation counting. Plas-

BS II

FIG. 2. DNA binding activity of T antigens to separated JCV BS I and II. Binding activity was determined by the McKay coimmunoprecipitation method (McKay, 1981; Lynch and Frisque, 1991) using end-labeled pMlTC(dlBam) digested with Ddel as a probe. MK denotes marker lanes (probe DNA from the same labeling reaction as was used in the binding reactions, the amount loaded is indicated above the lanes). Fragments bearing JCV BS I or BS II are indicated. T antigens present in the nuclear extracts used in the binding reactions are indicated above the lanes; no T antigen was present in the control extract from untransformed BHK-21 cells.

TAMS AND FRISQUE

244 TABLE 3

BINDING PREFERENCEOF MUTANT JCV T ANTIGENSFORSV40 DNA Relative DNA binding activity T antigen or control JCV-T SV40-T BHK-21 R131-T S145-T H149-T L157-T Cl 59-T 1162-T R168-T SHLCI-T HLC-T HL-T

JCV BSl+ll 100 980 0 193 190 173 200 137 123 301 83 166 171

sv40 BS I + II + Illa 300 1390 0 453 462 327 468 166 247 720 132 254 348

DNA binding ratio (SV4O/JCV) 3.0 1.4 2.4 2.4 1.9 2.3 1.2 2.0 2.4 1.6 1.5 2.0

a DNA binding is calculated relative to JCV-T binding to JCV combined sites; values are 3.0 times larger than those found in Table 2 due to lesser binding activity of XV-T toward JCV DNA (see Table 2, footnote b).

mid DNA standards were included on all gels and were used to determine the quantity of replicated DNA detected.

RESULTS JCV and SV40 differ greatly in their lytic activities despite sharing a high degree of nucleic acid identity. Previous work in our laboratory has shown that the T antigens of these viruses do not bind to the viral DNA identically, and that these binding differences may be responsible in part for the dissimilarity observed in viral DNA replication (Lynch and Frisque, 1990, 1991). The SV40 T-antigen sequences that bind DNA have been identified, and a comparison of those sequences to the homologous JCV sequences revealed five amino acid differences between the two proteins in a region that is particularly important for DNA binding (SV40, aa 147166) (Table 1). To begin to assess the contribution of these nonconserved residues to JCV’s lytic behavior, each was altered to match the corresponding SV40 amino acid, and DNA binding and promotion of DNA replication by the mutant T antigens was measured.

DNA binding activity The binding of the mutant JCV T antigens to JCV and SV40 DNA fragments containing the linked or separated BS I and BS II sequences was measured by a modified McKay assay. JCV T antigen bound DNA less efficiently than did the SV40 protein; binding to the JCV and SV40 combined binding sites was reduced lo-

and 5-fold, respectively (Table 2 and Fig. 1). Both the JCV and SV40 T antigens bound SV40 sequences preferentially over JCV sequences. Radioactivity in the appropriate band of marker DNA (20 pg total labeled DNA) was equivalent to 127% of the radioactivity precipitated by JCV T antigen when JCV DNA sequences were used, but to only 42% of the radioactivity precipitated when SV40 DNA sequences were used, indicating that the JCV protein bound 3-fold more SV40 DNA than it did JCV DNA. To determine the relative level of DNA binding to the two origins by SV40 T antigen, the data for SV40 DNA sequences in Table 2 were adjusted to take into account the 3-fold difference in DNA binding of the JCV T-antigen sample (to which all SV40 DNA binding data in the table have been normalized); SV40 T antigen bound 1.4-fold more SV40 DNA than JCV DNA. All mutant JCV T antigens bound to JCV and SV40 DNA (Table 2 and Fig. 1); some mutations altered DNA binding activity of the proteins. Binding of the mutant proteins to the combined JCV binding sites relative to that of the wild-type JCV T antigen ranged from 839/o (SHLCI-T) to 301% (R168-T), and binding to the combined SV40 sites ranged from 44% (SHLCI-T) to 240% (R168-T). The DNA binding activities of the JCV T antigens carrying multiple alterations (SHLCI-T, HLC-T, and HL-T) were not the sum of the activities of the T antigens with the component individual mutations, and no simple pattern of contribution from their constituent substitutions was apparent. The JCV T antigens bound only to BS I when BS I and II were separated (Fig. 2 and Table 2). The binding data for the T antigens to isolated BS I were similar to those obtained when the combined sites were employed, indicating that binding to BS II did not contribute greatly to the activity observed with the combined site probes. The binding of T antigen to BS II is essential for initiation of DNA replication; therefore, additional attempts were made to detect an interaction between the JCV T antigen and origin. Modified McKay assays employing different NaCl concentrations, nonspecific competitor DNA compositions and concentrations, and protein concentrations were performed. No binding to JCV BS II (even in the absence of BS I) was detected for the JCV T antigen obtained from the transformed BHK-21 line (CM 1~13) under any of these conditions. JCV T antigen from POJ cells bound weakly to BS II under all conditions employed; it bound to origin sequences best when the nonspecific blocking agent in the reactions was 17 pg/ml salmon sperm DNA plus 17 pg/ml poly(dl-dC), and when the concentration of calf thymus DNA in the wash buffer was 20 pg/ml. The steadystate level of T antigen in POJ cells is considerably higher than that in cM1 cl3 cells (K. Lynch and J. Tavis, unpublished observations), so the failure to detect bind-

JCV T-ANTIGEN

MUTANTS

245

5130-5130

2319-

-2319

876-

,876

5130

-5243

.2936

2319

-1427 876

FIG. 3. DNA replication of rnutant JCV DNAs in PHFG cells at Days 0, 4, 7, and 14 post-transfection. DNA replication activity was determined by the Dpnl DNA replication assay and detected by Southern blotting. MKl and MK2 are markers [lo ng pM lTC(dlBam) digested with EcoRl and Bg/l, and 10 ng pSV4ORlB digested with EcoRl and Aval, respectively], Molecular weights of marker bands are indicated; replicated JCV and SV40 DNAs migrate at 5130 and 5243 bp, respectively. The viral sequences transfected into the cultures and the day post-transfection samples were collected are indicated above the lanes; CT indicates cultures that were transfected with calf thymus DNA. The SV40 Day 7 sample was diluted 1 O-fold before loading.

ing to BS II sequences with extracts from transformed BHK-21 cells may be due to insufficient amounts of T antigen. Higher levels of T antigen could be tested using greater amounts of nuclear extract, but the increased protein concentration raised nonspecific DNA binding to unacceptable levels (K. Lynch, unpublished observations). Differential post-translational modification of the Tantigens in the BHK-2 1 cells relative to that in the POJ cells also could account for the failure to detect binding to BS II, however, there is no evidence for such differences. Extensive attempts to measure binding to BS II by gel mobility shift and filter binding assays were also unsuccessful due to interference from cellular proteins in the nuclear extracts (data not shown). The preference for binding to SV40 DNA over JCV DNA was altered for the JCV T-antigen mutants. Table 3 lists the binding efficiencies of the T antigens normalized to binding of the wild-type JCV T antigen to JCV DNA, and lists ratios of DNA binding (SV4O/JCV sequences) for each T antigen. In all cases the JCVT-antigen mutants exhibited a reduced preference for SV40

over JCV DNA relative to that of wild-type JCV T antigen. Altering the JCV T antigen to resemble its SV40 counterpart in the putative DNA binding domain made the mutants’ preference for binding substrates more similar to that of the SV40 T antigen. Increased nonspecific DNA binding relative to that of the wild-type JCV T antigen was observed while attempting to measure binding to isolated BS 11for R13 lT, S145-T, and SHLCI-T, but this increase was not apparent in binding assays when BS I was present in the reactions. Increased nonspecific binding has been noted for an SV40 T antigen mutant that was unable lo bind specifically to origin sequences (T22, histidine 203 to glutamic acid; Mohr et al., 1989). DNA replication:

Primary human fetal glial cells

An essential early step in the initiation of polyomavirus replication is the specific recognition and binding of BS II by T antigen. As the JCV and SV40 T antigens do not bind to DNA identically, the JCV mutants were tested for DNA replication activity in PHFG cells, a cell

TAVIS AND FRISQUE

246 TABLE 4

DNA REPLICATIONACTIVITV OF JCV T-ANTIGEN MUTANTS RELATIVETO JCV IN PRIMARYHUMAN FETAL GLIALAND CV-1 CELLS~

DNA JCV SV40CJj Ml (B-B) Calf thymus Ml (R131-T) Ml (S145-T) Ml (H149-T) Ml (L157-T) Ml (C159-T) Ml (1162-T) Ml (R168-T) Ml (SHLCI-T) Ml (HLC-T) Ml (HL-T) JCV (FBS)“,” Ml (HL-T) (FBS)‘oe

PHFG (Day 14)

cv- 1 (Dw7)

100b 340,000 0 0 113+ 7 90+ 16 26+ 7 220*22 35+ 9 37f 3 0 30f 5 192 f 24 72+34

100b >3,000,000 0 0 238k 20 246k 74 28k 2 522k150 14f 6 40+ 8 0 21f 2 328k 54 69+ 22 22 42

a Values are the average of two experiments + the standard error. ’ DNA detected for JCV (100%) was 12 ng in PHFG cells and 0.037 ng in CV-1 cells. c Results are from one experiment only for SV40, JCV (FBS), and M 1 (HL-T) (FBS) samples. dThe data for SV40 in CV-1 cells was obtained at Day 3 posttransfection. e Cells were maintained in DME supplemented with FBS instead of BCS.

type permissive for both JCV and SV40. All mutants except M 1(R168-T) replicated their DNA in PHFG cells (Fig. 3 and Table 4). Viral DNA replication was efficient in PHFG cells, and increasing amounts of DNA were detected with each time point. Because DNA continued to accumulate in these permissive cells, it is likely that all mutants, except M 1(R168-T), are viable. The DNA replication activity of some of the mutants was altered compared to that of wild-type JCV; at day 14 replication varied from 26% [M 1(H149-T)] to 220% [Ml (L157-T)]. The possibility that reduced activity was the result of a temperature-sensitive defect in some of the mutant T antigens was investigated by conducting a Dpnl assay at 33” in PHFG cells; no significant difference was observed in the mutants’ replication at the two temperatures (data not shown). SV40 DNA replicated vigorously, with an activity 3400 times that of JCV DNA at Day 14. This greater activity was due in part to SV4O’s shorter lytic cycle and more rapid cellto-cell spread. DNA replication:

CV-1 cells

DNA replication was assayed in CV-1 cells, which are permissive for SV40 but not forJCV (Feigenbaum et

a/., 1987; Lynch and Frisque, 1990, 1991) to determine if wild-type or mutant JCV DNAs could replicate in these simian cells. All JCV T-antigen mutants except M 1(R168-T) replicated their DNA in CV-1 cells (Fig. 4). DNA replication was inefficient in CV-1 cells; at Day 7 post-transfection the level of DNA detected for JCV was only 6% that detected for JCV in PHFG cells. None of the DNAs generated viable virus following transfection of CV-1 cells, and DNA replication peaked at Day 7. The level of replication of the mutants was variable, and ranged from 14% [Ml(C159-T)] to 522% [Ml(Ll57-T)] relative to that of JCV (Table 4). SV40transfected cells displayed extensive cytopathic effect by Day 4 post-transfection, and the entire culture was lysed by Day 7. SV40 DNA replication was greater than 30,000 times that of JCV at 3 days post-transfection; again the SV40 activity was magnified by secondary infection. JCV DNA replication was detected for the first time in CV-1 cells; previous attempts (Feigenbaum et al., 1987; Lynch and Frisque, 1990, 1991) were unsuccessful due to insufficiently sensitive assays. The greater sensitivity of the Dpnl assay employed here was due to a number of alterations: DNA was extracted from more cells, longer transfection times were used, and the probes for detecting Southern blotted DNA were labeled to a higher specific activity. However, the largest increase (4. to 5-fold) resulted from the use of BCS instead of FBS to supplement the cul-

FIG. 4. DNA replication of mutant JCV DNAs in CV-1 cells at Day 7 post-transfection. DNA replication activity was determined by the Dpnl DNA replication assay and detected by Southern blotting. MK denotes the marker lane [l ng pMlTC(dlBam) digested with EcoRl and Bgll]. Molecular weights of marker bands are indicated; replicated JCV DNA migrates at 5130 bp. The viral sequences transfected into the cultures are indicated above the lanes; CT indicates the culture that was transfected with calf thymus DNA. Samples from cultures supplied with medium containing FBS instead of BCS are indicated by “(FBS)“.

JCV T-ANTIGEN

5132 5130

2936 2319

876 802

FIG. 5. Replication of mutant M 1(SV40) DNAs in CV-1 cells at Day 3 post-transfection. DNA replication activity was determined by the Dpnl DNA replrcation assay and detected by Southern blotting. MKl and MK2 are markers [0.2 ng pMlTC(dlBam) digested with EcoRl and Bgll. and 0.2 ng pMl(SV40) digested with EcoRl and Awl, respectively]. Molecular weights of marker bands are indicated; JCV and Ml(SV40) DNAs migrate at 5130 and 5132 bp, respectively. Viral sequences transfected into the cultures are indicated above the lanes; CT indicates calf thymus DNA. JCV samples from Day 3 (d3) and Day 7 (d7) post-transfection are shown. ICV DNA replication peaks in CV-1 cells on Day 7, Ml(SV40) DNA replication peaks on Day 3.

ture medium (Fig. 4 and Table 4). If BCS stimulates greater activity from the early JCV promoter-enhancer, it could elevate the level of T antigen in transfected cells and potentially increase DNA replication. This possibility was investigated through chloramphenicol acetyltransferase (CAT) assays; the use of BCS instead of FBS did not affect the activity of the early promoterenhancers in CV-1 cells (data not shown). However, the steady-state level of T antigen in CV-1 cells may have been altered by BCS in other ways, such as alterations to mRNA or protein stability. DNA replication

of Ml (SV40) mutants

The coding sequences for five of the mutant proteins were transferred into the hybrid virus M 1(SV40) to assess their interactions with the SV40 regulatory signals present in this virus. The mutations were chosen based on their effects on replication from the JCV origin, and included two that reduced DNA replication (Cl 59-T and SHLCI-T), one that exhibited no detectable activity (R168-T), and two that promoted increased activity (L157-T and HLC-T). DNA replication in CV-1 cells was detected for M 1(SV40) and all M 1(SV40) mutants except MS(R168-

247

MUTANTS

T) (Fig. 5 and Table 5). The activity was very low; the peak level obtained by Ml (SV40) was 1.8% that of JCV in CV-1 cells. DNA replication of the mutants relative to that of the parental Ml (SV40) ranged from 1 10% [MS(C159-T)] to 412% [MS(HLC-T)]. Replication peaked at Day 3 for M 1(SV40) and M 1(SV40) mutants, versus Day 7 for JCV and JCV mutants, so temporal control of replication mapped to the regulatory sequences, not to T antigen. The slower DNA replication profile induced by the JCV regulatory signals is consistent with the prolonged JCV lytic cycle. The effects of the mutant T antigens on DNA replication from the JCV or SV40 origin of replication [present in either JCV or Ml(SV40) as the parent virus] are shown in Table 5. Those mutant T antigens that increased DNA replication when in JCV (L157-T and HLC-T) also increased replication in Ml(SV40); the mutant T antigen that did not allow detectable replication in JCV (R168-T) also did not do so in Ml(SV40). However, the two mutant T antigens that decreased DNA replication when in JCV (Cl 59-T and SHLCI-T) did not decrease replication in M 1(SV40). Whatever detrimental effect these mutations had on T antigen interactions with the JCV replication origin was not apparent in their interactions with the SV40 origin. DISCUSSION The JCV lytic cycle is highly restricted in vitro relative to that of SV40, and this restriction includes the central TABLE5 DNA

REPLICATION ACTIVITY OF JCV AND M 1 (SV40) T-ANTIGEN MUTANTS IN CV-1 CELLS RELATIVE TO PARENT VIRUS

Replication activity relative to parent virus? T antigen or control

Ml (SV40)

JCV

XV-T (in Ml (SV40)) JCV-T (in JCV) Calf thymus DNA Ll 57-Te Cl 59.Te Rl 68.Te SHLCI-Te H LC-T”

loo* 5560k2000d 0 217k 35 110+ 5 0 139+ 17 412+ 58

N.D.C loo* 0 522*150 14f 6 0 21f 2 328k 54

a Values are the average of two experiments f the standard error. DNA replication peaks for M 1 (SV40) and JCV DNAs on Days 3 and 7 post-transfection, respectively. * DNA detected for M 1 (SV40) (100%) was 0.0019 ng and for JCV (100%) was 0.037 ng. c N.D.. not done. d Relative DNA replication at peak time points (ICV Day 7/Ml (SV40) Day 3). e These T antigens would have the prefix “MS” when in Ml (SV40) and “Ml ” when in JCV [e.g., MS (L157-T) and Ml (L157-T)].

248

TAMS AND FRISQUE

point of the cycle, DNA replication. To begin to understand the molecular basis for this restriction, we focused on the first step in initiation of replication, binding of T antigen to the origin of DNA replication. Previous work in our laboratory has indicated that the JCV and SV40 T antigens interact with the viral DNA differently, and that these differences may be responsible, in part, for JCV’s inefficient DNA replication. In the present study 10 JCV T antigen mutants were constructed, 9 of which increased homology between JCV and SV40 in sequences homologous to the SV40 DNA binding domain. It was expected that some of these mutants would display increased DNA binding and replication activities. The binding activity of the mutant proteins was changed in two ways. First, binding efficiency of most mutant T antigens was enhanced; relative to wild-type JCV T antigen, 7 of 10 mutant proteins were more efficient at binding JCV DNA, and 4 were more efficient at binding SV40 DNA. Second, DNA substrate preference of the mutant JCV T antigens was altered; like SV40 T antigen, the mutant proteins displayed a reduced preference for SV40 versus JCV sequences. The strong preference of wild-type JCV T antigen for SV40 sequences over its own is likely due to a particular conformation of the binding domain, as it can be disrupted by alterations to many individual amino acids. Wild-type JCV T antigen binds BS II weakly (Lynch and Frisque, 1991; J. Tavis, unpublished observations). Our inability to detect binding of the mutant JCV T antigens to JCV BS II was probably due to the low levels of T antigen present in the transformed BHK-21 cell lines used as protein sources. Since binding of purified SV40 T antigen to SV40 BS II is stimulated by ATP (Dean et a/., 1987b; Deb and Tegtmeyer, 1987; Borowiec and Hurwitz, 1988; Mastrangelo et a/., 1989) attempts were made to enhance JCV T antigen binding by adding 4 mlLI ATP to the binding reactions. Under these conditions nonspecific DNA binding was greatly increased, and it completely masked specific DNA binding (data not shown). The use of crude nuclear extracts rather than purified protein may account for our inability to reproduce the ATP effect. The amino acids altered in the JCV T antigen probably do not form protein-DNA contacts during sequence-specific DNA binding. This speculation is based on three observations. First, both SV40 and JCV T antigens recognize the same pentanucleotide sequence, and as this motif has been conserved through evolution, it is likely that protein sequences recognizing it also have been maintained. Six of the seven residues that were altered are not conserved between JCV and SV40. Second, alteration of contact points might

be expected to disrupt binding function; however, all mutant JCV T antigens bound the viral DNAs. Finally, Simmons et a/. (1990b) have speculated that aa 153, 155, and 204 of the SV40 T antigen form sequencespecific DNA contacts; these positions are conserved between JCV and SV40 and have not been altered here. Our data suggest that the mutations to the JCV T antigen may have altered the conformation of the DNA binding domain and that this may have changed the protein-DNA interaction. Simmons and co-workers (1990a,b) proposed a model for SV40 T antigen DNA binding in which four major amino acid sequence elements coordinate DNA binding. Two of these elements are thought to determine sequence specific binding, and they may directly contact the DNA: “A” (SV40, aa 147-l 59, particularly subregion “Al ‘I, aa 152-l 55), and “B2” (aa 203207). A third element (“Bl “, aa 183-l 87) is proposed to mediate nonspecific binding. The fourth region (“B3”, aa 2 15-219) is believed to be required for binding to BS II but not to BS I. Regions Al, Bl, B2, and B3 are identical between JCV and SV40. Our data can be interpreted within this model. The mutated residues are within sequences roughly homologous to the A element, but do not correspond to any of the probable contact residues located within subregion Al. If the JCV and SV40 A elements function similarly, the sites we altered would contribute to the structure of the binding domain and would not contact the DNA. Furthermore, this model may offer an explanation for the poor binding activity of SHLCI-T. In this case SV40 sequences form one complete binding element (A), and JCV sequences form the remaining elements (Bl, B2, and 83); the heterologous binding elements may not cooperate well while interacting with the DNA. The relative effect of each of the mutations on DNA replication activity was similar in both cell types tested, despite the differences in viability of the viruses and in levels of DNA synthesized. The only mutants with significantly different replication patterns in the two cell types were M 1(R 13 1-T) and M 1(Sl45-T). Replication by these viral DNAs was equivalent to that of wild-type JCV DNA in PHFG cells, but was elevated in CV-1 cells. JCV does not undergo a productive infection of CV-1 cells, but the block to its lytic cycle is not entirely the result of inefficient transcription during the early phase of the viral replication cycle, as has been proposed by Feigenbaum and co-workers (1987). Although limited, JCV DNA replication in CV-1 cells indicates that the JCV early signals and replication origin do function in CV-1 cells (Fig. 4). Productive infection of these cells by the hybrid virus SV40(Ml), which contains the SV40 protein coding sequences and the JCV regulatory region, confirms this observation (Chuke et a/., 1986).

JCV T-ANTIGEN

The inability of JCV to produce infectious virions in CV1 cells suggests that either DNA replication is too inefficient to sustain a productive infection, or that an additional block occurs later in the cycle. M 1(SV40) DNA replication was also detected in CV1 cells, demonstrating for the first time a productive interaction between the JCV T antigen and SV40 origin sequences. However, this interaction was very inefficient; the JCV T antigen promoted DNA replication from the SV40 origin at only about 2% of the level seen with the JCV origin in CV-1 cells. Therefore, it is probably valid to consider the JCV T antigen to be essentially restricted to JCV origin sequences for promotion of DNA replication. None of the mutations to the JCV T antigen DNA binding domain markedly relieved this restriction. Overall, the pattern of DNA replication promoted by the set of mutant proteins did not correlate with their level of DNA binding (compare Tables 2 and 4); the DNA replication activities of the Ml(SV40) mutants also did not correlate with the DNA binding of the mutant T antigens to SV40 sequences (compare Tables 2 and 5). Our binding data reflect binding to BS I, not to BS II, yet we suspect that the alterations to DNA binding affinity of the mutant proteins to BS II is probably similar to that to BS I, and that these potential alterations to BS II binding affinity are not directly responsible for the alterations observed in DNA replication. The SV40 T antigen sequences that affect binding to BS II differently from that to BS I are aa 215-219 (Simmons et a/., 1990a) and aa 260-270 (H&s et al., 1990); our mutations did not affect the homologous JCV sequences. Given the multiprotein complex that assembles around T antigen during replication initiation, the alterations to the JCV T-antigen DNA binding domain may have changed its conformation or alignment when bound to the DNA, and this in turn may have affected the assembly or function of the replication apparatus around T antigen. If this is the case, the DNA binding activity of the JCV T antigen would contribute to its restricted DNA replication activity, but that contribution would not be a direct or simple one.

ACKNOWLEDGMENTS We thank Brigitte Bollag for the cell lines cMlcl3 and cSVcl3. Frank A. White III for the analysis of the effect of serum on CAT expression induced by the JCV and SV40 early promoter-enhancer elements, Jennifer J. Swenson for assistance with the temperature sensitivity assay, Ann Marie Daniel for preparation of the PHFG cultures, and Dr. Kevin 1. Lynch for the POJ nuclear extracts and for many helpful discussions. This work was supported by Public Health Service Grant CA-38789 from the National Cancer Institute. J.T. was supported in part by a National Science Foundation graduate fellowship.

MUTANTS

249

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