Jun 3, 1986 - Edith Trost Sorensen2, Kenneth L.Powell4.7,. Jane E.Walsh-Arrand38 and John ... Aarhus, Aarhus C, Denmark, 3Imperial Cancer Research Fund, Lincoln's. Inn Fields, London WC2A ... Communicated by B.E.Griffin. We have ...
The EMBO Journal vol.5 no.8 pp. 1959- 1966, 1986
Identification of an Epstein-Barr virus-coded thymidine kinase
Edward Littler1, Jesper Zeuthen2 5, Alison A.McBride36, Edith Trost Sorensen2, Kenneth L.Powell4.7, Jane E.Walsh-Arrand38 and John R.Arrand38 'Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester, M20 9BX, UK, 2Institute of Human Genetics, University of Aarhus, Aarhus C, Denmark, 3Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, and 4Department of Microbiology, University of Leeds, Leeds LS2 9JT, UK 5Present address: Novo Research Institute, Novo Alle, Bagsvaerd, Denmark 6Present address: Department of Virology, Royal Postgraduate Medical School, London W12 OHS, UK 7Present address: Department of Biochemical-Virology, Wellcome Research Laboratories, Beckenham, Kent, UK 8Present address: Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, UK Communicated by B.E.Griffin
We have demonstrated the presence of an Epstein-Barr virus (EBV)-coded thymidine kinase (TK) by producing biochemically transformed, TK-positive mammalian cell lines using either microinjection of whole EBV virions or calcium phosphate-mediated transfection of the Sall-B restriction endonuclease fragment of EBV DNA. Analysis of these cell lines showed that: (i) EBV DNA was present in the cell lines, (ii) sequences from the SailI-B restriction endonuclease fragment of EBV were expressed, (iii) a TK activity was present and (iv) a protein with antigenic cross-reactivity with the herpes simplex virus (HSV) TK was produced. The identity of the EBV TK gene was determined by demonstrating that a recombinant plasmid, which expressed the protein product of the BXLF1 open reading frame as a fusion protein, could complement TK- strains of E. coli. A comparison of the predicted amino acid sequences of the TK proteins of EBV and HSV-1 revealed significant regions of homology. Key words: Epstein-Barr virus/thymidine kinase/expression/ transformed cells Introduction The members of the herpes virus group induce at least four wellcharacterised replication-related enzyme activities in infected cells, a DNA polymerase, a ribonucleotide reductase, an alkaline exonuclease and a thymidine kinase (TK) (Cheng and Ostrander, 1976; Hoffman and Cheng, 1978; Huang, 1975; Keir et al., 1966; Cohen, 1972; Henry et al., 1978; Ogino et al., 1977). In the case of Epstein-Barr virus (EBV) there is considerable evidence for the induction of a novel, virus-specified DNA polymerase and an alkaline exonuclease (Datta et al., 1980; Grossberger and Clough, 1981; Ooka et al., 1979; Cheng et al., 1980; Clough, 1979) but the evidence for an EBV-coded TK is controversial (see for example Colby et al., 1981). Evidence for the existence of an EBV TK is provided by the observations that induction of Raji TK- cells by superinfection with the P3HR-1 strain of EBV or microinjection of P3HR-1 DNA into Raji TK - cells leads to the expression of a TK activity (Roubal and Klein, 1981; Stinchcombe and Clough, 1984; IRL Press Limited, Oxford, England
Graessmann et al., 1980). It was also observed that chemical induction of a TK- derivative of the P3HR-1 cell line led to the synthesis of EBV early and late antigens and also to the incorporation of [3H]thymidine into viral DNA (Ooka and Calender, 1980; Ooka et al., 1983). Additional circumstantial evidence for the presence of an EBV-coded TK is suggested by studies into the properties of the nucleoside analogues 9-(2-hydroxyethoxymethyl)guanine (acyclovir), 9-(1,3-dihydroxy-2-propoxy-methyl)guanine (DHPG) and arabinofuranosyl thymine (araT). Acyclovir has been shown to be a potent anti-EBV chemotherapeutic agent (Pagano and Datta, 1982; Sullivan et al., 1982; Pagano, 1983; Emberg and Andersson, 1986) whilst DHPG and araT have been shown to prevent EBV replication in vitro (Ooka et al., 1983; Lin et al., 1984). In herpes simplex virus (HSV)-infected cells the activation of these nucleoside analogues to their cytotoxic nucleotides is dependent upon the virus-coded TK for the initial phosphorylation to the nucleoside monophosphates (Furman et al., 1979). The remaining phosphorylation steps are accomplished by host-specified enzymes. By analogy this argues for the existence of an EBV TK. Support for this concept is provided by the production of nucleoside monophosphates of acyclovir, DHPG and araT by EBV genome-containing cells but not by EBV-negative lymphoid cell lines (Ooka et al., 1983; Colby et al., 1981). The major difficulty in definitively proving the existence of an EBV-coded TK is the lack of a fully permissive tissue culture system for EBV replication. Consequently, analysis of virus products is restricted to EBV-transformed cell lines which contain only small amounts of virus-specified protein. Similarly it is not possible to generate conditional lethal mutants of EBV which would allow identification of virus-coded functions. We preliminarily reported the establishment of biochemically transformed cell lines by microinjection of whole EB virions into mouse 3T3 TK- cells (Zeuthen, 1983). We now describe in detail the analysis of one such cell line and a subsequent line which was biochemically transformed to the TK+ phenotype by calcium phosphate-mediated transfection of a discrete region of the EBV genome. We demonstrate that these cell lines express limited regions of the EBV genome, contain a TK enzymatic activity and possess a protein which has antigenic cross-reactivity with the HSV TK protein. We show that a protein which exhibits antigenic cross-reactivity with the HSV TK enzyme is present in Raji cells which have been chemically induced to express EBV early antigens. Finally, we describe the construction of plasmids expressing the BXLF1 open reading frame of EBV as a fusion protein which can complement the defect in TK-deficient strains of E. coli.
Results To determine whether EBV contains a gene which could function operationally as a TK, whole EBV (B95-8) was microinjected into approximately 100 3T3 TK- cells. Five days after injection the cells were selected in HAT medium and resistant cells grown into monolayers. One of the major phenotypic
1959
E.Littler et al.
S X I G D R H
A B C E FG1G2 H I J D
I7-
ft
O..
4w
2
Is
a_ 5
3
LM(Sal IB)
3T3(EBV)
IJ
G1LG2 FNMK
A
I
I
I I I -1 II
--W
C
I
I
1
--'Y H
L I I~ ~I ~~~~~
1 II
II
1111
I1
E
B I
I
1 1
I
C
II 1 1 11
1
F QUPO MSL E ZRK a
H
Dhet
EcoRI
Nhet
BamHI
I
B
G
D
cb
e
TXV d
1
A
B95-8 EBV DNA
Sal lB --0
-I
I
20
40
60
80
100
110
6
Megadaltons Fig. 1. Hybridisation of cellular poly(A)+ RNA to cloned fragments of EBV DNA. In panels 1, 2 and 3 DNAs containing individual cloned EcoRI fragments of EBV DNA were cleaved with either EcoRI plus BamHI (lanes A-C) or EcoRI alone (lanes D-J). In panels 4 and 5 plasmid pSal-B containing the SallB fragment of EBV was digested with BamHI and Sall (lane S) and plasmids containing the BamHI-X, -I, -G, -D, -R and -H fragments of EBV were digested with BamHI (lanes X-H). The products were separated on 0.8% agarose gels. Ethidium bromide-stained gels are shown in panels 1 and 4. After transfer to nitrocellulose membranes the filters were hybridised to partially degraded, in vitro labelled poly(A)+ RNA as described by Arrand and Rymo (1982). RNA was obtained from 3T3 TK- parental cells (panel 2) 3T3 TK+ (EBV-microinjected) cells (panel 3); and LM TK+ (Sal-B-transformed) cells (panel 5). Panel 6 summarises the pattern of RNA expression detected in both transformed cell lines, relative to the EcoRI and BamHI restriction endonuclease maps of B95-8 EBV DNA. Fragments hybridizing to RNA probes are shown in solid bars. Also shown is the SalI-B fragment of B95-8 EBV DNA.
1960
EBV
characteristics of the cell line was that the cells grew very slow-
ly, were often multinucleate and had long cytoplasmic extensions.
The cells
were
seeded onto slides and stained for EBNA with
appropriate positive and negative control cells (Raji and BJAB). The biochemically transformed cells were negative for EBNA and this characteristic was maintained after many months in culture (data not shown). This result is similar to that of Graessmann et al. (1980) who could not detect EBNA expression in human or rat fibroblasts following microinjection of P3HR-l EBV DNA. To compare the relative growth rates of the microinjected 3T3 cells, the 3T3 TK- parental cells and wild type 3T3 TK+ cells, a simple growth curve was made. The results clearly showed that the EBV-injected cells grew more slowly in HAT medium than wild type 3T3 TK + cells. The 3T3 TK- cells failed to multiply and died after 3 days (data not shown). This suggests that the TK activity in the EBV-injected cells is less efficient than the endogenous cellular TK activity and may indicate that the enzyme activity is of EBV origin. Expression of the EBVgenome in biochemically transformed cells Expression of EBV sequences in transformed cell lines can be rapidly identified by hybridizing in vitro labelled cytoplasmic RNA to Southern blots of cloned EBV DNA fragments (Arrand and Rymo, 1982). We applied this technique to analyse the expression of the EBV genome in the microinjected 3T3 TK+ cell line. The results are shown in Figure 1. RNA from both the EBVmicroinjected TK+ cell line (panel 3) and the parental 3T3 TKcell line (panel 2) showed homology to the plasmid vector sequences. The basis of this cross-reaction is unclear. In these experiments vector DNA co-migrated with EBV BamHI fragments
I and K and parts of BamHI-A and E and thus any EBV-specific
hybridization to these fragments would be masked. In addition, sequences contained within the EcoRI fragments D and GI and the BamHI fragments F, Q, W and Y were found to hybridize to RNA probes from both the parental and microinjected cell lines. This is in accordance with the previous findings that RNA from all cell types examined, whether EBV infected or not, shows homology with sequences within EcoRI-D and -GI and BamHIF and -Q (Arrand et al., 1983) and that an EBV BamHI-W, -Y probe exhibits very extensive cross-homology with mouse 3T3 cellular sequences (Peden et al., 1982). However, specific hybridization was detected between RNA from the microinjected 3T3 TK + cell line and the BamHI-V and -X fragments (panel 3, lane C) and to the EcoRI-H fragment (panel 3, lane H). These three fragments which show EBVspecific hybridization are directly adjacent to one another on the EBV restriction enzyme map suggesting that this region of the genome may contain a transcriptional unit which codes for a TK enzyme. These results are summarised graphically in Figure IF. The EBV genomic fragments BamHI-X and -V and EcoRI-H are contained within the Sall-B fragment of EBV DNA (Dambaugh et al., 1980 - see panel 6). We therefore attempted to transform biochemically a mouse LMTK- cell line with plasmid DNA containing the Sall-B restriction enzyme fragment of EBV by calcium phosphate-mediated transfection. The resulting HATresistant cell lines once again grew only slowly and had an altered morphology. Expression of EBV sequences was examined as described above using Southern blots of the SalB-related BamHI fragments (see panel 6). The results in panel 5 show that this cell line again contains RNA which hybridizes with plasmid vecsequences. However EBV-speciflc expression was found from the whole of the original SalI-B fragment of EBV. A hybridiza-
tor
thymnidine kinase
tion control consisting of the EBV BamHI fragments R and H (which are not contained within Sail-B) did not react with the probe. Long-term maintenance of EBVDNA in biochemically transfomned cell lines To determine which EBV DNA sequences were maintained in the TK+-transformed cell lines, high mol. wt DNA from cells maintained for at least 20 passages was digested with BamHI or EcoRI and analysed on Southern blots using either EBV genomic DNA or the Sal-B fragment of EBV as probes. In DNA from both the 3T3TK- and LMTK- cell lines there was no evidence of any hybridization with either the genomic EBV probe or with the Sail-B probe. However, in both respective transformed cell lines the high mol. wt DNA contained most, if not all, of the original input EBV DNA sequences (data not shown). TK activity in cell lines biochemically transformed by EBV Although growth of the EBV-transformed, phenotypically TK+ cell lines in HAT medium suggested that a TK was being expressed, it was possible that growth of these cell lines in HAT was due to an alteration in the dihydrofolate reductase which is blocked by the aminopterin component of the HAT selection. This would result in a restoration of thymidylate synthetase activity, thus allowing de novo synthesis of thymidine. To determine if any TK activity was present the biochemically transformed cell lines and their TK- parents were grown in the presence of [3H]thymidine and the incorporation into cellular DNA determined. The results are shown in Figure 2a. It is obvious that the phenotypically TK+-transformed cell lines contained high levels of TK activity. The peak levels were seen 2 days after addition of labelled thymidine and were > 40 times higher than in the parental TK- cell lines. The incorporation of [3H]thymidine into nuclei of cells has been considered an adequate demonstration of a thymidine kinase activity (Butyann and Spear, 1981). We therefore incubated the TK-transformed cells with [3H]thymidine and subsequently exposed them to photographic emulsion. The transformed cells could clearly be seen to incorporate [3H]thymidine into their nuclei (Figure 2b). Analysis of biochemically transformed cells using anti-HSV TK serum We have shown that antisera specific for HSV-encoded proteins can be used as powerful reagents to probe for the presence of cross-reactive proteins in EBV-transformed cell lines (E.Littler, E.K.Wagner, K.G.Draper, K.L.Powell, A.A.McBride and J.R. Arrand, in preparation). We examined the EBV-microinjected and Sall-B transformed TK+ cell lines using a high-titre mouse anti-HSV TK serum in an indirect immunofluorescence test. The results are shown in Figure 3. Both of the biochemically transformed cell lines contained a protein which cross-reacted with the HSV TK antiserum (Figure 3B and D) whilst the parental TK- cell lines did not (Figure 3A and C). Thus the ability to survive HAT selection, the expression of a specific region of the EBV genome and the appearance of a TK activity in the transformed cell lines correlated with the appearance of a protein which cross-reacts with an antiserum against the HSV TK protein. To determine if such a cross-reactive protein is present in EBVpositive lymphoblastoid cell lines a number of such cell lines were similarly examined by indirect immunofluorescence (Figure 3E and F). The EBV-positive cell line Raji which had been induced using TPA and sodium butyrate, showed a reactivity with the anti-HSV TK serum. Similarly treated EBV-negative B-cell lines (such as Ball- 1) showed no reactivity. A low reactivity was pres-
1961
E.Littler et al.
a 300
C: .4
0 Q
,, 200E
1no
.0 x
E o 100-
U
0-I 0
b
ltk
F bT
-
-. .. .;, i4
N .
v
-.~ .ip ^
O
e
.
C
1 2 Days of incubation
3
>
Fig. 2.(a). Detection of TK activity in cell lines. Cell lines; (U U) 3T3 TK+ (EBV-injected); (0-0) LM TK+ (Sall-B transformed); (O-O) 3T3 were seeded at low density (5 x 105 cells/dish) in MEM containing 1 ICi [3H]thymidine/ml. Cells were harvested at 1, 2 and 3 days and the incorporation of [3H]thymidine into high mol. wt DNA was determined by TCA precipitation followed by scintillation counting. The results were normalised for protein concentration. (b) Cells (A) 3T3 TK-; (B) 3T3 TK+ (EBV-injected); (C) LMTK-; and (D) LM TK+ (Sall-B transformed); were grown on coverslips as above, harvested and fixed. The coverslips were immersed in photographic emulsion and after 3 days developed and fixed. After counterstaining with 0.01 % saffron the coverslips were examined by light microscopy. Extensive incorporation of [3H] into the cell nuclei is apparent in panels B and D.
TK-; (0-0) LMTK-;
1962
EBV thymidine kinase
Fig. 3. Immunofluorescent staining with anti-HSV TK serum of transformed cells or of EBV-infected cells. Cells, either 3T3 TK- (A); 3T3 TK+ (EBVinjected) (B); LM TK- (C); LM TK+ (SalI-B transformed) (D); Raji (E); or Raji induced as described in Materials and methods (F); were washed and fixed in acetone. The fixed cells were reacted first with a mouse serum specific for the HSV-1 TK protein and then with FITC-conjugated anti-mouse secondary antibody. Fluorescing cells were observed using indirect u.v. microscopy. Most cells in B or D reacted with the TK antiserum although the LM TK+ (Sall-B transformed) cell line reacted more weakly than the 3T3 TK' (EBV-injected) cells. Approximately 10% of the Raji-induced cells reacted with the anti-HSV TK serum.
ent in EBV producer cell lines B95-8 and P3HR- 1 (data not
shown). Expression of EBV-coded thymidine kinase activity in bacteria The previous results suggest that the Sall-B restriction endonuclease fragment of EBV contains a TK gene. Baer et al. (1984) noticed that the BamHI-X fragment of EBV contained an open reading frame (BXLF1) with limited amino acid homology to the HSV TK. The BamHI-X fragment of EBV maps within the Sall-B fragment and both the 3T3 and LM TK+ transformants express this region of the EBV genome. We therefore expressed the BXLF1 open reading frame of EBV in E. coli to determine if plasmids expressing this open reading frame could complement bacterial TK- strains. The construction of the plasmid is described in Materials and methods. The results are shown in Table I. Recombinant plasmids expressing the HSV
TK and EBV BXLF1 complemented the TK- E. coli whilst plasmid pUC8 and a plasmid containing the BXLF1 in the reverse orientation did not. The frequencies of transformation of the TK- E. coli using both the HSV TK construct and EBV construct were approximately the same. To characterise further the products expressed in the recombinant plasmids, proteins coded by the plasmids were expressed using in vitro transcription/translation. The products were analysed using SDS-polyacrylamide electrophoresis. The results are shown in Figure 4. A comparison of the polypeptides expressed by the various recombinants showed that additional proteins were apparent. In HSV the size of the major species was approximately 40 kd whilst in EBV a doublet of 70 kd was observed. An estimate of the mol. wts of both proteins based upon their predicted amino acid sequence would give values of 44 and 67 kd,
respectively. 1963
E.Littler et al.
m
Table I. Complementation of TK- E. coli with herpes virus TK genes Plasmid
pUC8 pUCX (EBV TK) pUCrX (EBV TK) pSK1
Concentration of 5-FU (/Ag/ml) 21 18 0 15
110 72 60 85
l15 70 5a
92
-
84 -
70
-
40 -
47
24 -
37 -
52
aColonies were very small. Competent TK- E. coli (C600 tdK) were transformed with 10 ng plasmid DNA and aliquots plated onto agarose containing 20 Atg/ml ampicillin, 25 pgg/ml uridine, 25 ttg/ml thymidine and varying amounts of 5-fluorouracil (5-FU) shown. After 4 days plates were scored for the number of colonies. Plasmid pUCX contains the EBV BXLF1 open reading frame in the correct orientation for expression as a fusion protein whilst plasmid pUCrX contains the BXLF1 open reading frame in the opposite orientation. Plasmid pSKI contains the HSV TK gene and has been described previously (Kaehler et al., 1984).
Analysis of the amino acid sequence of TK proteins Baer et al. (1984) have reported a 'limited' amino acid homology between HSV TK and EBV BXLF1. More recently Gentry (1985) identified a conserved nucleotide binding site in both HSV and vaccinia virus TK proteins whilst Darby et al. (1986) identified both the nucleotide and nucleoside binding sites in HSV-1 TK. The latter authors also defined a cysteine residue in HSV TK which is a component of the nucleotide and nucleoside binding sites. We compared the HSV-1 TK amino acid sequence with the predicted amino acid sequence of the EBV BXLF1 open reading frame. Alignment of the two sequences was based upon their highly conserved nucleotide binding sites which resulted in the BXLF1 protein having an additional 243 amino acid residues N-terminal to the start of the HSV TK protein. Figure 5 shows an alignment of the HSV-1 TK sequence with the EBV BXLF1 sequence from amino acid 244 to the end of the open reading frame at amino acid 607. This comparison demonstrates significant homology between the HSV-1 TK sequence and the EBV BXLF1 open reading frame (27% identical amino acids and a further 17% conservative amino acid changes) suggesting a functional similarity between the proteins. Discussion Much previous evidence for an EBV-coded TK has been largely indirect and inconclusive (Colby et al., 1981; Pagano and Datta, 1982). The analysis of TK- derivatives of EBV-containing lymphoblastoid cell lines and the observation of the antiviral effect of nucleoside analogues on EBV replication suggests the presence in EBV-positive cell lines of a TK activity which does not resemble the normal host cell enzyme (Ooka et al., 1983; Lin et al., 1984; Stinchcombe and Clough, 1984). One of the characteristic properties of the HSV-coded TK gene is its ability to transform biochemically TK- cell lines (Munyon et al., 1971, 1972; Wigler et al., 1977; Bacchetti and Graham, 1977; Wilkie et al., 1979). We have shown here that whole EBV DNA and in particular the Sall-B fragment of the genome has a similar property. This suggests that the TK+ phenotype of the biochemical transformants is the result of the presence of EBV DNA sequences but not the result of EBV replication. Furthermore our studies of the expression of EBV genes in the TK + transformants suggested that the Sall-B fragment of EBV DNA is likely to contain the gene(s) involved in the induction of this novel TK. Following our preliminary report of this (Zeuthen, 1983), Baer et al. (1984) noted a limited amino acid homology between the HSV-2 TK protein and the predicted protein pro-
1964
a b c de f
200 94 V.A
4
69 46 *
Fig. 4. Expression of recombinant TK genes by in vitro transcription/translation of plasmid DNA. (a) pUC8; (b) pBR322; (c) no plasmid DNA; (d) pSKI (containing the HSV TK gene); (e) pUCrX (containing the EBV BXLF1 open reading frame in the incorrect orientation for expression) and (f) pUCX (containing the EBV BXLF1 open reading frame in the correct orientation for expression) were in vitro transcribed and translated (Amersham, Ltd) using 50 gCi [35S]methionine to label the products. Labelled polypeptides were analysed by SDS-polyacrylamide gel electrophoresis and autoradiography. Mol. wt markers are shown (m).
duct of the BXLF1 open reading frame of EBV, which is contained in the SalI-B fragment. We suggest that the ability of recombinant plasmids which express the protein product of BXLF1 to complement the defect in TK- strains of E. coli in the same manner as HSV TK provides conclusive evidence that this open reading frame contains a gene which codes for a protein with TK activity. Comparison of the amino acid sequences of the EBV and HSV TK proteins suggests that common features are contained within the two proteins. The lack of extensive amino acid sequence homology between the two enzymes is not an unusual feature of protein conservation. A number of examples of alteration in amino acid sequence whilst retaining protein structure and function are well characterised (Ollis et al., 1985; Ploegman et al., 1978; Dickerson and Geis, 1983). The EBV and HSV TKs are substantially larger than the TKs of vaccinia virus or eukaryotes. This may be explained by the observation that eukaryotic (and vaccinia) TKs are exclusively thymidine kinases (Kwoh and Engler, 1984) whereas the HSV and EBV enzymes are different in that they are deoxynucleoside kinases with a broad substrate specificity (Jamieson and SubakSharpe, 1974; Chen and Prusoff, 1978; Ooka et al., 1983). In addition there is a considerable amino acid sequence in the EBV TK which is proximal to the first methionine of the HSV TK sequence. Similar phenomena have been observed when comparing a number of HSV and varicella zoster virus homologous genes (Davison and McGeoch, 1986).
EBV thymidine kinase M
NVLNLDDAQDTRQAKAQRKES * * ** ** *.* ** . **
MRVPIVTHLTNHVPVIKPAC[SL
*.*..I
FLEGAP
**** MASYPCHQHASAFDQAARS RGHSNRRTALRPRRQQEATEVRLEQKMPTLLLRVY I DGPH 1 Nucleotide *
GVGKTTML1NHLKAVFGDLTIV *-**** *
*-
*
*
*
VPEPMRYWTHVYENAIKAMHKNVTRARHGREDTSAE
**
*****
**
*-
*
GMGKTTTTJQLLVALGSRDDIVYVPEPMTYW _ Biniding Site
*
*
*
*
**
QVLGASETIANIYTTQHRLDQGEISAG
ESGARSVAPLDCWIL FMDRHLLSASVVF * * CQMKFTTPFRVCASRKRSLLV ** * * * * * * * * ** * * I*** ** * DAAVVMTSAQITMGMPYAVTDAVPAPHVGGEAG SSHAPPPALTLIIFDRHPIAA
VLA
, _
PLMLLRSQLLSY *
**.
**
SD *
*
IQ **
VLATFTA *** * *1
FVAjLIPP
LLCYPAARYLMQSMTPQAVLA Nucleoside Binding Site --
~~~(1)
DPGDTIVWMKLNVEENMRRLKKRGR * * ** **-* ** * .***TLPGTNIVLGALPEDRHI DRLAK RQR
KHES GLDAGYLK SVNDAYHAVYCAWLLTQ *
PGE
* **
*
*
RLDLAML
YFAPED IVKVCAGLTTI TTVCHQSHT * * * * *G * * *T* *.LLAN T V RY LG_PG GSWW ED W GQL S G T AV PP QGA ___ E_
***I
**
AAIRRVYG
PIIRSGVAEKYKNSIFSVLKEVIQPFRADAVLLEVCLAFTRTLAYLQFVLV * * * * * * * * * * * ** * * * ** * **.*
PQSNAG
PRPHIGDTLFTL
FRAPE
LLAPNGDLYNVFAWALDVLAKRLRPMH
DDLP
GCWTEIYMQALKNPAIRSQFFDWA
*
* *
*
VFILDYDQSPAGCRD
* *
*
*
*
DLSEFQ *
GLSKVISDFER *
* **.*
*
GNRD *
ALLQLTSGMQTHVTTPGSIPTICDLARTFAREMGEAN
(2) Fig. 5. Comparison of amino acid sequences of HSV-l TK (lower) and the predicted amino acid sequence of the BXLF1 open reading frame of EBV (upper) from residue 244 to the terminus. LDNA (Stockwell, 1982) and HOMOL (Taylor, 1984) sequence handling programs were used in this analysis. Identical amino acid residues are indicated by a * between the two sequences, conservative amino acid changes are indicated by a Amino acids in the HSV-1 sequence which have been shown to vary either between strains of HSV-1 or between HSV-1 and HSV-2 are underlined. The nucleotide and nucleoside binding sites are shown in boxes (Gentry, 1985; Darby et al., 1986). The alanine residue (1) determines the ability of the HSV-1 TK to utilise the nucleoside analogue BVdU as a substrate whilst the cysteine residue, (2) forms part of, or is near to, the nucleoside and nucleotide binding sites (Darby et al., 1986). There is 26% identity between the two sequences (with an additional 17% conservative changes), 39% identity between the nucleotide binding sites (with an additional 30% conservative changes) and 35% identity at the nucleoside binding site (with an additional 15% conservative changes). 28% of the differences between the two sequences occur at positions which show variations between HSV-1 strains or between HSV-1 and HSV-2. -
In conclusion, we have provided biochemical, immunological and genetic evidence that the product of the BXLF1 open reading frame of EBV codes for a protein with a TK activity similar to that of HSV. Clinically these findings provide some rational basis for the current practice of treating EBV infections with cytotoxic nucleoside analogues such as acyclovir (Ernberg and Andersson, 1986; Sullivan et al., 1982).
Materials and methods Cell lines The EBV-negative B-lymphoblastoid cell line Ball-I (Hiraki et al., 1977) and the EBV-positive lymphoblastoid cell lines Raji (Pulvertaft, 1965); and B95-8 (Miller and Lipman, 1973) were maintained as described previously (Arrand et al., 1983). Mouse 3T3 and LM cells were maintained in Dulbecco's Minimal Essential Medium (MEM) with 10% foetal calf serum. The TK- derivative of 3T3 (a gift from Dr Steffen Junker) and LM TK- cell lines were maintained in the same medium supplemented with 30 pg/ml of bromodeoxyuridine (BUdR) to eliminate any possible TK+ revertants.
Microinjection Microinjection of somatic monolayer cells was performed in a manner similar to that described by Graessman et al. (1980). The microinjection apparatus consisted of a Carl Zeiss Jena microsurgical manipulator where only the right-hand manipulator was slightly modified. The pipette holder was made of a 6-cm long bronze tube to avoid vibration of the micropipette which was connected to a pressure system (50 ml syringe) by a polyethylene tube (Celis et al., 1980) Borosilicate capillaries rinsed in 95% alcohol and air-dried were drawn to micropipettes (tip diameter - 1 /Am) by a mechanical puller. The micropipetters
were -y-sterilized for 1 h (total irradiation 55 000) and stored in a sterile glass canister. Monolayer cells were treated with EDTA for 3 min at room temperature, trypsin was added and the culture was incubated for 3 min at 37°C. The activity of the trypsin was stopped by adding growth medium. -y-Sterilized 3 x 3 mm glass slides were placed in a Petri dish (60 x 15 mm) and a drop of cell suspension was applied to each slide. The Petri dish was placed in a C02-incubator (5% C02) to enable the cells to attach themselves to the slide prior to addition of - 5 ml medium. Microcapillaries were loaded with approximately 10-3 ml of 100x concentrated B95-8 virus suspension and connected to the syringe used to inject single cells. The amount of material injected per cell was estimated by injection of either carrier-free [32P]orthophosphate (Ris, Denmark) or [1251]human serum albumin to be in the order of 10-11 in/cell in agreement with previous estimates (Celis et al., 1980). Cells were placed in a Petri dish containing about 5 ml of medium and viewed with a phase-contrast microscope at a 140 x magnification. The cells were focused and the tip of the pipette brought into focus with the cells. The tip was brought into a position just outside the target cell and it was found to be sufficient to touch the cell in order to perform the injection. The flow through the pipette was regulated by the syringe. After a cell had been injected, the pipette was raised by means of the tangent screw and moved to the next cell. After injection, the slide was placed in a Petri dish with fresh medium and maintained in a C02-incubator.
Plasmids B95-8 EB virion DNA (a generous gift from L.Rymo) was cleaved with Sail, fractionated on sucrose gradients and the large fragments (A, B and C) were cloned in the SalI site of cosmid Homer I (Chia et al., 1982) as described previously (Arrand et al., 1981).
1965
E.Littler et al. The plasmid expressing the HSV TK (pSKl) has been described previously (Kaehler et al., 1984). Plasmid pUCX expressing the EBV BXLF1 open reading frame as a fusion protein was produced by cloning the BamHI-X restriction endonuclease fragment of EBV in the BamHI site of pUC8. This results in the first eight amino acids of the lacZ gene product preceding the first methionine of the BXLF1 open reading frame polypeptide. Calcium phosphate-mediated DNA-transfection Mouse LMTK- cells were transformed using I jg of Sall-B plasmid DNA and 10 ag cellular carrier DNA as described by Graham and van der Eb (1973). HAT selection was applied 2 days after transformation and resistant colonies were picked after 2-3 weeks.
Immunofluorescence Indirect immunofluorescence of EBV-injected 3T3 TK+ or transformed LM TK+ cells was performed as described previously (E.Littler, E.K.Wagner, K.G.Draper, K.L.Powell, A.A.McBride and J.R.Arrand, in preparation). Hyperimmune serum to HSV-1 TK was obtained from Balb C mice which had been immunized with purified TK protein (Banks et al., 1984). TK assays Mouse LM TK- and 3T3 TK- cells and their corresponding TK+ transformants were analysed for TK activity by plating in MEM containing 10% dialysed foetal calf serum and 1 PCi/mn [3H]thymidine (Amersham, Ltd). To assay for TK activity cells were washed with phosphate-buffered saline (PBS) and scraped into Eppendorf tubes. After mixing with cold 10% trichloroacetic acid (TCA) the cellular DNA was precipitated on ice for 10 min and washed five times with fresh 10% TCA. [3H]Thymidine incorporation was measured by scintillation counting in Aquasol (New England Nuclear Ltd) and normalized for protein concentration of the cell extracts. For direct examination of [3H]thymidine incorporation into nuclear DNA cells were grown on glass coverslips. They were washed with PBS and incorporated [3H]thymidine was detected by direct autoradiography as described by Butyann and Spear (1981). Nuclear staining was examined by light microscopy. High mol. wt DNA and Southern blot analysis High mol. wt DNA was prepared as described previously (Arrand et al., 1983). Conditions for restriction endonuclease cleavage, agarose gel electrophoresis and Southern blotting have been described previously (Arrand and Rymo, 1982). RNA extraction and end-labelling Cytoplasmic polyadenylated RNA was extracted and 32P-labelled in vitro as described by Arrand and Rymo (1982). Selection of TK-expressing E. coli The TK- derivative of E. coli (C600 tdk) has been described by Kaehler et al. (1984). Competent cells were transformed with 10 ng plasmid DNA using the colony transformation method of Hanahan (1983). Transformed bacteria were plated onto LM agar containing 20 Ag/ml ampicillin, 25 ytg/ml thymidine, 25 ttg/ml uridine and varying concentrations of 5-fluorouracil (5-FU). Colonies took approximately 3 days to appear on plates containing 5-FU. In vitro transcriptionltranslation 5 Ag plasmid DNA was used for in vitro transcription/translation using a kit obtained from Amersham International.
Acknowledgements The excellent technical assistance of Yvonne Connolly is gratefully acknowledged. We thank Michael Strauss for providing the E. coli C600 tdk strain and plasmid pSK- 1. This work was supported in part by the Cancer Research Campaign (UK), the Danish Cancer Society, the Danish Natural Science Research Council and the Medical Research Council (UK). We are grateful to B.E.Griffin for critical reading of the manuscript.
References Arrand,J.R. and Rymo,L. (1982) J. Virol., 41, 376-389. Arrand,J.R., Rymo,L., Walsh,J.E., Bjorck,E., Lindahl,T. and Griffim,B.E. (1981) Nucleic Acids Res., 9, 2999-3104. Arrand,J.R., Walsh-Arrand,J.E. and Rymo,L. (1983) EMBO J., 2, 1673-1683. Bacchetti,S. and Graham,F.L. (1977) Proc. Natl. Acad. Sci. USA, 74, 1590-1594. Baer,R., Bankier,A.T., Biggin,M.D., Deininger,P.L., Farrell,P.J., Gibson,T.J., Hatful,G., Hudson,G.S., Satchwell,S.C., Seguin,C., Tuffnell,P.S. and Barrell,B.G. (1984) Nature, 310, 207-211. Banks,L.M., Vaughan,P.J., Meredith,D. and Powell,K.L. (1984) J. Gen. Virol., 65, 1625-1670. Butyann,R. and Spear,P.G. (1981) J. Virol., 37, 459-472.
1966
Celis,J.E., Kaltoft,K. and Bravo,R. (1980) In Celis,J.E., Graessmann,A. and Loyter,A. (eds), Transfer of Cell Constituents into Eukaryotic Cells. Plenum Press, pp. 1-28. Chen,M.S. and Prusoff,W.M. (1978) J. Biol. Chem., 253, 1325-1327. Cheng,Y.C. and Ostrander,M. (1976) J. Biol. Chem., 251, 2605-2610. Cheng,Y.C., Ehn,J.Y., Hoffman,P.J. and Glaser,R. (1980) Virology, 100, 334-338. Chia,W., Scott,M.R. and Rigby,P.W. (1982) Nucleic Acids Res., 10, 2503 - 2520. Clough,W. (1979) Biochemistry, 18, 4517-4521. Cohen,G.H. (1972) J. Virol., 9, 408-418. Colby,B.M., Furman,P.A., Shaw,J.E., Ellion,G.B. and Pagano,J.S. (1981) J. Virol., 38, 606-611. Dambaugh,T., Beisel,C., Hummel,M., King,W., Fennewald,S., Cheung,A., Heller,M., Raab-Traub,N. and Kieff,E. (1980) Proc. Natl. Acad. Sci. USA, 77, 2999-3003. Darby,G., Larder,B.A. and Inglis,M.M. (1986) J. Gen. Virol., 67, 753-758. Datta,A.K., Feighney,R.J. and Pagano,J.S. (1980) J. Biol. Chem., 255, 5120-5125. Davison and McGeoch (1986) J. Gen. Virol., in press. Dickerson,R.E. and Geis,I. (1983) Hemoglobin: Structure, Function, Evolution and Pathology. Benjamin/Cummings, Menlo Park, California, p. 72. Ernberg,I. and Andersson,J. (1986) J. Gen. Virol., in press. Furman,P.A., St.Clair,M.H., Fyfe,J.A., Ridedout,J.L., Keller,P.M. and Elion,G.B. (1979) J. Virol., 32, 72-77. Gentry,G.A. (1985) Proc. Natl. Acad. Sci. USA, 82, 6815-6819. Graessmann,A., Wolf,H. and Bornkamm,G.W. (1980) Proc. Natl. Acad. Sci. USA, 77, 433-436. Graham,F.L. and Van der Eb,A.J. (1973) Virology, 52, 456-467. Grossberger,D. and Clough,W. (1981) Biochemistry, 20, 4049-4055. Hanahan,D. (1983) J. Mol. Biol., 166, 557-580. Henry,B.E., Glaser,R., Hewetson,J. and O'Callaghan,D.J. (1978) Virology, 89, 262 -271. Hiraki,S., Myoshi,I., Musuji,H., Kubonishi,I., Matsuda,Y., Nakayoma,T., Kishimoto,M., Chen,P. and Kimura,I. (1977) J. Natl. Cancer Inst., 59. 93-94. Hoffman,P.J. and Cheng,Y.C. (1978) J. Biol. Chem., 253, 3557-3562. Huang,E.S. (1975) J. Virol., 16, 298-310. Jamieson,A.T. and Subak-Sharpe,J.H. (1974) J. Gen. Virol., 24, 481-492. Kaehler,R., Strauss,M. and Kiessling,U. (1984) Biomed. Biochim. Acta, 11, K25 -K29. Kwoh,J. and Engler,J. (1984) Nucleic Acids Res., 12, 3959-3971. Keir,H.M., Subak-Sharpe,H., Shedden,W.I.H., Watson,D.H. and Wildy,P. (1966) Virology, 30, 154-157. Lin,J.-C., Smith,M.C. and Pagano,J.S. (1984) J. Virol., 50, 50-55. Miller,G. and Lipman,M. (1973) Proc. Natl. Acad. Sci. USA, 70, 190-194. Munyon,W., Kraiselburd,E., Davis,S. and Mann,J. (1971) J. Virol., 7, 813-820. Munyon,W., Bauchstraum,R., Paoletti,E., Kraiselburd,E. and Mann,J. (1972) Virology, 49, 683-689. Ogino,T., Otsuka,T. and Takahishi,M. (1977) J. Virol., 21, 1232-1235. Ollis,D., Brick,P., Hamiin,R., Xuong,N.G. and Steitz,T.A. (1985) Nature, 313, 762-766. Ooka,T. and Calender,A. (1980) Virology, 104, 219-233. Ooka,T., Lenoir,G. and Daillie,J. (1979) Virology, 29, 1-10. Ooka,T., Calender,A., De Turenna,M. and Daillie,J. (1983) J. Virol., 46, 187-195. Pagano,J.S. (1983) J. Antimicrob. Chemother., 12, 1-10. Pagano,J.S. and Datta,A.K. (1982) Am. J. Med., 73, 18-26. Peden,K., Mounts,P. and Hayward,G.S. (1982) Cell, 31, 71-80. Ploegman,J.M., Drent,G., Kalk,K. and Hol,W.G. (1978) J. Mol. Biol., 123, 557-594. Pulvertaft,R.J.V. (1965) J. Clin. Pathol., 18, 261-273. Roubal,J. and Klein,G. (1981) Intervirology, 15, 43-48. Stinchcombe,T. and Clough,W. (1984) Biochemistry, 24, 2027-2033. Stockwell,P.A. (1982) Nucleic Acids Res., 10, 115-125. Sullivan,J.L., Byron,K.S., Brewster,F.E., Sakamoto,K., Shaw,J.E. (1982) Am. J. Med., 73, 262-266. Taylor,P. (1984) Nucleic Acids Res., 12, 447-456. Wigler,M., Silverstein,S., Leed,L.S., Pellicer,A., Cheng,Y.C. and Axel,R. (1977) Cell, 11, 223-232. Wilkie,N.M., Clements,J.J., Boll,W. Lonsdale,D. and Weismann,C. (1979) Nucleic Acids Res., 7, 850-877. Zeuthen,J. (1983) J. Cancer Res., Clin. Oncol., 106, 1 -11. Received on 18 March 1986; revised on 3 June 1986
