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Cloning and inducible synthesis of poliovirus nonstructural proteins
Gene, 117 (1992) 185-192
© 1992 Elsevier Science Publishers B.V. All fights reserved. 0378-1119/92/$05.00
185
GENE 06540
Cloning and inducible synthesis of poliovirus nonstructural proteins (Poliovirus gene expression; T7 RNA polymerase; membrane permeability; recombinant DNA; lyric toxic products)
J u a n L a m a , R o s a r i o G u i n e a , F r a n c i s c o M a r t i n e z - A b a r c a a n d Luis C a r r a s c o Centro de Biologia Molecular. Universidad Aut6noma de Madrid. Canto Blanco 28049 Madrid (Spain)
Received by A. Kohn: 16 December 1991; Revised/Accepted: 27 January/2 February 1992; Received at publishers: 1 April 1992
SUMMARY
The poliovirus nonstructural protein-encoding genes have been cloned and expressed in Escherichta colt using the inducible system described by Studier and Moffat [J. Mol. Biol. 189 (1986) 113-130] and Studier [J. Mol. Biol. 219 (1991) 37-44]. The two genes encoding the poliovirus proteases, 2Apr° and 3C pr°, were cloned together with their flanking regions in order to test the ability of the polyprotein precursors synthesized to cause proteolytic cleavage and generate mature forms. Both proteases were synthesized and showed activity upon induction in this system. Previously, it had not been possible to produce the three poliovirus nonstructural proteins, 2B, 2C and 3A, and some of their precursors, 2C3AB, 2C3A and 3AB, at high levels in E. colt cells. We report the cloning of their genes using PCR techniques and their efficient expression from pET vectors upon induction with IPTG (isopropyl-//-D-thiogalactopyranoside). Moreover, some of these proteins, e.g., 3A'B, 3A and 2B, are quite toxic for E. coli cells and lysed them upon production. Our results demonstrate the usefulness of this inducible system using the pET vectors to express these toxic poliovirus proteins.
INTRODUCTION
The use of sub-genomic cDNA clones of poliovirus h~s permitted studies manipulating the genome of this important human pathogen (Wimmer et al., 1987; Semler et al., 1988). Since poliovirus has only one open reading frame, the mature viral proteins are derived from a precursor by
Correspondence to: Dr. L. Carrasco, Centre de Biologia Molecular, Universidad Aut6noma de Madrid, Canto Blanco 28049 Madrid (Spain). Tel. (34- !)397 8450; Fax (34-1) 397 4799.
Abbreviations: A, absorbance; 2A,2B,2C,3A,3B(3BVpg),3C, poliovirus nonstructural proteins (see INTRODUCTION); 2A pr°, 3C pr°, poliovirus proteases; aa, amino acid(s); Ap, ampicillin; bp, base pair(s); cDNA, DNA complementary to RNA; Cm, chloramphenicol; DTI', dithiothreitol; EtdBr, ethidium bromide; IPTG, isopropyi-p-D-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; LB, Luria-Bertani (medium); nt, nucleotide(s); oligo, oligodeoxydbonucleotide; PA, polyacrylamide; PAGE, PA-gel electrophoresis; PCR, polymerase chain reaction; pro, protease; Rif, rifampicin; SDS, sodium dodecyl sulfate; Sm, streptomycin; [ ], denotes plasmid-carrier state.
proteolytic cleavage accomplished by the two virusencoded proteases (proteins 2Apr° and 3cpr°), that are active as precursors and are able to cleave themselves to render the final mature protein (Carrasco and Castrillo, 1987; Krliusslich and Wimmer, 1988; Palmenberg, 1990). The seven nonstructural polypeptides are designated as 2A pr°, 2B, 2C, 3A, 3B vPg, 3C pr° and 3D p°l (Wimmer etal., 1987). Apart from the two proteases 2Apr° and 3C pr°, protein 3B is the genome-bound protein in the 5'end of the viral RNA (3B vPg) (Flanegan et al., 1977; Takegami etal., 1983), whereas protein 3D p°l is the virus RNA-dependent RNA polymerase (Van Dyke and Flanegan,1980; Semler et al., 1988; Plotch et al., 1989). As an initial step towards understanding the function of poliovirus proteins we have cloned and expressed them in E. coll. Since some of the poliovirus nonstructural proteins are thought to be toxic for E.coli, we expressed them in the inducible system described by Studier and Moffat (1986). This system makes use of vectors containing the gene of interest under the control of the ~10 promoter of phage T7. The E. coil ceils carry in the chromosome the integrated T7 RNA polymerase gene
186 under the control o f the lacUV5 promoter t h a t is inducible u p o n addition of I P T G . Thus, upon induction with I P T G the T7 R N A polymerase is synthesized a n d then transcribes the gene under study (Studier and Moffat,1986; Studier et al., 1990).
RESULTS AND DISCUSSION (a) General
methods
Fragments from poliovirus type 1 e D N A were cloned behind the T7 promoter following s t a n d a r d techniques (Maniatis et al., 1982). Plasmids that express polyproteins containing active 2A pr° (pT710.1D2AB and p T 7 . 1 D 2 A B ) (Table I) were constructed by digesting vectors p E T 3 X A or
p E T 3 A (Rosenberg et al., 1987) with BamHl a n d ligating them to the 2.5-kb BamHI poliovirus fragment ( s p a n n i n g nt 2 0 9 9 - 4 6 0 0 of the poliovirus genome) from vector p T 7 X L D (a derivative of p T 7 p V 1 - 5 containing the full e D N A of poliovirus type 1) (Van der W e f t et al., 1986). Plasmid p T 7 . 3 A B C containing the complete coding region for 3C pr° was constructed by partial digestion o f p E T 3 B (Rosenberg et al., 1987) with Nhel a n d ligation to the 1.4 kb Xbal fragment (nt 4 8 8 6 - 6 3 0 4 ) obtained from p T 7 X L D . The rest o f the constructs were carried out by P C R amplification. Oligos were designed to hybridize with the desired regions o f poliovirus type-! c D N A t h a t contain the c o d i n g region for mature or precursor polypeptides (Fig. 1). A m plified products were purified using the G e n e C l e a n TM kit. D N A s were submitted to single or double restriction en-
TABLE 1
Characteristics of the constructs Plasmids
nt f r o m Complete poliovirusa poliovirus proteins b
Incomplete poliovirus proteins ~
Additional aa at N terminus d
Additional aa at C terminusd
Proteolytic processing e
Molecular sizes of expectedproductsr (kDa)
pT7.3ABC
4886-6304
3A, 3B and 3C
2C (74 aa from C terminus) 3D (105 aa from N terminus)
- 1 Met - I Ala in place of Set2s3 of poliovirus2C protein
19 aa from vector
four sites (3Cp r ° )
- Precursor A2C3ABCA3D - Processed products: 14 products 2.5-46
pT710.ID2AB 2099-4600
ID, 2A and 2B
I C (127 aa from C terminus) 2C(158aafrom N terminus)
- 260 aa from protein 10 of TT. -IArg
19 aa from vector
one site (2Am°)
-T7.AICID2ABA2C 123 - T7.AICID 75 - 2ABA2C 48
pTT.ID2AB
2099-4600 ID, 2A and 2B
IC (127 aa from C terminus) 2C (158 aa from N terminus)
- !1 aa from protein 10 ofT7 - 1 Arg
19 aa from vector
one site (2Apr°)
- 31CID2ABA2C - 31CID - 2AB32C
pTT.2A
3386-3832 2A
--
! Met
~
--
17
pT7.2B
3833-4123 2B
--
1 Met
--
--
10
pT7.2C
4124-5110
--
! Met
m
--
38
pT7.2C3A
4124-5371 2C and 3A
--
! Met
m
--
48
pTT.2C3AB
4124-5437
--
! Met
_
m
50
pTT.3A
5111-5371 3A
--
i Met
--
--
10
pT7.3AB
5111-5437 3A and 3B
--
1 Met
--
m
12
2C
2C, 3A and 3B
96 49 48
" The nt cloned from the poliovirus genome are shown, starting from No. 1 at the 5'end. b The poliovirusproteins indicated; they are named according to the poliovirus nomenclature. Only the proteins whose complete genes are contained in the construct are shown, irrespective of whether the mature proteins appear or not. The fragments containing the poliovirus proteases 2A or 3C also contain additional sequences that encode truncated poliovirus proteins. d The aa that have been added to the eDNA of the wild-type virus are shown. The number of sites for the proteolytic cleavage recognized by proteases 2A or 3C are shown. f The expected molecular sizes of the precursor and processed products are indicated (see also Fig, 1).
187
~, l0 t,886
A pT7.3ABC
[ A3D
3C
2099
4600
I 2.
pT710.1D2AB
3386
3832
pTT.2A
3833
t~123
sy .
p1"7.2B
5371
z,12z, pTT.2C3A
"~!t
p'I'7.2C3AB
-•*i
2;
2C
I
SA
2C
I
3A
5ti37
.
Is.
pTT,3A
p'I'7.3AB 3"2A 3"28 ~_
a.
L4 ,B I,C I '° I - I - I Primer S'.2A No'el CCCGGGCAT~Tnr,(~ATTC~f~A4~A~CAAAAC met Polio 2A (3386.3403)
3"2G 3"3A3'3B
L-
&..
,c
4~..L
i-II,cl
,°
S'.2A 5".28 5".2C Pdmer 3".2A 3B 8c/I GGGCCCTGAT(~AI"lrAI"I'C,TT~CATC,~r.I"r~rT(~
PrlmerS".2B NOel
stmstm Polio2A(3832.3815)
Primer 3".2B BamHI
GGCCCGGATCC,Z ~ TTA'rr~--~TT~-ATI~A(~ATAA~TA stop stop Polio2B (4123-4104) Pdmer 3".2C Ndel GGCCCATATG.U.A'~.AI"lr/'~AAA~AAAr-/"-~T~r-ATA~A CCC,GGGCATAT~f:~T(~A~A~'r'T~r4'rr~ AAOJ~A~ st0p stop Polio.(S110-5090) met POlio2C (4124-4144) Pd~:~3'.SA
CCCGGGCATAT~i~AT~A(~CAATTAI~ATA~A~; met Polio2B (3833-3853) Primer 5".2C
Pdmer S'.SA Ndel CCCGGGOA~GGACCACTCOAGTATAAAGAC met Polio3A (5111-5131)
GGG(:CCTGAT~.AI"I'Ar.Tr4r,Tt~rf~r.At~r-AAACA(~TT stop stop P~io 3A (5371-5352) Primer 8"3B 8c~1 GGGCCCTGATCATr,~I"rGTACCTTTGCTGTCCGAAT stop stop Polio38 (5437-$4171
Fig. !. Poliovirus genes and oligo primers. (A) Construction of recombinant plasmids. Schematic representation of the constructs used in this work. The Ol0 promoter and TO terminator from gene i0 of bacteriophage T7 and the cloned region between them are depicted. Shaded boxes indicate the codon for N-terminal Met and hatched boxes the translation stqp codons. The nt from poliovirus eDNA included in each construct are indicated, pT7. ID2AB is identical to pT7.10. I D2AB except that the first vector" fuses the poliovirus genes to eleven codons from gene 10 of bacteriophage T7 and the second one to 260 codons from the same gene. The constructs are not drawn to scale. (B) Primers used for PCR amplification. The region that hybridizes with poliovirus cDNA, the restriction enzyme sites and the codons to initiate or stop translation are shown. In the upper part, the eDNA of poliovirus has been represented showing the regions complementary to the primers.
zyme digestions and then were gel-purified. Inserts were ligated to pET3B vector digested with NdeI, in the case of vector pTT.2C, or NdeI+BamHI, for the rest of the constructs, and used to transform DH5 E. coil cells. In the case of the pT7.2A construct, an additional step was necessary to add EcoRI linkers to the amplified product to allow cloning in pBR322. Then, the 2A-encoding gene was extracted by partial digestion with NdeI+BclI and ligated
to pET3B vector digested with the same enzymes. DNA from positive clones was used to transform competent BL2 I(DE3)pLysS cells (Studier et al., 1990). In the case of pT7.2B, 3A and 3AB, plasmid DNA from BL21(DE3) pLysS cells was extracted and the region that was amplified by PCR was completely sequenced by the dideoxy method (Smith, 1980). To induce expression, single clones were grown overnight in LB medium in the presence of 100
188
#g Ap/ml and 34 #g Cm/mL Then, cultures were diluted 100-200-fold in M9 medium (Maniatis et al., 1982) supplemented with 0.2~ glucose and antibiotics. Induction was fired by addition of 0.5 mM IPTG when cultures reached A66o of 0.6-0.8. Rif was used at 150 #g/ml.
6304 (protein 3D p°~)(Table I; Fig. 1). The viral polymerase 3 D p°l w a s not cloned because a number of recent reports have described its cloning in several systems (Neufeld et al., 1991; Richards et al., 1987; Morrow et al., 1987) including the one we have used in the present work (Plotch et al., 1989). Two strategies of cloning were followed: the first was aimed at cloning the proteases 2 A pr° o r 3Cpr° and utilized larger fragments (obtained by directly cutting with appropriate restriction enzymes), encompassing the pro coding region and additional flanking sequences that encode adjacent proteins, in order to analyze the cleavage capabilities of both proteases in this system. The second approach
(b) Description of the constructions used Recombinant plasmids were constructed containing the different regions ofthe poliovirus genome depicted in Fig. 1. The regions of the poliovirus genome cloned in the present work encompass almost all of the poliovirus nonstructural region starting at nt 2099 (protein VP 1) and finishing at nt
A
B pET3XA
PULSE(min) R I F (rain) IPTG
kDa 97-~
0 --
90 +
9 0 90 9 0 0 30 0 + + -
pT?. 10.1D2AB
pT7. TD2 AB
0
90 90 90 9 0
0
-
--t"
-
0 +
30 +
0 -
90 90 90 90 0 30 0 +
+
+
-
1
..... .,,.,
2
kDa --123 "-- 7 5
38-
26-
----32 17-
Fig, 2. Synthesis of polyproteins containing active 2Apr°. (A). Cells BL21(DE3)[pLysS] containing the indicated plasmids were grown as described in general methods. Cells were induced with 0.5 mM IPTG at zero time ( + ) and at the indicated times were pulsed for 10 min at 37 ° C with 2/~Ci/ml of [35S]methionine, Then, cells were pelleted and resuspended in lysis buffer (2~ SDS/10~ glycerol/100 mM DTI'/160 mM Tris.HCl pH 6.8/0.033% bromophenol blue) and submitted to 15~ PAGE. The gels were fixed, treated with IM salicilic acid and exposed to X-AR films (Kodak). In some cases Rif was added (150/~g/ml) at the indicated times post-induction. Molecular size markers are shown, (Panel B) BL21(DE3) cells carrying pLysS (lane 1) or pLysS and pTT,10.1D2AB (lane 2) were induced for 90 rain with 0.5 mM IPTG and Rif (150 pg/ml) from 30 min post-induction. Then, extracts were obtained in lysis buffer, separated by a 15% PAGE, transferred to nitrocellulose membranes and reacted with antiserum against 260 aa from protein 10 of phage "1"7.The reaction was visualized with a second antibody labeled with peroxidase. The molecular size of the products are shown.
189 together (Fig. 2A). The induction of the expression of pT7.10. ID2AB with IPTG and Rif gives rise to two major polypeptides, neither migrates with pl0.T7 32-kDa protein synthesized from pET3XA, and an array of additional proteins produced in lesser amounts. The apparent Mr of these polypeptides correspond to those expected for the fusion protein T7D1CID2ABD2C (123 kDa) and the processed proteins (75 kDa and 48 kDa) (Table I). In addition, a 120-kDa protein (Fig. 2A, arrowhead on the left) is synthesized in the presence of IPTG alone, both with pT7.10.1D2AB and pT7.1D2AB. This protein may correspond to #-galactosidase (118 kDa). The production of the proteins encoded in plasmid pTT.ID2AB (Fig. 2A) shows that the precursor protein AI C1D2ABA2C is synthesized in addition to several processed precursors (Table I). The precursor AIC 1D2ABA2C is a major product when Rills added at zero time (Fig. 2A). The products synthesized by pTT. 10.1D2AB were further analyzed by immunoblotting with an antibody prepared against gene 10 protein of phage TT. Fig. 2B shows that pl0T7 is present in the precursor proteins indicating that poliovirus proteins are made as fusion polypeptides with
utilized PCR and was aimed at obtaining individual mature poliovirus proteins with an additional methionine at the N terminus. Since all poliovirus proteins contain an Nterminal Gly, it is probable that this additional Met is removed in most cases giving rise to the genuine poliovirus mature protein. (c) Cloning of the poliovirus proteases 2A pr° and 3C p'° The expression of the poliovirus proteins encoded by the fragment containing nt 2099 to 4600 is shown in Fig. 2. This fragment was cloned to generate a fusion protein with 260 aa from the gene 10 protein of phage T7 (pl0TT)(construction pT?.I0.1D2AB), or with only 11 aa of that protein (construction pT7.1D2AB). Plasmid pET3XA was used as a control to show the efficient induction of the truncated 32-kDa protein of phage T7 (Rosenberg et al., 1987), by IPTG alone, by Rif, or by both IPTG and Rif. In agreement with Studier and Moffatt (1986), induction of the 32-kDa synthesis is very eificient and almost no background of E.coli protein synthesis is detected when cells are labeled with [3SS]methionine, particularly when both compounds (IPTG and Rif) are present 0 !
om eL, G
pT75ABC TIME(h)
0
pET3B
1 3 5 1 5
=
o ._11~ mix Q.
¢j --
bJ l-
w
<¢
!.
55kD
- 5 5 kDa
i"
blo
-i! Ib
5C
~ - 3C
---5C
:SAB
....i
A
B
C
Fig. 3. Synthesis of poliovirus polyprotein containing active 3C pr°. (Panel A) Bacteria BL21(DE3)[pLysS] containing pT?.3ABC or pET3B were induced with 0.5 mM IPTG and Rif was added 30 rain later. At the times (h) post-induction shown, cells were labeled with [3SSlmethionine (see legend to Fig. 2) and total extracts were analyzed by 0.1% SDS-15 % PAGE. The gel was fixed, treated and exposed to X-AR films (Kodak) as indicated in Fig. 2. Proteins synthesized by the pT7.3ABC clone were compared to proteins that appeared in HeLa cells infected with poliovirus (panel B) or with the proteins synthesized in a reticulocyte system from transcripts of the same vector made in vitro with T7 RNA polymerase (panel C). The molecular weight of the larger recombinant product and the position of bacterial #-lactamase (bla) and poliovirus proteins 3C and 3AB are shown.
190 most of the Rif-resistant translation products, whereas only a protein corresponding in size to 3C pr° (20 kDa), increased in amount during the chase. Addition of 10 mM ZnCl 2, an inhibitor of 3C pr°, led to the accumulation of precursor polypeptides. Finally, the proteolytic capacity of 3C pr° made by the bacteria was tested in a cell-free system. A recombinant bacterial clone expressing 2C3AB (see section d) was used to provide a substrate (Baum et al., 1991). After induction in the presence of Rif, this protein was labeled with [35S]methionine, a lysate was made and mixed with a cold lysate containing 3 C pr°. Fig. 4B shows that the 3C pr° proteolytic site present in 2C3AB (50 kDa) is cleaved upon addition of this lysate, giving rise to the appearance of the 2C poliovirus protein (38 kDa). Addition of an extract made from bacteria expressing plasmid pET3B did not generate the 2C band.
pl0T7. The fact that the more abundant product synthesized is sfghtly larger than the 32-kDa pl0T7 protein and is recognized by an antibody against this polypeptide (Fig. 2B) is in agreement with the idea that premature termination generates this product. Plasmid pET3B (Rosenberg et al., 1987) was used to express the second pofovirus pro 3Cpr° and the flanking proteins. This fragment was cloned in the Nhel site present in this plasmid. The synthesis of the proteins encoded in the poliovirus fragment encompassing nt 4886-6304 is shown in Fig. 3. Induction with IPTG plus Rif generates the precursor A2C3ABCA3D and a wide array of processed proteius. All these polypeptides are compared with the proteins synthesized in poliovirus-infected cells (Fig. 3B). The polypeptides made in E.coli bearing the construction pT7.3ABC were compared with the proteins synthesized by a reticulocyte lysate primed with the mRNA made in vitro. The two larger polypeptides are also present in the reticulocyte lysate, although the cleavage of the large precursor is very inefficient in this system. Several assays were conducted to test the proteolytic activity of 3C pr°. Pulsechase experiments (Fig. 4A) showed the disappearance of
(d) Synthesis of individual poliovirus nonstructural proteins cloned by PCR As an initial step for the isolation in reasonable quantitites of individual poliovirus nonstructural proteins, or combinations of some of them, they were cloned by PCR
B
A OmM
M CHASE (rain)
Zinc
C
lOmM
Zinc
M
M
0 10 20 45 100180100. 0 to 20 45 100100 ~,.-, - 9 7 ( P I ) Ill-
kDa A2C3ABC A3D, 52 " " 3ABCA3r)
,46
7 2 (3CO)
~
~
~l~l~l~l~i)--
II1~
Wam~l~w~
'=--. ,~I~
2C3AB
- s~ (30)
....
.
blo m31 ,,.,,,_
atoll!In
am-
ImX
o~.,
TIME(h)
pET.3B pTT.3ABC 0 I 2.5 0 | 2.5
m
O ~
'~ , , 4
,~
26 (1C)
.... ' , - ~ m m
- 17 ( 2 A ) 3A8,12 ----
~ .
.
.
.
12 (3AS)
Fig. 4, Pulse-chase analysis of the polyprotein containing 3C. (Panel A) BL21(DE3)pLysS cells containing pT73ABC were induced with 0.5 mM IPTG and Rif (added at 30 min post-induction). Cells were pulsed with [3SS]methionine (25 pCi/ml) for 45 s after 45 min of induction, and then diluted with 3 vols. of growth medium supplemented with 2 mM methionine/150 pg Rif per ml/50 pg Sm per ml/0 or 10 mM ZnCh. At the indicated times aliquots were removed and analyzed by 0.1% SDS-15% PAGE gel. The apparent Mr and position of some synthesized products are shown (left margin). As molecular size markers (right side), labeled protein extracts from poliovirus-infected HeLa cells were used (lane M). (Lane C) Control to show the lack of radioactive proteins when 2 mM cold methionine/150/Ag Rif per ml/50/ag Sm per ml is present during the radioactive pulse and after 180 min chase. (Panel B) In vitro assay for 3Cpr°, Bacteria producing protein 2C3AB (see Fig. 6 or 8) were labeled for 15 min with [3SS]methionine after 90 min of induction with IPTG and Rif (added at 30 min post-induction). Then, the bacteria were treated with lysozyme and mixed with a non-radioactive extract from bacteria expressing 3C (pT7.3ABC) or containing the parental vector (pET3B) that had been induced for 2 h with IPTG alone. After 0, 1 or 2.5 h of incubation at 37°C the samples were analyzed by 0.1~ SDS-15~ PAGE together with proteins from poliovirus infected HeLa cells (lane M). The position of 2C and 2C3AB proteins are indicated.
191 2A
2B
P. 0 30 30*
3A
0 303n.
3AB
P. 0 30 30.
2C
P. 0 30 30-
2C3A
P, 0 30 30.
2C3AB
P. 0 30 3_0.
2A ( s t a i n i n g )
P. 0 3030.
0
1
2
3
I'.1. k D a
-66
~.
-45
#,,,,Q
2c-
-m-
:
36-- ~
zc
* m . ~ - ~ m m 4 . %_., , q.~. N~, 'N
'
=4-
ai~ -31
am
m
',OD.,-,'
......~ - 2 1
Fig, 5, Labeling of recombinant PCR clones. BL21(DE3)[pLysS] cells containing the plasmid pTT.2A, 2B, 2C, 2C3A, 2C3AB, 3A or 3AB were i n d u c e d with iPTG. Before addition of IPTG (zero time) or 30 min after, the cells were labeled as described in Fig. 2 and submitted to 0. !% SDS-PAGE. Some cultures were treated with Rif (150 mg/ml) from 20 min post-induction and labeled also at 30 min post-induction (30 + ), Extracts with 2A, 2B, 3A or 3AB proteins were electrophoresed on 20% PA gels, the other samples were electrophoresed in 15% gels. Proteins from poliovirus-infected cells were used as markers (P). The positions of the recombinant proteins are marked by arrows. Analysis of total proteins (Coomassie blue staining) from induced bacteria producing protein 2A (from pT7.2A) is indicated as 2A staining. Total extracts were obtained at the times post-induction shown (only with IPTG) electrophoresed in a 20% polyacrylamide gel and stained with Coomassie blue. The position of protein 2A and molecular size markers are shown.
using the primers depicted in Fig. lB. Induction of synthesis of these proteins with IPTG plus or minus Rif is shown in Fig. 5. The individual proteins 2A, 2B, 2C, 3A and the precursors 2C3AB, 2C3A and 3AB are all efficiently produced in bacteria• Remarkably, 2A was produced as a single protein at much higher levels than those obtained when the protein was part of a larger polyprotein (compare Fig. 2A with Fig. 5). Substantial levels of 2A pr° can be detected by gel staining with Coomassie blue, indicating that this protein was efficiently produced when the PCR cloning techniques were used. This also applies to the other
C. 4.
2A o
4.
C. 4.
3A -
4.
C. 3AB
C•
4.
4.
-
4.
2C .
4"
poliovirus nonstructural proteins, i.e., 2B, 2C, 2C3A, 2C3AB, 3A and 3AB. To our knowledge these results show the most efficient synthesis achieved until now with any of these polypeptides in E. coil These proteins were further characterized with antibodies raised against synthetic peptides of proteins 2A, 2C and 3A. A major band is detected by immunoblotting the polypeptides synthesised by clone 2C3AB with antibodies raised against 2C (Fig. 6). This putative precursor also reacts with antibodies against 3A. A number of additional smaller proteins also reacted with antibodies against 2C. At least some of these proteins are
C,
2C3A
c.
4.
-
4"
4.
2C3A c. 2C3AB c. 2C3AB "
4"
4.
"
4.
4"
"
4.
IPTG
I
iu.2A
oGA
I
~3A
c~2C
c~2C
~3A
(x2C
(x3A
Fig. 6. Immunoblot analysis of 2A, 2C, 2C3A, 2C3AB, 3A and 3AB. Cells BL21(DE3) containing plasmids pLysS and pET3B (lanes C) or pLysS together with the T7 expression plasniid containing the poliovirus gene indicated, were induced with 0.5 mM IPTG ( + ) or not ( - ). After 45 rain of induction crude extracts were separated by 0.1% SDS-PAGE, transferred to nitrocellulose membranes and reacted with specific rabbit antisera (a). Reaction was developed with a second goat antibody labeled with peroxidase or in the case of 2A with a second unlabeled goat antibody and [ X2Sl]protem A. The position of recombinant poliovirus proteins is shown by arrowheads.
192 ,1 ......... '-~rly seen after induction with IPTG and Rif and
..... ,iith [3SS]methionine (Fig. 5). The characterization of proteins 3AB and 3A was carried out by immuneblotting with antibodies against 3A (Fig. 6). A single band corresponding to 3AB, or 3A was specifically detected in the clones carrying the respective constructions upon induction with IPTG, but not after induction of bacteria containing the parental plasmid pET3B (Fig. 6). We noticed that the synthesis of some of the individual poliovirus proteins in E. coil was highly toxic and induced cell lysis upon induction. This effect was most dramatic with the clone bearing the construct encoding protein 3AB. Other proteins, e.g., 3A and 2B, were also lyric, whereas the synthesis of proteins 2A pr°, 2C, 2C3A, or 2C3AB was tolerated for long periods of time (results not shown). (e) Conclusions
(1) The inducible E. coil system described by Studier et al. (1990) to express toxic genes has been used to efficiently synthesize poliovirus nonstructural proteins. Both proteases 2Apr° and 3C pr° were cloned with flanking regions of the poliovirus genome in order to produce longer polypeptides containing 2A pr° or 3C pr° and to analyze the activity of the enzymes on these precursors. Both proteases showed activity and produced a number of proteolytic products. (2) To date, the poliovirus proteins 2B, 2C and 3A have not been produced in bacteria perhaps due to their potential toxicity. We made use of the PCR to clone each of these proteins and some of their precursors. Each protein cloned corresponded exactly to the genuine poliovirus protein, containing only the extra Met at the N terminus. Upon induction with IPTG or IPTG and Rif each protein was synthesized very efficiently. With this work all the poliovirus nonstructural proteins have been cloned and produced in bacteria.
ACKNOWLEDGEMENTS
The expert technical assistance of Ms. M. Chorro and Mr. M.A Sanz is acknowledged. Dr. E. Wimmer is acknowlwdged for providing antisera against proteins 2A and 2C and Dr. P. Sarnow for the antiserum against 2A. Drs. B,A. Moil'at and F.W. Studier are acknowledged for kindly providing us with the pET vectors used in this work. J.L. is the holder era Caja l~adrid fellowship. R.G. and F.M.A. are holders of F.P.I. fellowships. Plan Nacional, project No. (BId 88-0233), (DGICYT PB90-0177) and Fundaci6n Ram6n Areces are acknowledged for financial support.
REFERENCES Baum, E.Z., Bebernitz, G.A., Palant, O., Mueller, T. and Plotch, S.J.: Purification, properties and mutagenesis of poliovirus 3C protease. Virology 185 (1991) 140-150. Carrasco, L. and Castrillo, J.L.: The regulation of translation in picornavirus-infectedcells. In: Carrasco, L.(Ed.), Mechanisms of Viral Toxicity in Animal Cells, CRC Press, Boca Raton, FL, 1987, pp. 115146. Flanegan, J.B., Pettersson, R.F., Ambros, V., Hewlett, M.J. and Baltimore, D. Covalent linkage of a protein to a defined nucleotide sequence at the 5'-terminus ofvirion and replicative intermediate RNAs of poliovirus. Prec. Natl. Acad. Sci. USA 74 (1977) 961-965. Kratlsslich, H. and Wimmer, E.: Viral proteinases. Annu. Rev. Biochem. 57 (1988) 701-7~4. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Morrow, C., Warren, B. and Lentz, M.: Expression of enzymatically active poliovirus RNA-dependent RNA polymeras¢ in Escherichia coll. Prec. Natl. Acad. Sci. USA 84 (1987) 6050-6054. Neufeld, K.L., Richards, d.C. and Ehrenfeld, E.: Expression and characterization of poliovirus proteins 3Bvps, 3Cpro and 3Dpol in recom= binant baculovirus-infected Spodopterafrugiperda cells. Virus Res. 19 (1991) 173-188. Pahnenberg, A.C.: Proteolytic processing of picomaviral polyprotein. Annu. Rev. Microbiol. 44 (1990) 603-623. Plotch, S.J., Palant, O. and Gluzman, Y.: Purification and properties of poliovirus RNA polymerase expressed in Escher~chia coll. J. Virol. 63 (1989) 216-225. Richards, O., lvanoff, L., Bienkowska, K., Butt, B., Petteway, S., Rothstein, M. and Ehrenfeld, E.: Formation of poliovims RNA polymerase 3D in Escherichia coli by cleavage of fusion proteins expressed from cloned viral eDNA. 161 (1987) 348-356. Rosenberg, A.H., Lade, B.N., Chui, D., Lin, S.W., Dunn, J.L and Studier, W.: Vectors for selective expresion of cloned DNAs by T7 RNA polymerase. Gene 56 (1987) 125-135. Semler, B.L., Kuhn, R.J. and Wimmer, E.: Replication of the poliovirus genome. In: Domingo, E., Holland, J.J. and Ahlquist, P. (Eds.), RNA Genetics. CRC Press, Boca Raton, FL, 1988, pp. 23-48. Smith, A.J.H.: DNA sequence analysis by primed synthesis. Methods Enzymol. 65 (1980) 560-580. Studier, F.W.: Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J. Mol. Biol. 219 (1991) 37-44. Studier, F.W. and Moffatt, B.A.: Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189 (1986) 113-130. Studier, F.W., Rosenberg, A.H., Dunn, J.L and Dubendorff, J.W.: Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymcl. 185 (1990) 60-89. Takegami, T., Kuhn, R.J., Anderson, C.W. and Wimmer, E.: Membranedependent midylylation of the genome-linked protein VPg of poliovirus. Prec. Natl. Acad. Sci. USA 80 (1983) 7447-7451. Van der Weft, S., Bradley, J., Wimmer, E., Studier, F.W. and Dunn, J.: Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. Prec. Natl. Acad. Sci. USA 83 (1986) 2330-2334. Van Dyke, T.A. and Flanegan, J.B.: Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase. J. Virol.35 (1980) 732-740. Wimmer, E., Kuhn, R., Pincus, S., Yong, C.F., Toyoda, H., Nicklin, M.LH. and Takeda, N.: Molecular events leading to picornavirus genome replication. J. Cell. Sci. Suppl. 7 (1987) 251-276.
© 1992 Elsevier Science Publishers B.V. All fights reserved. 0378-1119/92/$05.00
185
GENE 06540
Cloning and inducible synthesis of poliovirus nonstructural proteins (Poliovirus gene expression; T7 RNA polymerase; membrane permeability; recombinant DNA; lyric toxic products)
J u a n L a m a , R o s a r i o G u i n e a , F r a n c i s c o M a r t i n e z - A b a r c a a n d Luis C a r r a s c o Centro de Biologia Molecular. Universidad Aut6noma de Madrid. Canto Blanco 28049 Madrid (Spain)
Received by A. Kohn: 16 December 1991; Revised/Accepted: 27 January/2 February 1992; Received at publishers: 1 April 1992
SUMMARY
The poliovirus nonstructural protein-encoding genes have been cloned and expressed in Escherichta colt using the inducible system described by Studier and Moffat [J. Mol. Biol. 189 (1986) 113-130] and Studier [J. Mol. Biol. 219 (1991) 37-44]. The two genes encoding the poliovirus proteases, 2Apr° and 3C pr°, were cloned together with their flanking regions in order to test the ability of the polyprotein precursors synthesized to cause proteolytic cleavage and generate mature forms. Both proteases were synthesized and showed activity upon induction in this system. Previously, it had not been possible to produce the three poliovirus nonstructural proteins, 2B, 2C and 3A, and some of their precursors, 2C3AB, 2C3A and 3AB, at high levels in E. colt cells. We report the cloning of their genes using PCR techniques and their efficient expression from pET vectors upon induction with IPTG (isopropyl-//-D-thiogalactopyranoside). Moreover, some of these proteins, e.g., 3A'B, 3A and 2B, are quite toxic for E. coli cells and lysed them upon production. Our results demonstrate the usefulness of this inducible system using the pET vectors to express these toxic poliovirus proteins.
INTRODUCTION
The use of sub-genomic cDNA clones of poliovirus h~s permitted studies manipulating the genome of this important human pathogen (Wimmer et al., 1987; Semler et al., 1988). Since poliovirus has only one open reading frame, the mature viral proteins are derived from a precursor by
Correspondence to: Dr. L. Carrasco, Centre de Biologia Molecular, Universidad Aut6noma de Madrid, Canto Blanco 28049 Madrid (Spain). Tel. (34- !)397 8450; Fax (34-1) 397 4799.
Abbreviations: A, absorbance; 2A,2B,2C,3A,3B(3BVpg),3C, poliovirus nonstructural proteins (see INTRODUCTION); 2A pr°, 3C pr°, poliovirus proteases; aa, amino acid(s); Ap, ampicillin; bp, base pair(s); cDNA, DNA complementary to RNA; Cm, chloramphenicol; DTI', dithiothreitol; EtdBr, ethidium bromide; IPTG, isopropyi-p-D-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; LB, Luria-Bertani (medium); nt, nucleotide(s); oligo, oligodeoxydbonucleotide; PA, polyacrylamide; PAGE, PA-gel electrophoresis; PCR, polymerase chain reaction; pro, protease; Rif, rifampicin; SDS, sodium dodecyl sulfate; Sm, streptomycin; [ ], denotes plasmid-carrier state.
proteolytic cleavage accomplished by the two virusencoded proteases (proteins 2Apr° and 3cpr°), that are active as precursors and are able to cleave themselves to render the final mature protein (Carrasco and Castrillo, 1987; Krliusslich and Wimmer, 1988; Palmenberg, 1990). The seven nonstructural polypeptides are designated as 2A pr°, 2B, 2C, 3A, 3B vPg, 3C pr° and 3D p°l (Wimmer etal., 1987). Apart from the two proteases 2Apr° and 3C pr°, protein 3B is the genome-bound protein in the 5'end of the viral RNA (3B vPg) (Flanegan et al., 1977; Takegami etal., 1983), whereas protein 3D p°l is the virus RNA-dependent RNA polymerase (Van Dyke and Flanegan,1980; Semler et al., 1988; Plotch et al., 1989). As an initial step towards understanding the function of poliovirus proteins we have cloned and expressed them in E. coll. Since some of the poliovirus nonstructural proteins are thought to be toxic for E.coli, we expressed them in the inducible system described by Studier and Moffat (1986). This system makes use of vectors containing the gene of interest under the control of the ~10 promoter of phage T7. The E. coil ceils carry in the chromosome the integrated T7 RNA polymerase gene
186 under the control o f the lacUV5 promoter t h a t is inducible u p o n addition of I P T G . Thus, upon induction with I P T G the T7 R N A polymerase is synthesized a n d then transcribes the gene under study (Studier and Moffat,1986; Studier et al., 1990).
RESULTS AND DISCUSSION (a) General
methods
Fragments from poliovirus type 1 e D N A were cloned behind the T7 promoter following s t a n d a r d techniques (Maniatis et al., 1982). Plasmids that express polyproteins containing active 2A pr° (pT710.1D2AB and p T 7 . 1 D 2 A B ) (Table I) were constructed by digesting vectors p E T 3 X A or
p E T 3 A (Rosenberg et al., 1987) with BamHl a n d ligating them to the 2.5-kb BamHI poliovirus fragment ( s p a n n i n g nt 2 0 9 9 - 4 6 0 0 of the poliovirus genome) from vector p T 7 X L D (a derivative of p T 7 p V 1 - 5 containing the full e D N A of poliovirus type 1) (Van der W e f t et al., 1986). Plasmid p T 7 . 3 A B C containing the complete coding region for 3C pr° was constructed by partial digestion o f p E T 3 B (Rosenberg et al., 1987) with Nhel a n d ligation to the 1.4 kb Xbal fragment (nt 4 8 8 6 - 6 3 0 4 ) obtained from p T 7 X L D . The rest o f the constructs were carried out by P C R amplification. Oligos were designed to hybridize with the desired regions o f poliovirus type-! c D N A t h a t contain the c o d i n g region for mature or precursor polypeptides (Fig. 1). A m plified products were purified using the G e n e C l e a n TM kit. D N A s were submitted to single or double restriction en-
TABLE 1
Characteristics of the constructs Plasmids
nt f r o m Complete poliovirusa poliovirus proteins b
Incomplete poliovirus proteins ~
Additional aa at N terminus d
Additional aa at C terminusd
Proteolytic processing e
Molecular sizes of expectedproductsr (kDa)
pT7.3ABC
4886-6304
3A, 3B and 3C
2C (74 aa from C terminus) 3D (105 aa from N terminus)
- 1 Met - I Ala in place of Set2s3 of poliovirus2C protein
19 aa from vector
four sites (3Cp r ° )
- Precursor A2C3ABCA3D - Processed products: 14 products 2.5-46
pT710.ID2AB 2099-4600
ID, 2A and 2B
I C (127 aa from C terminus) 2C(158aafrom N terminus)
- 260 aa from protein 10 of TT. -IArg
19 aa from vector
one site (2Am°)
-T7.AICID2ABA2C 123 - T7.AICID 75 - 2ABA2C 48
pTT.ID2AB
2099-4600 ID, 2A and 2B
IC (127 aa from C terminus) 2C (158 aa from N terminus)
- !1 aa from protein 10 ofT7 - 1 Arg
19 aa from vector
one site (2Apr°)
- 31CID2ABA2C - 31CID - 2AB32C
pTT.2A
3386-3832 2A
--
! Met
~
--
17
pT7.2B
3833-4123 2B
--
1 Met
--
--
10
pT7.2C
4124-5110
--
! Met
m
--
38
pT7.2C3A
4124-5371 2C and 3A
--
! Met
m
--
48
pTT.2C3AB
4124-5437
--
! Met
_
m
50
pTT.3A
5111-5371 3A
--
i Met
--
--
10
pT7.3AB
5111-5437 3A and 3B
--
1 Met
--
m
12
2C
2C, 3A and 3B
96 49 48
" The nt cloned from the poliovirus genome are shown, starting from No. 1 at the 5'end. b The poliovirusproteins indicated; they are named according to the poliovirus nomenclature. Only the proteins whose complete genes are contained in the construct are shown, irrespective of whether the mature proteins appear or not. The fragments containing the poliovirus proteases 2A or 3C also contain additional sequences that encode truncated poliovirus proteins. d The aa that have been added to the eDNA of the wild-type virus are shown. The number of sites for the proteolytic cleavage recognized by proteases 2A or 3C are shown. f The expected molecular sizes of the precursor and processed products are indicated (see also Fig, 1).
187
~, l0 t,886
A pT7.3ABC
[ A3D
3C
2099
4600
I 2.
pT710.1D2AB
3386
3832
pTT.2A
3833
t~123
sy .
p1"7.2B
5371
z,12z, pTT.2C3A
"~!t
p'I'7.2C3AB
-•*i
2;
2C
I
SA
2C
I
3A
5ti37
.
Is.
pTT,3A
p'I'7.3AB 3"2A 3"28 ~_
a.
L4 ,B I,C I '° I - I - I Primer S'.2A No'el CCCGGGCAT~Tnr,(~ATTC~f~A4~A~CAAAAC met Polio 2A (3386.3403)
3"2G 3"3A3'3B
L-
&..
,c
4~..L
i-II,cl
,°
S'.2A 5".28 5".2C Pdmer 3".2A 3B 8c/I GGGCCCTGAT(~AI"lrAI"I'C,TT~CATC,~r.I"r~rT(~
PrlmerS".2B NOel
stmstm Polio2A(3832.3815)
Primer 3".2B BamHI
GGCCCGGATCC,Z ~ TTA'rr~--~TT~-ATI~A(~ATAA~TA stop stop Polio2B (4123-4104) Pdmer 3".2C Ndel GGCCCATATG.U.A'~.AI"lr/'~AAA~AAAr-/"-~T~r-ATA~A CCC,GGGCATAT~f:~T(~A~A~'r'T~r4'rr~ AAOJ~A~ st0p stop Polio.(S110-5090) met POlio2C (4124-4144) Pd~:~3'.SA
CCCGGGCATAT~i~AT~A(~CAATTAI~ATA~A~; met Polio2B (3833-3853) Primer 5".2C
Pdmer S'.SA Ndel CCCGGGOA~GGACCACTCOAGTATAAAGAC met Polio3A (5111-5131)
GGG(:CCTGAT~.AI"I'Ar.Tr4r,Tt~rf~r.At~r-AAACA(~TT stop stop P~io 3A (5371-5352) Primer 8"3B 8c~1 GGGCCCTGATCATr,~I"rGTACCTTTGCTGTCCGAAT stop stop Polio38 (5437-$4171
Fig. !. Poliovirus genes and oligo primers. (A) Construction of recombinant plasmids. Schematic representation of the constructs used in this work. The Ol0 promoter and TO terminator from gene i0 of bacteriophage T7 and the cloned region between them are depicted. Shaded boxes indicate the codon for N-terminal Met and hatched boxes the translation stqp codons. The nt from poliovirus eDNA included in each construct are indicated, pT7. ID2AB is identical to pT7.10. I D2AB except that the first vector" fuses the poliovirus genes to eleven codons from gene 10 of bacteriophage T7 and the second one to 260 codons from the same gene. The constructs are not drawn to scale. (B) Primers used for PCR amplification. The region that hybridizes with poliovirus cDNA, the restriction enzyme sites and the codons to initiate or stop translation are shown. In the upper part, the eDNA of poliovirus has been represented showing the regions complementary to the primers.
zyme digestions and then were gel-purified. Inserts were ligated to pET3B vector digested with NdeI, in the case of vector pTT.2C, or NdeI+BamHI, for the rest of the constructs, and used to transform DH5 E. coil cells. In the case of the pT7.2A construct, an additional step was necessary to add EcoRI linkers to the amplified product to allow cloning in pBR322. Then, the 2A-encoding gene was extracted by partial digestion with NdeI+BclI and ligated
to pET3B vector digested with the same enzymes. DNA from positive clones was used to transform competent BL2 I(DE3)pLysS cells (Studier et al., 1990). In the case of pT7.2B, 3A and 3AB, plasmid DNA from BL21(DE3) pLysS cells was extracted and the region that was amplified by PCR was completely sequenced by the dideoxy method (Smith, 1980). To induce expression, single clones were grown overnight in LB medium in the presence of 100
188
#g Ap/ml and 34 #g Cm/mL Then, cultures were diluted 100-200-fold in M9 medium (Maniatis et al., 1982) supplemented with 0.2~ glucose and antibiotics. Induction was fired by addition of 0.5 mM IPTG when cultures reached A66o of 0.6-0.8. Rif was used at 150 #g/ml.
6304 (protein 3D p°~)(Table I; Fig. 1). The viral polymerase 3 D p°l w a s not cloned because a number of recent reports have described its cloning in several systems (Neufeld et al., 1991; Richards et al., 1987; Morrow et al., 1987) including the one we have used in the present work (Plotch et al., 1989). Two strategies of cloning were followed: the first was aimed at cloning the proteases 2 A pr° o r 3Cpr° and utilized larger fragments (obtained by directly cutting with appropriate restriction enzymes), encompassing the pro coding region and additional flanking sequences that encode adjacent proteins, in order to analyze the cleavage capabilities of both proteases in this system. The second approach
(b) Description of the constructions used Recombinant plasmids were constructed containing the different regions ofthe poliovirus genome depicted in Fig. 1. The regions of the poliovirus genome cloned in the present work encompass almost all of the poliovirus nonstructural region starting at nt 2099 (protein VP 1) and finishing at nt
A
B pET3XA
PULSE(min) R I F (rain) IPTG
kDa 97-~
0 --
90 +
9 0 90 9 0 0 30 0 + + -
pT?. 10.1D2AB
pT7. TD2 AB
0
90 90 90 9 0
0
-
--t"
-
0 +
30 +
0 -
90 90 90 90 0 30 0 +
+
+
-
1
..... .,,.,
2
kDa --123 "-- 7 5
38-
26-
----32 17-
Fig, 2. Synthesis of polyproteins containing active 2Apr°. (A). Cells BL21(DE3)[pLysS] containing the indicated plasmids were grown as described in general methods. Cells were induced with 0.5 mM IPTG at zero time ( + ) and at the indicated times were pulsed for 10 min at 37 ° C with 2/~Ci/ml of [35S]methionine, Then, cells were pelleted and resuspended in lysis buffer (2~ SDS/10~ glycerol/100 mM DTI'/160 mM Tris.HCl pH 6.8/0.033% bromophenol blue) and submitted to 15~ PAGE. The gels were fixed, treated with IM salicilic acid and exposed to X-AR films (Kodak). In some cases Rif was added (150/~g/ml) at the indicated times post-induction. Molecular size markers are shown, (Panel B) BL21(DE3) cells carrying pLysS (lane 1) or pLysS and pTT,10.1D2AB (lane 2) were induced for 90 rain with 0.5 mM IPTG and Rif (150 pg/ml) from 30 min post-induction. Then, extracts were obtained in lysis buffer, separated by a 15% PAGE, transferred to nitrocellulose membranes and reacted with antiserum against 260 aa from protein 10 of phage "1"7.The reaction was visualized with a second antibody labeled with peroxidase. The molecular size of the products are shown.
189 together (Fig. 2A). The induction of the expression of pT7.10. ID2AB with IPTG and Rif gives rise to two major polypeptides, neither migrates with pl0.T7 32-kDa protein synthesized from pET3XA, and an array of additional proteins produced in lesser amounts. The apparent Mr of these polypeptides correspond to those expected for the fusion protein T7D1CID2ABD2C (123 kDa) and the processed proteins (75 kDa and 48 kDa) (Table I). In addition, a 120-kDa protein (Fig. 2A, arrowhead on the left) is synthesized in the presence of IPTG alone, both with pT7.10.1D2AB and pT7.1D2AB. This protein may correspond to #-galactosidase (118 kDa). The production of the proteins encoded in plasmid pTT.ID2AB (Fig. 2A) shows that the precursor protein AI C1D2ABA2C is synthesized in addition to several processed precursors (Table I). The precursor AIC 1D2ABA2C is a major product when Rills added at zero time (Fig. 2A). The products synthesized by pTT. 10.1D2AB were further analyzed by immunoblotting with an antibody prepared against gene 10 protein of phage TT. Fig. 2B shows that pl0T7 is present in the precursor proteins indicating that poliovirus proteins are made as fusion polypeptides with
utilized PCR and was aimed at obtaining individual mature poliovirus proteins with an additional methionine at the N terminus. Since all poliovirus proteins contain an Nterminal Gly, it is probable that this additional Met is removed in most cases giving rise to the genuine poliovirus mature protein. (c) Cloning of the poliovirus proteases 2A pr° and 3C p'° The expression of the poliovirus proteins encoded by the fragment containing nt 2099 to 4600 is shown in Fig. 2. This fragment was cloned to generate a fusion protein with 260 aa from the gene 10 protein of phage T7 (pl0TT)(construction pT?.I0.1D2AB), or with only 11 aa of that protein (construction pT7.1D2AB). Plasmid pET3XA was used as a control to show the efficient induction of the truncated 32-kDa protein of phage T7 (Rosenberg et al., 1987), by IPTG alone, by Rif, or by both IPTG and Rif. In agreement with Studier and Moffatt (1986), induction of the 32-kDa synthesis is very eificient and almost no background of E.coli protein synthesis is detected when cells are labeled with [3SS]methionine, particularly when both compounds (IPTG and Rif) are present 0 !
om eL, G
pT75ABC TIME(h)
0
pET3B
1 3 5 1 5
=
o ._11~ mix Q.
¢j --
bJ l-
w
<¢
!.
55kD
- 5 5 kDa
i"
blo
-i! Ib
5C
~ - 3C
---5C
:SAB
....i
A
B
C
Fig. 3. Synthesis of poliovirus polyprotein containing active 3C pr°. (Panel A) Bacteria BL21(DE3)[pLysS] containing pT?.3ABC or pET3B were induced with 0.5 mM IPTG and Rif was added 30 rain later. At the times (h) post-induction shown, cells were labeled with [3SSlmethionine (see legend to Fig. 2) and total extracts were analyzed by 0.1% SDS-15 % PAGE. The gel was fixed, treated and exposed to X-AR films (Kodak) as indicated in Fig. 2. Proteins synthesized by the pT7.3ABC clone were compared to proteins that appeared in HeLa cells infected with poliovirus (panel B) or with the proteins synthesized in a reticulocyte system from transcripts of the same vector made in vitro with T7 RNA polymerase (panel C). The molecular weight of the larger recombinant product and the position of bacterial #-lactamase (bla) and poliovirus proteins 3C and 3AB are shown.
190 most of the Rif-resistant translation products, whereas only a protein corresponding in size to 3C pr° (20 kDa), increased in amount during the chase. Addition of 10 mM ZnCl 2, an inhibitor of 3C pr°, led to the accumulation of precursor polypeptides. Finally, the proteolytic capacity of 3C pr° made by the bacteria was tested in a cell-free system. A recombinant bacterial clone expressing 2C3AB (see section d) was used to provide a substrate (Baum et al., 1991). After induction in the presence of Rif, this protein was labeled with [35S]methionine, a lysate was made and mixed with a cold lysate containing 3 C pr°. Fig. 4B shows that the 3C pr° proteolytic site present in 2C3AB (50 kDa) is cleaved upon addition of this lysate, giving rise to the appearance of the 2C poliovirus protein (38 kDa). Addition of an extract made from bacteria expressing plasmid pET3B did not generate the 2C band.
pl0T7. The fact that the more abundant product synthesized is sfghtly larger than the 32-kDa pl0T7 protein and is recognized by an antibody against this polypeptide (Fig. 2B) is in agreement with the idea that premature termination generates this product. Plasmid pET3B (Rosenberg et al., 1987) was used to express the second pofovirus pro 3Cpr° and the flanking proteins. This fragment was cloned in the Nhel site present in this plasmid. The synthesis of the proteins encoded in the poliovirus fragment encompassing nt 4886-6304 is shown in Fig. 3. Induction with IPTG plus Rif generates the precursor A2C3ABCA3D and a wide array of processed proteius. All these polypeptides are compared with the proteins synthesized in poliovirus-infected cells (Fig. 3B). The polypeptides made in E.coli bearing the construction pT7.3ABC were compared with the proteins synthesized by a reticulocyte lysate primed with the mRNA made in vitro. The two larger polypeptides are also present in the reticulocyte lysate, although the cleavage of the large precursor is very inefficient in this system. Several assays were conducted to test the proteolytic activity of 3C pr°. Pulsechase experiments (Fig. 4A) showed the disappearance of
(d) Synthesis of individual poliovirus nonstructural proteins cloned by PCR As an initial step for the isolation in reasonable quantitites of individual poliovirus nonstructural proteins, or combinations of some of them, they were cloned by PCR
B
A OmM
M CHASE (rain)
Zinc
C
lOmM
Zinc
M
M
0 10 20 45 100180100. 0 to 20 45 100100 ~,.-, - 9 7 ( P I ) Ill-
kDa A2C3ABC A3D, 52 " " 3ABCA3r)
,46
7 2 (3CO)
~
~
~l~l~l~l~i)--
II1~
Wam~l~w~
'=--. ,~I~
2C3AB
- s~ (30)
....
.
blo m31 ,,.,,,_
atoll!In
am-
ImX
o~.,
TIME(h)
pET.3B pTT.3ABC 0 I 2.5 0 | 2.5
m
O ~
'~ , , 4
,~
26 (1C)
.... ' , - ~ m m
- 17 ( 2 A ) 3A8,12 ----
~ .
.
.
.
12 (3AS)
Fig. 4, Pulse-chase analysis of the polyprotein containing 3C. (Panel A) BL21(DE3)pLysS cells containing pT73ABC were induced with 0.5 mM IPTG and Rif (added at 30 min post-induction). Cells were pulsed with [3SS]methionine (25 pCi/ml) for 45 s after 45 min of induction, and then diluted with 3 vols. of growth medium supplemented with 2 mM methionine/150 pg Rif per ml/50 pg Sm per ml/0 or 10 mM ZnCh. At the indicated times aliquots were removed and analyzed by 0.1% SDS-15% PAGE gel. The apparent Mr and position of some synthesized products are shown (left margin). As molecular size markers (right side), labeled protein extracts from poliovirus-infected HeLa cells were used (lane M). (Lane C) Control to show the lack of radioactive proteins when 2 mM cold methionine/150/Ag Rif per ml/50/ag Sm per ml is present during the radioactive pulse and after 180 min chase. (Panel B) In vitro assay for 3Cpr°, Bacteria producing protein 2C3AB (see Fig. 6 or 8) were labeled for 15 min with [3SS]methionine after 90 min of induction with IPTG and Rif (added at 30 min post-induction). Then, the bacteria were treated with lysozyme and mixed with a non-radioactive extract from bacteria expressing 3C (pT7.3ABC) or containing the parental vector (pET3B) that had been induced for 2 h with IPTG alone. After 0, 1 or 2.5 h of incubation at 37°C the samples were analyzed by 0.1~ SDS-15~ PAGE together with proteins from poliovirus infected HeLa cells (lane M). The position of 2C and 2C3AB proteins are indicated.
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Fig, 5, Labeling of recombinant PCR clones. BL21(DE3)[pLysS] cells containing the plasmid pTT.2A, 2B, 2C, 2C3A, 2C3AB, 3A or 3AB were i n d u c e d with iPTG. Before addition of IPTG (zero time) or 30 min after, the cells were labeled as described in Fig. 2 and submitted to 0. !% SDS-PAGE. Some cultures were treated with Rif (150 mg/ml) from 20 min post-induction and labeled also at 30 min post-induction (30 + ), Extracts with 2A, 2B, 3A or 3AB proteins were electrophoresed on 20% PA gels, the other samples were electrophoresed in 15% gels. Proteins from poliovirus-infected cells were used as markers (P). The positions of the recombinant proteins are marked by arrows. Analysis of total proteins (Coomassie blue staining) from induced bacteria producing protein 2A (from pT7.2A) is indicated as 2A staining. Total extracts were obtained at the times post-induction shown (only with IPTG) electrophoresed in a 20% polyacrylamide gel and stained with Coomassie blue. The position of protein 2A and molecular size markers are shown.
using the primers depicted in Fig. lB. Induction of synthesis of these proteins with IPTG plus or minus Rif is shown in Fig. 5. The individual proteins 2A, 2B, 2C, 3A and the precursors 2C3AB, 2C3A and 3AB are all efficiently produced in bacteria• Remarkably, 2A was produced as a single protein at much higher levels than those obtained when the protein was part of a larger polyprotein (compare Fig. 2A with Fig. 5). Substantial levels of 2A pr° can be detected by gel staining with Coomassie blue, indicating that this protein was efficiently produced when the PCR cloning techniques were used. This also applies to the other
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poliovirus nonstructural proteins, i.e., 2B, 2C, 2C3A, 2C3AB, 3A and 3AB. To our knowledge these results show the most efficient synthesis achieved until now with any of these polypeptides in E. coil These proteins were further characterized with antibodies raised against synthetic peptides of proteins 2A, 2C and 3A. A major band is detected by immunoblotting the polypeptides synthesised by clone 2C3AB with antibodies raised against 2C (Fig. 6). This putative precursor also reacts with antibodies against 3A. A number of additional smaller proteins also reacted with antibodies against 2C. At least some of these proteins are
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Fig. 6. Immunoblot analysis of 2A, 2C, 2C3A, 2C3AB, 3A and 3AB. Cells BL21(DE3) containing plasmids pLysS and pET3B (lanes C) or pLysS together with the T7 expression plasniid containing the poliovirus gene indicated, were induced with 0.5 mM IPTG ( + ) or not ( - ). After 45 rain of induction crude extracts were separated by 0.1% SDS-PAGE, transferred to nitrocellulose membranes and reacted with specific rabbit antisera (a). Reaction was developed with a second goat antibody labeled with peroxidase or in the case of 2A with a second unlabeled goat antibody and [ X2Sl]protem A. The position of recombinant poliovirus proteins is shown by arrowheads.
192 ,1 ......... '-~rly seen after induction with IPTG and Rif and
..... ,iith [3SS]methionine (Fig. 5). The characterization of proteins 3AB and 3A was carried out by immuneblotting with antibodies against 3A (Fig. 6). A single band corresponding to 3AB, or 3A was specifically detected in the clones carrying the respective constructions upon induction with IPTG, but not after induction of bacteria containing the parental plasmid pET3B (Fig. 6). We noticed that the synthesis of some of the individual poliovirus proteins in E. coil was highly toxic and induced cell lysis upon induction. This effect was most dramatic with the clone bearing the construct encoding protein 3AB. Other proteins, e.g., 3A and 2B, were also lyric, whereas the synthesis of proteins 2A pr°, 2C, 2C3A, or 2C3AB was tolerated for long periods of time (results not shown). (e) Conclusions
(1) The inducible E. coil system described by Studier et al. (1990) to express toxic genes has been used to efficiently synthesize poliovirus nonstructural proteins. Both proteases 2Apr° and 3C pr° were cloned with flanking regions of the poliovirus genome in order to produce longer polypeptides containing 2A pr° or 3C pr° and to analyze the activity of the enzymes on these precursors. Both proteases showed activity and produced a number of proteolytic products. (2) To date, the poliovirus proteins 2B, 2C and 3A have not been produced in bacteria perhaps due to their potential toxicity. We made use of the PCR to clone each of these proteins and some of their precursors. Each protein cloned corresponded exactly to the genuine poliovirus protein, containing only the extra Met at the N terminus. Upon induction with IPTG or IPTG and Rif each protein was synthesized very efficiently. With this work all the poliovirus nonstructural proteins have been cloned and produced in bacteria.
ACKNOWLEDGEMENTS
The expert technical assistance of Ms. M. Chorro and Mr. M.A Sanz is acknowledged. Dr. E. Wimmer is acknowlwdged for providing antisera against proteins 2A and 2C and Dr. P. Sarnow for the antiserum against 2A. Drs. B,A. Moil'at and F.W. Studier are acknowledged for kindly providing us with the pET vectors used in this work. J.L. is the holder era Caja l~adrid fellowship. R.G. and F.M.A. are holders of F.P.I. fellowships. Plan Nacional, project No. (BId 88-0233), (DGICYT PB90-0177) and Fundaci6n Ram6n Areces are acknowledged for financial support.
REFERENCES Baum, E.Z., Bebernitz, G.A., Palant, O., Mueller, T. and Plotch, S.J.: Purification, properties and mutagenesis of poliovirus 3C protease. Virology 185 (1991) 140-150. Carrasco, L. and Castrillo, J.L.: The regulation of translation in picornavirus-infectedcells. In: Carrasco, L.(Ed.), Mechanisms of Viral Toxicity in Animal Cells, CRC Press, Boca Raton, FL, 1987, pp. 115146. Flanegan, J.B., Pettersson, R.F., Ambros, V., Hewlett, M.J. and Baltimore, D. Covalent linkage of a protein to a defined nucleotide sequence at the 5'-terminus ofvirion and replicative intermediate RNAs of poliovirus. Prec. Natl. Acad. Sci. USA 74 (1977) 961-965. Kratlsslich, H. and Wimmer, E.: Viral proteinases. Annu. Rev. Biochem. 57 (1988) 701-7~4. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Morrow, C., Warren, B. and Lentz, M.: Expression of enzymatically active poliovirus RNA-dependent RNA polymeras¢ in Escherichia coll. Prec. Natl. Acad. Sci. USA 84 (1987) 6050-6054. Neufeld, K.L., Richards, d.C. and Ehrenfeld, E.: Expression and characterization of poliovirus proteins 3Bvps, 3Cpro and 3Dpol in recom= binant baculovirus-infected Spodopterafrugiperda cells. Virus Res. 19 (1991) 173-188. Pahnenberg, A.C.: Proteolytic processing of picomaviral polyprotein. Annu. Rev. Microbiol. 44 (1990) 603-623. Plotch, S.J., Palant, O. and Gluzman, Y.: Purification and properties of poliovirus RNA polymerase expressed in Escher~chia coll. J. Virol. 63 (1989) 216-225. Richards, O., lvanoff, L., Bienkowska, K., Butt, B., Petteway, S., Rothstein, M. and Ehrenfeld, E.: Formation of poliovims RNA polymerase 3D in Escherichia coli by cleavage of fusion proteins expressed from cloned viral eDNA. 161 (1987) 348-356. Rosenberg, A.H., Lade, B.N., Chui, D., Lin, S.W., Dunn, J.L and Studier, W.: Vectors for selective expresion of cloned DNAs by T7 RNA polymerase. Gene 56 (1987) 125-135. Semler, B.L., Kuhn, R.J. and Wimmer, E.: Replication of the poliovirus genome. In: Domingo, E., Holland, J.J. and Ahlquist, P. (Eds.), RNA Genetics. CRC Press, Boca Raton, FL, 1988, pp. 23-48. Smith, A.J.H.: DNA sequence analysis by primed synthesis. Methods Enzymol. 65 (1980) 560-580. Studier, F.W.: Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J. Mol. Biol. 219 (1991) 37-44. Studier, F.W. and Moffatt, B.A.: Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189 (1986) 113-130. Studier, F.W., Rosenberg, A.H., Dunn, J.L and Dubendorff, J.W.: Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymcl. 185 (1990) 60-89. Takegami, T., Kuhn, R.J., Anderson, C.W. and Wimmer, E.: Membranedependent midylylation of the genome-linked protein VPg of poliovirus. Prec. Natl. Acad. Sci. USA 80 (1983) 7447-7451. Van der Weft, S., Bradley, J., Wimmer, E., Studier, F.W. and Dunn, J.: Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. Prec. Natl. Acad. Sci. USA 83 (1986) 2330-2334. Van Dyke, T.A. and Flanegan, J.B.: Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase. J. Virol.35 (1980) 732-740. Wimmer, E., Kuhn, R., Pincus, S., Yong, C.F., Toyoda, H., Nicklin, M.LH. and Takeda, N.: Molecular events leading to picornavirus genome replication. J. Cell. Sci. Suppl. 7 (1987) 251-276.