160s Biochemical SocietvTransactions ( 1 992) 20 cloning,
Ikprraaioa,
.ad
Yutagur.aia
of
S I V ,
Protainaao in 0 . coli BERNADETTE R. WILDERSPIN.
CORR, MARK RICHARDSON+ and ANDREW
F.
Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, Malet Street, London, WClE 7HX, UK. 'British Bio-technology Ltd., Watlington Road, Oxford, OX4 5LY, UK. Biological and biochemical characterisation of non-human AIDS model systems is essential for understanding the mechanism of retrovirus-induced innnune deficiency diseases. The highly conserved sequence homology between several of the proteins in human inununodeficiency virus (HIV-1/-2) and macaque simian innnunodeficiency virus (SIV,,) may permit such infected monkeys to be used in the testing and evaluation of potential drugs against the diseasel. Retroviral aspartic proteinases (PRs) are virally encoded enzymes responsible for the proteolytic maturation process that converts the nascent virus into a mature and infectious ford-' - hence an ideal target for the design of antiviral drugs. The human and simian PRs show approximately 80% amino acid sequence similarity with the highest degree of homology in the active site cleft5tC (see Ti1). In order to evaluate the degree of functional homology between HIV-1/-2 PR and S I V , PR initial work involved the production of large quantities of S I V , PR namely by bacterial expression. A 560bp segment of the S I V , gag-pol that encodes both the transframe protein and the first 25 amino acids of reverse transcriptase was PCR amplified from DNA extracted from a standard S I V , isolate (11/88 32H) and supplied to us courtesy of soluble Neil Almond at NIBSC (pNIBSC-6). Two intracellular systems involving a g1utathione-Stransferase (GST) fusion were initially chosen for expression (Pharmacia)'. The PR gene was cloned into the Sma I/Eco RI sites of pGEX-2T to give pGP-2T and into the B g l II/Eco RI sites of pGEX-3X to give pGP3X. Both are under the control of a tac promoter and recognition sites for site-specific nucleases, thrombin (-2T) and factor Xa (-3X), allows cleavage of the 26-kDa GST domain from the 47-kDa fusion proteins. The vectors were grown up and the fusion expressed in the E.coli strain TG1. Alternative inducible but insoluble expression systems were also employed. A synthetic 950bp horseradish peroxide (HRP) gene was cloned into the Nde I / B m HI sites of pGC517 another IPTG-inducible vector - to give pSD18. The B g l II/Bal I fragment of SIV, PR was subsequently inserted into Bg1 II/Pvu I1 cut pSD18 to give pOGS445. This resulted in the PR being located in the centre of the HRP gene thus facilitating from its initiating methionine and termination codons. Growth and expression in the E.coli strain HWlllO was to result in the production of the 54-kDa HRP-PR-HRP fusion in the form of insoluble inclusion bodies (IBs). In addition, the HRP-PR-HRP fragment was removed using Nde I/Bam HI sites from pOGS445 and cloned into the NdeI/BamHI sites of pJLA503 to give pOGS457. In this case the fusion was under the control of a A PR/PL promoter in the E.coli strain HW87. SIV, PR was thus expressed by growing pGP-ZT, pGP-3X and pOGS445 in the appropriate E.coli strain to mid-log phase (OD550m = 0.4) at 37OC and inducing with 2mM IPTG. For pOGS457, cells were grown at 3OoC to OD550m = 1.0 and induced by heat-shocking at 6OoC for 90 seconds and continuing incubation at 42OC thereafter. The cells were subsequently harvested at various times post-induction (p.i.) and centrifuged. The resultant cell pellets were resuspended in a tenth volume of SDS-loading buffer, boiled at 100°C for 5-10 minutes and ran on 20% homogeneous SDS-PAGE gels. These gels were coomassie/silver stained or used for electroblotting onto PVDF membrane. The latter was probed with a sheep anti-HIV-1 PR polyclonal antibody (1 in 500 dilution) and protein bands visualized using a donkey anti-sheep-HRP conjugated antibody with substrate 0.5mg/ml 3'-3diaminobenzidine, 0.5% hydrogen peroxide (1OOvols). To demonstrate specific SIV, PR activity, p.i. samples of the cells were resuspended in a tenth volume of ice-cold lysis buffer (50mM Tris, 5mM NaC1,
-
5mM EDTA, 1mM DTT, 1mM PMSF, 1E-64, O.Ol+g/ml aprotinin, 200pg/ml lysozyme, 10% glycerol, pH 7.8) and incubated at O°C for 30 minutes. After addition of 0.5% NP-40 and lOfig/ml DNase, the cells were sonicated at 4OC (3 x 30 second pulses). The resultant crude lysate was used directly virus-like particle (VLP) assays. At pH 6.0, lOOpl/ml VLP (80kDa substrate) was incubated at 37OC for one hour with various amounts of the lysate and subsequently ran on a 20% SDS gel. The proteins were blotted and visualised using rabbit anti-VLP antibody and an anti-rabbit-HRP conjugated antibody. Activity was noted by the presence of 53-kDa and 27-kDa product bands'. RIV-1 PQITLWQRPLVTIKIGGQLKEALLDTCADDTVLEELPGRWK RIV-2 PQFSLWKRPWTAYIEGQPVEVLLDTCADDSIVAGIELGNNS sIM.c PQFSLWRRPWTAHIEGQPVEVLLDTCADDSIVTGIELGPHYT 1 10 20 30 40 IIIV-1 RIV-2 S-C
PKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPWIIG PKIVGGIGGFINTKEYKNVEIEVL~TIMTGDTPINIFG
PKIVGGIGGFINTKEYKNVKIEVLGKRIKGTI~GDTPINIFG 50 60 70 80
RIV-1 RNLLTQIGCTLNF RIV-2 RNILTALGUSLNL SIVnuc RNLLTALGMSLNF 90 Figure 1. Amino acid sequence alignment of human and simian retroviral proteinases. The residues in boldtype represent the conserved catalytic triad in the active site (-DTG-). Expression of the PR using the two soluble systems, pGP-2T and pGP-3X, resulted in retarded growth of the cells. This effect was lessened slightly by the addition of 20% glucose to the culture, suggesting that the PR was autoprocessing itself from the GST fusion to give the active form in the cytoplasm, thus having a toxic effect on the cells. Similar results were obtained when a further two strains of E.coli were used for the expression. The formation of insoluble IBs by the HRP system was to solve these toxicity problems. For both pOGS445 and pOGS457, coomassie and silver stained gels showed and a strong band at 43-kDa (HRP and part of SIV,,) 35-kDa (HRP), with a weak band at 11-kDa. These bands were of increased intensity with increasing time p.i.. From the western blots all three bands were also visible, but the 11-kDa protein was much more intense than the others. On no occasion was the the complete 54-kDa fusion observed. The VLP assay blots only showed notable activity for pOGS457 and none for pOGS445, perhaps because A PR/PL promoter is stronger than tac and hence produces larger amounts of the PR allowing activity to be detected. Low levels of SIVmac PR have thus been expressed and specific activity recorded. Further work is aimed at designing and synthetizing PR mutants to completely evaluate the suitability of SIV,,, infected monkeys as animal models for anti-HIV drug testing. This work was supported by the MRC.
1. Kanki, P.J; Uc Lane, U.F; King, N.W; Letvin, N.L; Hunt, R.D; Sehgal, P: Daniel, M.D; Desrosiers, R.C; Essex, M. (1985) Science 228, 1189-1201. 2. Krausslich, H-G; Schneider, H; Zybarth, G; Carter, C.A; Wimmer, E. (1988) J. Virol. 62(ll), 4393-4397. 3. Graves, M.C; Lim, J.J; Heimer, E.P; Kramer, R.A. (1988) PNAS USA 8 5 , 2449-2453. 4. Jacks, T; Madhani, H.D; Maslarz, F . R ; Varmus, H.E. (1988) Cell 51, 447-458. 5 . Pearl, L.H. and Taylor, W.R. (1987) Nature 328, 482. 6. Blunddl, T.L. (1990) Retroviral Proteinases (Pearl, L.H. od.), p.79-92, The Macmillan Press Ltd. 7 . Smith, D.B. and Johnson, K.S. (1989) Gene 67, 31. 8.Burn8, N.R: Craig, S; Lee, S . R ; Richardson, S.M.H; Stenner, N; Adams, S.E; Kingsman, S.M; Kingsman, A.J. (1990) J. M o l . Biol. 216, 207-211.
Ikprraaioa,
.ad
Yutagur.aia
of
S I V ,
Protainaao in 0 . coli BERNADETTE R. WILDERSPIN.
CORR, MARK RICHARDSON+ and ANDREW
F.
Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, Malet Street, London, WClE 7HX, UK. 'British Bio-technology Ltd., Watlington Road, Oxford, OX4 5LY, UK. Biological and biochemical characterisation of non-human AIDS model systems is essential for understanding the mechanism of retrovirus-induced innnune deficiency diseases. The highly conserved sequence homology between several of the proteins in human inununodeficiency virus (HIV-1/-2) and macaque simian innnunodeficiency virus (SIV,,) may permit such infected monkeys to be used in the testing and evaluation of potential drugs against the diseasel. Retroviral aspartic proteinases (PRs) are virally encoded enzymes responsible for the proteolytic maturation process that converts the nascent virus into a mature and infectious ford-' - hence an ideal target for the design of antiviral drugs. The human and simian PRs show approximately 80% amino acid sequence similarity with the highest degree of homology in the active site cleft5tC (see Ti1). In order to evaluate the degree of functional homology between HIV-1/-2 PR and S I V , PR initial work involved the production of large quantities of S I V , PR namely by bacterial expression. A 560bp segment of the S I V , gag-pol that encodes both the transframe protein and the first 25 amino acids of reverse transcriptase was PCR amplified from DNA extracted from a standard S I V , isolate (11/88 32H) and supplied to us courtesy of soluble Neil Almond at NIBSC (pNIBSC-6). Two intracellular systems involving a g1utathione-Stransferase (GST) fusion were initially chosen for expression (Pharmacia)'. The PR gene was cloned into the Sma I/Eco RI sites of pGEX-2T to give pGP-2T and into the B g l II/Eco RI sites of pGEX-3X to give pGP3X. Both are under the control of a tac promoter and recognition sites for site-specific nucleases, thrombin (-2T) and factor Xa (-3X), allows cleavage of the 26-kDa GST domain from the 47-kDa fusion proteins. The vectors were grown up and the fusion expressed in the E.coli strain TG1. Alternative inducible but insoluble expression systems were also employed. A synthetic 950bp horseradish peroxide (HRP) gene was cloned into the Nde I / B m HI sites of pGC517 another IPTG-inducible vector - to give pSD18. The B g l II/Bal I fragment of SIV, PR was subsequently inserted into Bg1 II/Pvu I1 cut pSD18 to give pOGS445. This resulted in the PR being located in the centre of the HRP gene thus facilitating from its initiating methionine and termination codons. Growth and expression in the E.coli strain HWlllO was to result in the production of the 54-kDa HRP-PR-HRP fusion in the form of insoluble inclusion bodies (IBs). In addition, the HRP-PR-HRP fragment was removed using Nde I/Bam HI sites from pOGS445 and cloned into the NdeI/BamHI sites of pJLA503 to give pOGS457. In this case the fusion was under the control of a A PR/PL promoter in the E.coli strain HW87. SIV, PR was thus expressed by growing pGP-ZT, pGP-3X and pOGS445 in the appropriate E.coli strain to mid-log phase (OD550m = 0.4) at 37OC and inducing with 2mM IPTG. For pOGS457, cells were grown at 3OoC to OD550m = 1.0 and induced by heat-shocking at 6OoC for 90 seconds and continuing incubation at 42OC thereafter. The cells were subsequently harvested at various times post-induction (p.i.) and centrifuged. The resultant cell pellets were resuspended in a tenth volume of SDS-loading buffer, boiled at 100°C for 5-10 minutes and ran on 20% homogeneous SDS-PAGE gels. These gels were coomassie/silver stained or used for electroblotting onto PVDF membrane. The latter was probed with a sheep anti-HIV-1 PR polyclonal antibody (1 in 500 dilution) and protein bands visualized using a donkey anti-sheep-HRP conjugated antibody with substrate 0.5mg/ml 3'-3diaminobenzidine, 0.5% hydrogen peroxide (1OOvols). To demonstrate specific SIV, PR activity, p.i. samples of the cells were resuspended in a tenth volume of ice-cold lysis buffer (50mM Tris, 5mM NaC1,
-
5mM EDTA, 1mM DTT, 1mM PMSF, 1E-64, O.Ol+g/ml aprotinin, 200pg/ml lysozyme, 10% glycerol, pH 7.8) and incubated at O°C for 30 minutes. After addition of 0.5% NP-40 and lOfig/ml DNase, the cells were sonicated at 4OC (3 x 30 second pulses). The resultant crude lysate was used directly virus-like particle (VLP) assays. At pH 6.0, lOOpl/ml VLP (80kDa substrate) was incubated at 37OC for one hour with various amounts of the lysate and subsequently ran on a 20% SDS gel. The proteins were blotted and visualised using rabbit anti-VLP antibody and an anti-rabbit-HRP conjugated antibody. Activity was noted by the presence of 53-kDa and 27-kDa product bands'. RIV-1 PQITLWQRPLVTIKIGGQLKEALLDTCADDTVLEELPGRWK RIV-2 PQFSLWKRPWTAYIEGQPVEVLLDTCADDSIVAGIELGNNS sIM.c PQFSLWRRPWTAHIEGQPVEVLLDTCADDSIVTGIELGPHYT 1 10 20 30 40 IIIV-1 RIV-2 S-C
PKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPWIIG PKIVGGIGGFINTKEYKNVEIEVL~TIMTGDTPINIFG
PKIVGGIGGFINTKEYKNVKIEVLGKRIKGTI~GDTPINIFG 50 60 70 80
RIV-1 RNLLTQIGCTLNF RIV-2 RNILTALGUSLNL SIVnuc RNLLTALGMSLNF 90 Figure 1. Amino acid sequence alignment of human and simian retroviral proteinases. The residues in boldtype represent the conserved catalytic triad in the active site (-DTG-). Expression of the PR using the two soluble systems, pGP-2T and pGP-3X, resulted in retarded growth of the cells. This effect was lessened slightly by the addition of 20% glucose to the culture, suggesting that the PR was autoprocessing itself from the GST fusion to give the active form in the cytoplasm, thus having a toxic effect on the cells. Similar results were obtained when a further two strains of E.coli were used for the expression. The formation of insoluble IBs by the HRP system was to solve these toxicity problems. For both pOGS445 and pOGS457, coomassie and silver stained gels showed and a strong band at 43-kDa (HRP and part of SIV,,) 35-kDa (HRP), with a weak band at 11-kDa. These bands were of increased intensity with increasing time p.i.. From the western blots all three bands were also visible, but the 11-kDa protein was much more intense than the others. On no occasion was the the complete 54-kDa fusion observed. The VLP assay blots only showed notable activity for pOGS457 and none for pOGS445, perhaps because A PR/PL promoter is stronger than tac and hence produces larger amounts of the PR allowing activity to be detected. Low levels of SIVmac PR have thus been expressed and specific activity recorded. Further work is aimed at designing and synthetizing PR mutants to completely evaluate the suitability of SIV,,, infected monkeys as animal models for anti-HIV drug testing. This work was supported by the MRC.
1. Kanki, P.J; Uc Lane, U.F; King, N.W; Letvin, N.L; Hunt, R.D; Sehgal, P: Daniel, M.D; Desrosiers, R.C; Essex, M. (1985) Science 228, 1189-1201. 2. Krausslich, H-G; Schneider, H; Zybarth, G; Carter, C.A; Wimmer, E. (1988) J. Virol. 62(ll), 4393-4397. 3. Graves, M.C; Lim, J.J; Heimer, E.P; Kramer, R.A. (1988) PNAS USA 8 5 , 2449-2453. 4. Jacks, T; Madhani, H.D; Maslarz, F . R ; Varmus, H.E. (1988) Cell 51, 447-458. 5 . Pearl, L.H. and Taylor, W.R. (1987) Nature 328, 482. 6. Blunddl, T.L. (1990) Retroviral Proteinases (Pearl, L.H. od.), p.79-92, The Macmillan Press Ltd. 7 . Smith, D.B. and Johnson, K.S. (1989) Gene 67, 31. 8.Burn8, N.R: Craig, S; Lee, S . R ; Richardson, S.M.H; Stenner, N; Adams, S.E; Kingsman, S.M; Kingsman, A.J. (1990) J. M o l . Biol. 216, 207-211.
