Methods for the Use of Poliovirus for Gene Delivery Casey D. Morrow,
Vectors
David C. Ansardi, and Donna C. Porter
1. Introduction Poliovirus is a member of the Picornaviridae family of viruses. Characteristic of all members of this family, the poliovirus genome consrsts of approx 7500 bp of RNA of the plus sensepolarity (1,2). Pol~ovn-usIS undoubtedly one of the most thoroughly characterized animal vu-uses. The three-dimensional structure of the entire vtrion is known (31, an infectious cDNA clone of the poliovirus genome has been generated (4,5), the entire nucleic acid sequence of poliovirus has been determined (I), and the cellular receptor that pohovirus uses to enter cells has been cloned and sequenced (6). The avatlability of a transgenic mouse expressing the poliovirus receptor has facilitated further description of the pathogenests of poliovirus (7,8). The expression of the poliovirus genome occurs by the translation of the entire open reading frame, resulting in a long polyprotein that is subsequently processed by virus-encoded proteinases 2A and 3C (9) (Fig. 1). The initial proteolytic processing results in the release of a capsid precursor protein, P 1, by the proteinase 2A (IO). The subsequent processing of the nonstructural precursor proteins P2 and P3 is accomplished by proteinase 3C (1 I), The capsid precursor Pl is efficiently processed by 3CProin the form of a fusion protein between 3CProand 3DPoi,called 3CD (12). Previous studies have established that processing of Pl by 3CD will occur efficiently even when the two proteins are expressed from individual recombinant vaccima vnuses (12,13). The availability of an infectious clone of poliovirus has provided an opportunity to utilize molecular biologic techniques to investigate various regions of the poliovirus genome that are required for replication. Early studies have demonstrated that the majority of the Pl region of poliovirus could be deleted as From
Methods fn Molecular Medrclne, Gene Therapy Protocols Edited by P Robbins Humana Press Inc , Totowa, NJ
103
104
Morrow, Ansardi, and Porter WAPSID-NON-CAPSI743
3386
5110
5’ vpg-~ne-Jlp:(A)~AA
3’ OPEN
READING
FRAME
POLYPROTEIN
n=60
I
WEEI VP4
A
cleavage
catalyzed
by 2A
A
cleavage
h
cleavage
catalyzed
by 3C
A
source
by 3CD
of cleavage
unknown
organization of poliovirus: The entire poliovirus genome has been cloned and the 7500 bp nucleic acid sequence determined (Z). The viral RNA genome contains a long 5’ nontranslated region (approx 7.50 nucleotides containing an internal ribosome entry site [IRES]). Translation begins at nucleotide 743 and results in the synthesis of a long polyprotein. The amino-terminal glycine of the polyprotein is covalently linked to a single molecule of a 14-carbon fatty acid, myristic acid (a tetradecanoic acid). The processing of the polyprotein occurs by two viral-encoded proteinases, 2A and 3C. Following translation of the 2A proteinase, the autocatalytic activity of this enzyme releases the capsid precursor Pl from the polyprotein (10). The capsid precursor is subsequently processed by the 3C proteinase in the form of a precursor, 3CD, to give the viral capsid proteins VPO, VP3, and VPl. Following encapsidation of the viral genomic RNA, VP0 is further processed by an as yet unknown mechanism, giving VP4 and VP2. Further processing of the P2 and P3 region polyproteins is carried out by the viral 3C proteinase. Processing of the P3 polyprotein results in the release of a viral-genome linked protein, VPg, which is found attached to all 5’ poliovirus RNA molecules, and is believed to be involved in replication and encapsidation of genomic RNA. The viral RNA-dependent RNA polymerase, 3D, is released from the polyprotein by the catalytic activity of 3C. Further details regarding the sequential cleavage of the P2 and P3 proteins and the kinetics of this reaction can be found in Lawson and Semler (29). The processing of the Pl polyprotein by the 3CD viral proteinase has been demonstrated to occur in trans using both in vitro and in vivo systems (12,13). Fig.
1. Genomic
catalyzed
Poliovirus Vectors
105 Sal I
Qln Lyr
Leu Lw
Aap Thr Tyr Gly Lw t 2A potmae ahavap *It*
Glu t Xho I
Tyr Val Thr Lya Alp t Snr 81
Lou Thr Thr Tyr Gly t 2A promae olrvsg. an*
Fig. 2. Schematic of a poliovirus replicon: In previous studies, we have described poliovirus replicons that express regions of the HIV-l genome (17,19,20). We have found that we can substitute foreign gene sequences between nucleotldes 949-3359 of the poliovirus genome. This allows us to substitutea sequenceof approx 2.5 kb of
genetic information to maintun the appropriate size of the poliovn-us genome. The plasmtd containing the poliovirus rephcon is linearizedusing the MI restriction endonuclease.Following in vitro transcription using T7 RNA polymerase,the RNA is transfectedinto cells. The replication of the RNA genomeresultsin the synthesisof a
polyprotein containing a VP4-foreign gene fusion protein that can be detected m the transfected cells using appropriate antibodies. Synthetic proteolytic cleavage sites for the 2A proteinase 1s engineered at the amino and carboxy termini of the foreign geneto express proteins that can be subsequently released from the poliovirus polyprotein (20).
long as the translational reading frame was maintained between the P2 and P3 regions (14-26). Replication of a chimeric genome in which foreign gene sequences were inserted into the poliovirus genome were observed as long as the translational reading frame was maintained between the inserted genes and the remainder of the poliovirus polyprotein (17,18). These chimeric genomes maintain the capacity for replication and are referred to as “replicons.” To date, however, there has been no demonstration that genomes that do not have the capacity to replicate (i.e., in which the translational reading frame is disrupted) can be complemented in truns by viral proteins. Previous studies from our laboratory have demonstrated that between 800 and 2600 bp of foreign gene sequences can be substituted in the poliovirus genome (I 7,1%21). A prototype replicon is presented in Fig. 2. In this replicon, the foreign gene is positioned between the VP4 coding sequencesand the 2A gene of poliovirus. The foreign gene is positioned such that the translational reading frame is maintained between the VP4-foreign protein 2A protein. We have constructed these replicons such that cleavage sites for the 2A proteinase are positioned at the amino and carboxy termini of the foreign protein. The expression of the foreign protein occurs upon proteolytic processing of the VP4-foreign protein fusion protein from the remainder of the polyprotein, as a result of the autocatalytic activity of the 2A proteinase. A second cleavage occurs by the 2A proteinase in tram
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Morrow, Ansardi, and Porter
to release the foreign protem, which contains only mimmal changes at the amino terminus (usually only two amino acid changes) and eight additional ammo actds at the carboxy terminus (20). We have used this vector to express a number of different proteins, most of which retain then native features The poliovirus rephcon cDNA IS positioned nnmediately downstream from the phage T7 promoter. The plasmid templates are first linearized at a unique Sal1 restriction site outside of the viral genome; the DNA templates are then used for in vitro transcription to generate an RNA copy. The transfection of the replicon RNA into tissue culture cells results in the replication of the RNA. The replication features for these genomes are similar to those seen m poliovirus-infected cells in that there is an asymmetric overproductron of the plus strand RNA. Since the poliovirus replicons do not encode a functional PI capsid precursor protem, the genomes do not have the capacity to spread to other cells Previous studies from this laboratory have demonstrated that poliovirus genomes containmg deletions in the Pl region, or replicons that contain foreign gene insertions in the PI region, could be encapsidated into poliovirions rf transfected into cells previously infected wtth a vaccima virus (VV-Pl), which expresses the Pl protein of poliovnus (19-22). The features of this complementation system are presented m Fig. 3. Upon transfectron of the replicon RNA into cells previously infected with VV-P 1, the replicon undergoes RNA rephcatron and expression of viral proteins. Previous studies from thus laboratory have demonstrated that transfection of poliovnus rephcons mto vaccima virus-infected cells actually results m enhanced replication and expression of viral proteins (23). Expression of the viral proteins from the chimerit genome results m the production of 3CD proteinase, which processes the Pl protein expressed by the recombinant vaccinia virus zn trans. Upon processing of the PI protein, assembly of subvlral intermediates of poliovirus as well as encapsidation of the chrmeric RNA genome occurs. Amplification of the encapsidated RNA genomes is accomplished by reinfection of cells with the encapsidated replicons and VV-Pl . Multiple serial passage of the encapsidated rephcon m the presence of VV-PI results in the production of high titer stocks of the encapsidated replicons. Removal of the vaccinia virus is accomplished using a combmation of detergents and nnmunoabsorptron with anti-vaccinia virus antibodies. The methods for production of the replicons, transfection into cells, and the generation of stocks of encapsidated replicons are described in the following sections.
2. Materials All general laboratory chemicals were purchased from Sigma (St. Louis, MO). Restriction enzymes were obtained from New England Biolabs (Beverly, MA). Tissue culture media and supplements were purchased from Gibco-BRL
Poliovirus Vectors
107 Replicon
vv
VWPl Replicates, RNA Replicates, Results
m
cDNA
infect Ceils with VWPl Transfect in Vitro Transcribed Repiicon RNA Results in Pl Expression in Poliovirus Protein Expression
;:w
3CD Protein
v&lvps-lm I
Cleavage, Assembly and Encapsidation of the Repiicon RNA Occurs
Release
Passage
From the Ceil
of Encapsidated Replicons of VV-Pl to Derive Stocks
in Presence
Fig. 3. Encapsidation of poliovirus replicons using VV-Pl : Schematic representation of the encapsidation of the replicons transfected into cells previously infected with a recombinant vaccinia virus W-P 1, which expresses the poliovirus capsid precursor protein. This system is based on the ability of the poliovirus capsid precursor protein, Pl, to be processed by the poliovirus protease 3CD, provided in trans from the replication of the replicon RNA. Once the Pl protein has been processed, the encapsidation of the replicon RNA ensues, resulting in a polio virion containing the encapsidated replicon.
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(Gaithersburg, MD). The [35S]Translabel (methiomne/cysteine) and methionine/ cysteine-free DMEM were purchased from ICN Biochemicals (Costa Mesa, CA). The T7 RNA polymerase was prepared in this laboratory by the method of Grodberg and Dunn (24); alternatively, the transcription krt Megascript from Ambton (Austin, TX) can be used. 2.7. Tissue Culture Cells and Viruses Two common cell lines, HeLa and BSC40 (a derivative of CV-1) were grown m Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (complete medium). The recombinant vaccinia virus VV-P 1, which expresses the poliovirus P1 capsid precursor protein, has been previously described (13). 3. Methods 3.7. Overview The first step in the expression of a foreign protein from a poliovirus rephcon is the subcloning of the gene of interest mto a modified poliovirus cDNA. We have fused foreign genes between nucleotides 949 and 3359 of the poliovirus genome. The gene is fused such that a synthetic 2A (tyrosme-glycine) cleavage site is positioned at the Junction between the VP4 gene (amino terminus) and the foreign protein, which also has a 2A cleavage site at the carboxy terminus. We have found that following the expression of this fusion protein, the viral proteinase 2Apr0 will process the synthetic 2A positioned between VP4 and the foreign protein, respectively, resulting m the full-length desired protein. The processed protein will then need to be evaluated to determine if it has maintained its native conformation (20) (see Fig. 2). Standard molecular biology techniques can be used to subclone the foreign gene of interest into the replicon cDNA; to facilitate cloning, we have constructed rephcon cDNAs that contain unique XhoI or Sac1 (5’) and SnuBI ( 3’) restriction sites. In our previous studies, we used DNA oligomers and PCR to create the appropriate restriction sites in the foriegn genes (20,21). Once a suitable replicon has been constructed, the following procedures should be carried out to obtain stocks of encapsidated replicons: 3.2. Preparations of Template and In Vitro Transcription The preparation of the template replicon DNA required for in vitro transcription is a critical step, Single, well-isolated E. coli colonies transformed with replicon DNA are picked from the plate and inoculated directly mto 200 mL of LB plus 50 mg/mL of ampicillin. After overnight growth, the E. coli are collected by centrifugation and processed using the alkalme lysis procedure (25). The plasmid DNA is purified by ultracentrifugation in CsCl, gradients (25). After centrifugation, the band containing the DNA is visualized by UV
Poliovirus Vectors
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and removed using a syringe. The ethidium is extracted from the DNA with water-saturated butanol and the DNA is dialyzed against TE buffer (10 mA4 Tris pH 7.85 mMEDTA) overnight. The DNA is then precipitated with 0.3M NaOAC and 3 vol of 100% ethanol at -7OOC overnight. The precipitate is collected by centrifugation, washed with 70% ETOH, dried, and resuspended in TE buffer; the DNA is quantitated by UV 260/280. The DNA (5 cl8>is linearized by Sal1 digestion, followed by successive phenol-chloroform (1: 1) and chloroform extractions. The DNA is precipitated with 0.3MNaOAC and 3 vol of 100% ethanol at-70°C for at least 2 h. The precipitate is collected by centrifugation, washed once with 70% ethanol, and dried. The in vitro transcription mixtures (100 pL) contain 5 ~18of linearized DNA template, 5X transcription buffer (100 mMTris pH 7.7,50 mMMgC12, 20 mM spermidine, 250 mA4 NaCl), 10 mM dithiothreitol, 2 mM each of GTP, UTP, ATP, and CTP, 40 U of recombinant RNasin (Promega, Madison, WI), and approx 5 m of purified T7 RNA polymerase per reaction mixture. After 90 min at 37OC,5% of the in vitro synthesized RNA was analyzed by native agarose gel electrophoresis. Under these conditions, the full-length RNA transcripts (approx 7.5 kb) comigrate with the 2.0-2.2 kb DNA markers obtained from Hind111 digest of phage h. We have found that the Megascript kit from Ambion works well for in vitro transcription of the replicon DNAs. The m vitro transcribed RNAs can be transfected immediately or frozen at -70°C overnight prior to use. The best results are obtained if the m vitro synthesized RNA is transfected without further processing (such as phenolchloroform extraction).
3.3. Transfecflon of rep/icon RNA The transfection of the replicon RNA into cells can be accomplished using a variety of established methods. In our laboratory, we have utilized DEAE-Dextran as a facilitator (mol wt 500 kDa, obtained from Sigma). For our transfections, HeLa cells were first infected with 10 plaque-forming units (PFU) per cell of VV-P 1. The amounts of virus and RNA used have been optimized for a single well of a six-well plate (Falcon, Los Angeles, CA, 3046). After 2 h of infection, the cells were transfected using a protocol adapted from the originally described method of Vaheri and Pagan0 (26). The cells are washed twice with phosphate buffered saline (PBS; calcium and magnesium-free). The cells are then incubated with approx 10 M of the in vitro synthesized RNA (approximately one-fifth of the in vitro reaction) with DEAE-Dextran at a concentration of 300 &mL in PBS at 37OC for approx 2 h. Although the cells have a tendency to round up from this procedure, they do not detach from the plate. Following two h incubation, cells are washed carefully with DMEM, followed by the addition of complete medium.
110 3.4. Serial Passage of Encapsidated
Morrow, Ansardi, and Porter Rep/icons
Transfection of the replmon RNA into cells previously infected with VV-P 1 results in expression of the appropriate poliovirus proteins (e.g., 3CD) for the proteolytic processing of the Pl protein expressed from VV-Pl (19-22). This results in the assembly of subviral particles and the subsequent encapsidation of the poliovirus repltcon RNA. The procedure for passaging the encapsidated replicons IS presented m Fig. 4. Twenty-four hours after transfection of the rephcon RNA into VV-Pl -infected cells, the cells are lysed on ice by the addttron of Trrton-X- 100 at a final concentration of 1%. The lysates are then treated with RNase A for 10 min at room temperature, and the extract IS clarified by low-speed centrifugation at 14,000g for 20 min. The supernatants are then adjusted to 0.25% SDS, and overlaid onto a 30% sucrose cushion (30% sucrose, 30 mMTris pH 8.0, 1% Triton X-100, and O.lMNaCl). Following centnfugation in a Beckman SW-55 TI rotor at 290,OOOgfor 1.5 h at 4”C, the supernatant is gently decanted and the pelleted virlons are washed with a buffer consrstmg of 30 mM Trrs pH 8.0,O. 1M NaCl. To avoid losses during the washing procedure, the pellet IS again recentrrfuged in the wash buffer for an additional 1.5 h using the Beckman SW 55 TI rotor at 290,OOOg.The pellets are then resuspended in complete medium and designated as passage one of the rephcons. For additional serial passage of the encapsidated replicons, BSC40 cells are first infected with 20 PFU/cell of VV-Pl. At 2 h postinfection, the cells are infected with passage one of the encapsidated replicons. At this stage, care IS needed to maintain the small volumes during the infection owing to the low titer of the encapsidated rephcons. After 24 h, the infection IS terminated by three successrve freeze-thaws, followed by sonication and clarification by a low-speed centrifugation at 14,000g for 20 min. Previous studies from this laboratory have determined that the low-speed centrifugation results in the removal of approx 90-95% of the vaccima virus from the cell lysate (19-221. The passage procedure is repeated by first infecting BSC40 cells with VVPl, followed by reinfection wrth the cell supernatant. After 5-6 serial passages, the cell monolayer takes on a cytopathic effect consistent with that of a poliovirus infection rather than a vaccima virus infection. This is easily visualized because pohovirus results in cellular destruction leading to the detachment of the cell monolayer from the culture flask, whereas vaccinia virus-infected cells do not readily detach. Once this type of cytopathrc effect IS observed, the stock of the encapsidated replicons can be increased by infecting larger amounts of BSC40 cells. Generally, this procedure is mitiated by increasing the size from one well of a 24-well plate to two and then four wells of a 24-well plate, followed by one well of a 6-well plate, leading eventually to the serial passage of stock m large tissue culture flasks.
Poliovirus Vectors Infect cells with W-P1 (Pl capsid protein expression)
24 hours later Detergent
lyse cells: Triton X-100 (1%) and SDS (0.25%) RNase A treatment Pellet virions: Sucrose cushion
Infect cells with W-P1 Infect cells with pelleted
replicons
24 hours later Freeze-thaw 3 times Pellet 20 minutes at 14,000 x g Remove lysate, use for passages
Infect cells with VV-Pl Infect cells with replicon
stocks
Metabolically label proteins lmmunoprecipitate viral proteins using specific antisera
Fig. 4. Replicon encapsidation and passaging protocol: The procedure for the serial passage and development of stocks of the encapsidated replicon is presented. The important feature of this system is that, once the replicon has been successfully encapsidated, it can be passaged in a manner similar to poliovirus as long as coinfecting VV-Pl is present. Once sufficient stocks of the replicon have been obtained, removal of VV-Pl by either detergent treatment or immunoadsorption results in a pure stock of the encapsidated replicons.
3.5. Detection of Encapsidated Rep/icons The detection of the encapsidated replicons can be accomplished by several methods. One method is to utilize radioactive probes specific for the inserted foreign gene sequence in the poliovirus genome. A simple dot blot procedure
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can be used to detect the presence of the replicons (see Section 3.7.). A second method is to use metabolic labeling followed by immunoprecipitation to assay for protein expression from the encapsidated replicon. In this case, we use antibodies specific for the foreign protem to be expressed or antibodies to the poliovirus 3D polymerase, if antibodies to the foreign protein are not available. It is important to note that because the poliovirus RNA is translated mto a single polyprotem that is then processed, a feature of the expression of foreign proteins using the replicons is that equivalent amounts of the foreign protein and the poliovirus rephcative proteins, such as the 3Dp0’ (or 3CD) protein, should be expressed. To metabolically label cells, the cells are infected with the stocks of encapsidated rephcons. At 6 h postmfection, the cells are starved for methiomne-cysteine by incubation in DMEM mmus methionine-cysteine for 30 mm. After starvation, 100 pCi of 35S-Translabel is added to these cultures for an additional 1 h. Cultures are then processed for mununoprecipitation of viral proteins by lysing the cells with a buffer consistmg of 150 mM NaCl, 10 mM Tris-pH 8.0, 1% Triton X-100, 1% sodium deoxychlate, and 0.2% sodium dodecyl sulfate (SDS, RIPA buffer). Upon lysis of the cells, the extract is centrifuged at 14,000g for 10 min to pellet any residual debris and the designated antibodies are added to the supernatants followed by incubation at 4OC for 24 h rocking. The immunoprecipitates are collected by addition of 100 &L of protein A Sepharose (10% wt/vol in RIPA buffer). After I h rocking at room temperature, the protein A Sepharose beads are collected by a brief centrifugation, washed four times with RIPA buffer, and the bound material is diluted by boiling for 5 min in gel sample buffer. The proteins can then be analyzed by SDS-PAGE; after fluorography the proteins can be visualized by autoradiography. In all casesthus far examined, we have found that the expression of the viral protems correlates with the capacity of the RNA to replicate m the infected cells. 3.6. Isolation of Encapsidated Poliovirus Rep/icons Devoid of Vaccinia Virus Once a sufficient stock of the encapsidated replicons IS obtained, it is important to concentrate these replicons and remove any residual vaccinia virus. This can be accomplished by adjusting the concentration of the supernatant to 1% Triton X- 100 and 0.25% SDS. The virus stock is then overlaid on a 30% sucrose cushion (30% sucrose, 30 mMTris pH 8.0, 1% Tnton X-100, and O.lMNaCl), and the encapsidated replicons are pelleted through the cushion in an SW 41 rotor at 160,OOOgfor 3 h at 4°C. Although the majority of the vaccima virus has been inactivated by the detergents, a residual amount of vaccinia virus, generally less than 0.1% of the input amount, is still detectable in the sample. To remove the residual vaccinia virus, the pelleted virions are resuspended in PBS (phos-
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phate-buffered saline) KI the presence of antivaccinia antibodres (Lee Biomolecular, San Diego, CA). The sample is incubated at 4°C for 24 h, followed by the addition of protein A Sepharose. The protein A Sepharose is collected by lowspeed centrifugatlon and the supernatant containing the encapsidated replicon is then used for further characterization. We have found that this procedure virtually eliminates any remaining vaccima virus from the preparations. 3.7. Estimation of the Titer of Encapsidated Poliovirus Replicons Since these repbcons have the capacity to infect a cell but lack capsid proteins, the replicons cannot form plaques, and therefore virus titers cannot be quantitated by traditional assay.To overcome this problem, we have devised a method to estimate the titer of the encapsidated rephcons by comparison with wild-type poliovirus of known titer. For these studies, RNA from a titered stock of poliovuus is isolated according to previously described procedures (27). The virion RNA is then precipitated in 2.5 vol of ethanol using 0.2M LiC12. The encapsldated replicon RNA is extracted using the same procedure. Following precipitation, the RNAs are washed consecutively wrth 80% ethanol and 100% ethanol, dried, resuspended in DEPC-treated H20, and serially diluted and spotted onto nitrocellulose paper (dot blot apparatus from Bio-Rad, Hercules, CA). For detection of poliovirus-specific RNA, a 503-bp riboprobe was generated complementary to nucleotrdes 67 l-l 174 of the poltovnus genome. The details for the construction of the plasmid to generate this riboprobe can be found in Choi et al. (I 7). The m vitro transcription of this plasmid results m a complementary RNA that will hybridize to the plus strand RNA of both wildtype poliovirus and the encapsidated chimeric RNA genomes. The in vitro transcriptions are performed under standard conditions using 5X transcription buffer (100 rmI4 Tris pH 7.7, 50 m&f MgC&, 20 mA4 spermidine, 250 rmI4 NaCl), 10 mM dithlothreitol, 2 miI4 each of ATP, GTP, and CTP, 32[P]-UTP, 40 U recombinant RNasin (Promega), and approx 5 pg of purified T7 RNA polymerase per reaction. The conditions for hybridization can be found in Choi et al. (I 7). To determine the relative titer of the encapsidated replicons, the radioactivity associated with each dilution was quantitated using a phosphorimager (or similar radiation scanner). A standard curve was generated in which the radioactive intensity is plotted vs the plaque-forming units of the wild-type poliovirus genome. Extrapolation of the radioactivity of the encapsidated replicon samples to the curve gives an estlmatlon of the approxtmate titer of the stocks of encapsidated replicons. The resulting titer is expressed in infectious units of replicons, since the infection of cells with the replicons does not lead to plaque formation owing to the absence of the Pl capsid gene, We have determined experimentally that the infectivity of equal
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amounts of infectious units of the encapsidated replicons correlates with similar amounts of plaque-forming units of wild-type pohov~rus. 3.8. Future Directions We have effectively encapsldated genomes that express a foreign gene of approx 3.0 kb (e.g., j3-galactosldase). We have grown such rephcons to titers of lo7 infectious U/mL using standard laboratory condltlons. Recent studies from other laboratories have described the use of internal ribosome entry sites (IRES elements) positioned within the poliovirus genome to construct polyclstromc replicons (28). This technology could easily be applied to these rephcons to produce two different proteins from the same replicon. The design of these constructs would be limited only by the upper limit for the encapsidation of the poliovirus RNA genome, which is probably around 8000 bp (40s 500 bp larger than the wild-type genome). Finally, one of the unique features of these encapsldated replicons is that it is now possible to use these replicons as a means to deliver foreign antigens or biologically active proteins to cells in the absence of an infectious virus. Once suitable stocks of the encapsldated replicons have been generated, the complementing vaccinia virus, VV-PI, can be effectively removed, resulting in a stock of encapsidated replicons that have the capacity to infect a cell and undergo one round of replication. Since these replicons do not encode a functional capsid protein, they can be viewed as noninfectious, thus greatly reducing concerns of disease caused by the vector used in delivery of the foreign gene. 4. Notes One of the critical features for the initiation and maintenance of the encapsidated genomes is to accurately titer the recombinant vaccima virus expressing the poliovirus capsid precursor protein (VV-Pl). It is important to titer on the particular cell type that will be used for the transfection and encapsidation process (i.e., HeLa or BSC40 cells). We have found that it 1s critical to maintain a multiplicity of infection of approx 20 PFU/cell so that every cell is infected. Great care has to be maintained during the passaging of the encapsidated genomes. In the early stagesof passaging, there is a low level of encapsldated genomes and it 1s imperative that low culture volumes are maintained to increase the multiplicity of infection of the encapsidated genomes. Since the inherent infectivity of poliovirus is somewhat low (approx 40-1000 particles per infectious units), it is important at early stages to optimize the conditions for infectivity. As the number of serial passagesincreases, the levels of encapsidated replicons increase, allowing for the expansion of the stock of the encapsidated replicons. Again, during the expansion, care has to be taken to avoid overdilution of the encapsidated replicons.
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Acknowledgments We thank Dee Martin for preparation of the manuscript, This work is supported in part by NIH grants AI25005, AI 15 128, AI28 147, DAMD I 7-94-J4403 (US Army), and a grant from the American Foundation for AIDS Research (AmFAR 50449-l 5-PG) to CDM.
References 1. Kitamura, N., Semler, B. L., Rothberg, P. G., Larsen, G. R., Adler, C. J., Dorner, A J., Emini, E. A., Hanecak, R , Lee, J. J., van der Werf, S., Anderson, C. W., and Wmuner, E. (198 1) Primary structure, gene organization and polypeptrde expression of poliovirus RNA. Nature 291,547-553. 2. Koch, F. and Koch, G. (1985) The Molecular Bzologv of Poliowrus. SprmgerVerlag Vienna. 3. Hogle, J. M., Chow, M , and Filman, D. J. (1985) Three-dimensional structure of poliovirus at 2.9 A resolution. Science 229, 1358-1365. 4. Racaniello, V and Baltimore, D. (198 1) Cloned poliovirus complementary DNA is infectious in mammalian cells. Science 214, 9 16-9 19. 5. Semler, B. L., Dorner, A. J., and Wimmer, E. (1984) Production of infectious poliovirus from cloned cDNA is dramatically increased by SV40 transcription and replication signals Nucleic Acids Res. 12,5 123-5 141. 6. Mendelsohn, C. L., Wunmer, E., and Racaniello, V. R. (1989) Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the unmunoglobulin superfamily. Cell 56, 855-865 7. Ren, R., Costantini, F. C., Gorgacz, E. J., Lee, J. J., and Racamello, V. R. (1990) Transgemc mice expressing a human poliovirus receptor. a new model for poliomyelitis. Cell 63, 353-362. 8. Ren, R. and Racaniello, V. R. (1992) Poliovirus spreads from muscle to the central nervous system by neural pathways. J Infect Dis. 166, 747-752. 9. Palmenberg, A. C. (1990) Proteolytic processing of picornaviral polyprotem. Ann. Rev. Microbial. 44, 603-623. 10. Toyoda, H., Nicklin, M. J. H., Murray, M. G., Anderson, C. W., Dunn, J. J., Studier, F. W., and Wimmer, E. (1986) A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein. Cell 45,761-770. 11. Hanecak, R., Semler, B. L., Anderson, C. W., and Wimmer, E. (1982) Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage of glutamine-glycine pairs. Proc. Natl. Acad. SCL USA 79,3973-3977. 12. Ypma-Wong, M. F., Dewalt, P. G., Johnson, V. H., Lamb, J. G., and Semler, B. L. (1988) Protein 3CD is the major poliovirus proteinase responsible for cleavage of the Pl capsid precursor. Vzrology 166,265-270. 13. Ansardi, D. A., Porter, D. C., and Morrow, C. D. (1991) Coinfection with recombinant vaccinia viruses expressing poliovirus Pl and P3 proteins results m polyprotein processing and formation of empty capsid structures. J. Virol 65, 2088-2092.
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14. Cole, C N. (1975) Defective interfermg (DI) particles of poliovirus. Prog Med Vzrol 20, 180-207 15. Hagino-Yamagishi, K. and Nomoto, A. (1989) In vitro construction of poliovirus defective interfering particles. J Vzrol 63,5386-5392 16. Kaplan, G and Racamello, V. R. (1988) Construction and characterization of pollovn-us subgenomic replicons. J Vzrol 62, 1687-I 696 17 Chow, W S., Pal-Ghosh, R., and Morrow, C. D. (1991) Expression of human lmmunodeficlency virus type 1 HIV-l gag, pol and env proteins from chimeric HIV-l-poliovirus mimrephcons. J Vzrol. 65,2875-2883 18. Percy, N., Barclay, W. S., Sullivan, M , and Almond, J. W. (1992) A poliovirus rephcon contammg the chloramphemcol acetyl-transferase gene can be used to study the replication and encapsidation of poliovirus RNA. J Viral 66,504&5046. 19. Porter, D. C., Ansardl, D. C , Chow,W S., and Morrow, C. D. (1993) Encapsldatlon of genetically engineered pollovu~s mm+repllcons which express HIV- 1 gag and pol proteins upon mfectlon. J Vzrol. 67,37 12-37 19 20 Porter, D. C , Ansardl, D. C., and Morrow, C. D (1995) Encapsldation of pohovlrus replicons encoding the complete human lmmunodeficlency virus type 1 gag gene using a complementation system which provides the Pl capsld protein zn tram J Vzrol 69, 1548-1555. 21 Ansardi, D C., Moldoveanu, Z., Porter, D. C., Walker, D P., Conry, R. M , LoBuglio, A F., McPherson, S , and Morrow, C. D (1994) Characterization of poliovirus replicons encoding carcmoembryonic antigens Cancer Res. 54, 6359-6363. 22. Ansardi, D C., Porter, D. C., and Morrow, C. D. (1993) Complementatlon
of a pohovirus defective genome by a recombinant vaccmia virus which provides P 1 capsld precursor m trans J Virol 67, 3684-3690. 23. Pal-Ghosh, R and Morrow, C. D (1993) A poliovnus mini-replicon containing an inactive 2A proteinase 1s expressed m vaccmla virus-infected cells. J, Vzrol 67,4621-4629. 24. Grodberg, J. and Dunn, J. J. (1988) OmpT encodes the Escherichza coii outer
membrane protease that cleaves T7 RNA polymerase during purification.
J
Bacterial. 170, 1245-1253. 25. Sambrook, J., Fritsch, E F., and Mamatis, T (1989) Molecular Cloning. A Labo-
ratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 26. Vaheri, A. and Pagano, J S. (1965) Infectious poliovn-us RNA: a sensitive method of assay. Vzrologv 27,434-436. 27 Rico-Hesse, R., Pallansch, M. A., Nottay, B. K., and Kew, 0. M. (1987) Geographic distribution of wild pohovirus type 1 genotypes. Vzrology 160,3 1 l-322 28 Alexander, L., Lu, H. H., and Wlmmer, E. (1994) Pol~ovuus contammg picornavirus type 1 and.or type2 internal ribosomal entry site elements: genetic hybrids and the expression of a foreign gene. Proc. Natl. Acad. Scz USA 91, 1406-1410. 29. Lawson, M. A. and Semler, B. L. (1992) Alternate poliovirus nonstructural protein processmg cascades generated by primary sites of 3C proteinase cleavage Virology 191,309-320.
Vectors
David C. Ansardi, and Donna C. Porter
1. Introduction Poliovirus is a member of the Picornaviridae family of viruses. Characteristic of all members of this family, the poliovirus genome consrsts of approx 7500 bp of RNA of the plus sensepolarity (1,2). Pol~ovn-usIS undoubtedly one of the most thoroughly characterized animal vu-uses. The three-dimensional structure of the entire vtrion is known (31, an infectious cDNA clone of the poliovirus genome has been generated (4,5), the entire nucleic acid sequence of poliovirus has been determined (I), and the cellular receptor that pohovirus uses to enter cells has been cloned and sequenced (6). The avatlability of a transgenic mouse expressing the poliovirus receptor has facilitated further description of the pathogenests of poliovirus (7,8). The expression of the poliovirus genome occurs by the translation of the entire open reading frame, resulting in a long polyprotein that is subsequently processed by virus-encoded proteinases 2A and 3C (9) (Fig. 1). The initial proteolytic processing results in the release of a capsid precursor protein, P 1, by the proteinase 2A (IO). The subsequent processing of the nonstructural precursor proteins P2 and P3 is accomplished by proteinase 3C (1 I), The capsid precursor Pl is efficiently processed by 3CProin the form of a fusion protein between 3CProand 3DPoi,called 3CD (12). Previous studies have established that processing of Pl by 3CD will occur efficiently even when the two proteins are expressed from individual recombinant vaccima vnuses (12,13). The availability of an infectious clone of poliovirus has provided an opportunity to utilize molecular biologic techniques to investigate various regions of the poliovirus genome that are required for replication. Early studies have demonstrated that the majority of the Pl region of poliovirus could be deleted as From
Methods fn Molecular Medrclne, Gene Therapy Protocols Edited by P Robbins Humana Press Inc , Totowa, NJ
103
104
Morrow, Ansardi, and Porter WAPSID-NON-CAPSI743
3386
5110
5’ vpg-~ne-Jlp:(A)~AA
3’ OPEN
READING
FRAME
POLYPROTEIN
n=60
I
WEEI VP4
A
cleavage
catalyzed
by 2A
A
cleavage
h
cleavage
catalyzed
by 3C
A
source
by 3CD
of cleavage
unknown
organization of poliovirus: The entire poliovirus genome has been cloned and the 7500 bp nucleic acid sequence determined (Z). The viral RNA genome contains a long 5’ nontranslated region (approx 7.50 nucleotides containing an internal ribosome entry site [IRES]). Translation begins at nucleotide 743 and results in the synthesis of a long polyprotein. The amino-terminal glycine of the polyprotein is covalently linked to a single molecule of a 14-carbon fatty acid, myristic acid (a tetradecanoic acid). The processing of the polyprotein occurs by two viral-encoded proteinases, 2A and 3C. Following translation of the 2A proteinase, the autocatalytic activity of this enzyme releases the capsid precursor Pl from the polyprotein (10). The capsid precursor is subsequently processed by the 3C proteinase in the form of a precursor, 3CD, to give the viral capsid proteins VPO, VP3, and VPl. Following encapsidation of the viral genomic RNA, VP0 is further processed by an as yet unknown mechanism, giving VP4 and VP2. Further processing of the P2 and P3 region polyproteins is carried out by the viral 3C proteinase. Processing of the P3 polyprotein results in the release of a viral-genome linked protein, VPg, which is found attached to all 5’ poliovirus RNA molecules, and is believed to be involved in replication and encapsidation of genomic RNA. The viral RNA-dependent RNA polymerase, 3D, is released from the polyprotein by the catalytic activity of 3C. Further details regarding the sequential cleavage of the P2 and P3 proteins and the kinetics of this reaction can be found in Lawson and Semler (29). The processing of the Pl polyprotein by the 3CD viral proteinase has been demonstrated to occur in trans using both in vitro and in vivo systems (12,13). Fig.
1. Genomic
catalyzed
Poliovirus Vectors
105 Sal I
Qln Lyr
Leu Lw
Aap Thr Tyr Gly Lw t 2A potmae ahavap *It*
Glu t Xho I
Tyr Val Thr Lya Alp t Snr 81
Lou Thr Thr Tyr Gly t 2A promae olrvsg. an*
Fig. 2. Schematic of a poliovirus replicon: In previous studies, we have described poliovirus replicons that express regions of the HIV-l genome (17,19,20). We have found that we can substitute foreign gene sequences between nucleotldes 949-3359 of the poliovirus genome. This allows us to substitutea sequenceof approx 2.5 kb of
genetic information to maintun the appropriate size of the poliovn-us genome. The plasmtd containing the poliovirus rephcon is linearizedusing the MI restriction endonuclease.Following in vitro transcription using T7 RNA polymerase,the RNA is transfectedinto cells. The replication of the RNA genomeresultsin the synthesisof a
polyprotein containing a VP4-foreign gene fusion protein that can be detected m the transfected cells using appropriate antibodies. Synthetic proteolytic cleavage sites for the 2A proteinase 1s engineered at the amino and carboxy termini of the foreign geneto express proteins that can be subsequently released from the poliovirus polyprotein (20).
long as the translational reading frame was maintained between the P2 and P3 regions (14-26). Replication of a chimeric genome in which foreign gene sequences were inserted into the poliovirus genome were observed as long as the translational reading frame was maintained between the inserted genes and the remainder of the poliovirus polyprotein (17,18). These chimeric genomes maintain the capacity for replication and are referred to as “replicons.” To date, however, there has been no demonstration that genomes that do not have the capacity to replicate (i.e., in which the translational reading frame is disrupted) can be complemented in truns by viral proteins. Previous studies from our laboratory have demonstrated that between 800 and 2600 bp of foreign gene sequences can be substituted in the poliovirus genome (I 7,1%21). A prototype replicon is presented in Fig. 2. In this replicon, the foreign gene is positioned between the VP4 coding sequencesand the 2A gene of poliovirus. The foreign gene is positioned such that the translational reading frame is maintained between the VP4-foreign protein 2A protein. We have constructed these replicons such that cleavage sites for the 2A proteinase are positioned at the amino and carboxy termini of the foreign protein. The expression of the foreign protein occurs upon proteolytic processing of the VP4-foreign protein fusion protein from the remainder of the polyprotein, as a result of the autocatalytic activity of the 2A proteinase. A second cleavage occurs by the 2A proteinase in tram
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to release the foreign protem, which contains only mimmal changes at the amino terminus (usually only two amino acid changes) and eight additional ammo actds at the carboxy terminus (20). We have used this vector to express a number of different proteins, most of which retain then native features The poliovirus rephcon cDNA IS positioned nnmediately downstream from the phage T7 promoter. The plasmid templates are first linearized at a unique Sal1 restriction site outside of the viral genome; the DNA templates are then used for in vitro transcription to generate an RNA copy. The transfection of the replicon RNA into tissue culture cells results in the replication of the RNA. The replication features for these genomes are similar to those seen m poliovirus-infected cells in that there is an asymmetric overproductron of the plus strand RNA. Since the poliovirus replicons do not encode a functional PI capsid precursor protem, the genomes do not have the capacity to spread to other cells Previous studies from this laboratory have demonstrated that poliovirus genomes containmg deletions in the Pl region, or replicons that contain foreign gene insertions in the PI region, could be encapsidated into poliovirions rf transfected into cells previously infected wtth a vaccima virus (VV-Pl), which expresses the Pl protein of poliovnus (19-22). The features of this complementation system are presented m Fig. 3. Upon transfectron of the replicon RNA into cells previously infected with VV-P 1, the replicon undergoes RNA rephcatron and expression of viral proteins. Previous studies from thus laboratory have demonstrated that transfection of poliovnus rephcons mto vaccima virus-infected cells actually results m enhanced replication and expression of viral proteins (23). Expression of the viral proteins from the chimerit genome results m the production of 3CD proteinase, which processes the Pl protein expressed by the recombinant vaccinia virus zn trans. Upon processing of the PI protein, assembly of subvlral intermediates of poliovirus as well as encapsidation of the chrmeric RNA genome occurs. Amplification of the encapsidated RNA genomes is accomplished by reinfection of cells with the encapsidated replicons and VV-Pl . Multiple serial passage of the encapsidated rephcon m the presence of VV-PI results in the production of high titer stocks of the encapsidated replicons. Removal of the vaccinia virus is accomplished using a combmation of detergents and nnmunoabsorptron with anti-vaccinia virus antibodies. The methods for production of the replicons, transfection into cells, and the generation of stocks of encapsidated replicons are described in the following sections.
2. Materials All general laboratory chemicals were purchased from Sigma (St. Louis, MO). Restriction enzymes were obtained from New England Biolabs (Beverly, MA). Tissue culture media and supplements were purchased from Gibco-BRL
Poliovirus Vectors
107 Replicon
vv
VWPl Replicates, RNA Replicates, Results
m
cDNA
infect Ceils with VWPl Transfect in Vitro Transcribed Repiicon RNA Results in Pl Expression in Poliovirus Protein Expression
;:w
3CD Protein
v&lvps-lm I
Cleavage, Assembly and Encapsidation of the Repiicon RNA Occurs
Release
Passage
From the Ceil
of Encapsidated Replicons of VV-Pl to Derive Stocks
in Presence
Fig. 3. Encapsidation of poliovirus replicons using VV-Pl : Schematic representation of the encapsidation of the replicons transfected into cells previously infected with a recombinant vaccinia virus W-P 1, which expresses the poliovirus capsid precursor protein. This system is based on the ability of the poliovirus capsid precursor protein, Pl, to be processed by the poliovirus protease 3CD, provided in trans from the replication of the replicon RNA. Once the Pl protein has been processed, the encapsidation of the replicon RNA ensues, resulting in a polio virion containing the encapsidated replicon.
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(Gaithersburg, MD). The [35S]Translabel (methiomne/cysteine) and methionine/ cysteine-free DMEM were purchased from ICN Biochemicals (Costa Mesa, CA). The T7 RNA polymerase was prepared in this laboratory by the method of Grodberg and Dunn (24); alternatively, the transcription krt Megascript from Ambton (Austin, TX) can be used. 2.7. Tissue Culture Cells and Viruses Two common cell lines, HeLa and BSC40 (a derivative of CV-1) were grown m Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (complete medium). The recombinant vaccinia virus VV-P 1, which expresses the poliovirus P1 capsid precursor protein, has been previously described (13). 3. Methods 3.7. Overview The first step in the expression of a foreign protein from a poliovirus rephcon is the subcloning of the gene of interest mto a modified poliovirus cDNA. We have fused foreign genes between nucleotides 949 and 3359 of the poliovirus genome. The gene is fused such that a synthetic 2A (tyrosme-glycine) cleavage site is positioned at the Junction between the VP4 gene (amino terminus) and the foreign protein, which also has a 2A cleavage site at the carboxy terminus. We have found that following the expression of this fusion protein, the viral proteinase 2Apr0 will process the synthetic 2A positioned between VP4 and the foreign protein, respectively, resulting m the full-length desired protein. The processed protein will then need to be evaluated to determine if it has maintained its native conformation (20) (see Fig. 2). Standard molecular biology techniques can be used to subclone the foreign gene of interest into the replicon cDNA; to facilitate cloning, we have constructed rephcon cDNAs that contain unique XhoI or Sac1 (5’) and SnuBI ( 3’) restriction sites. In our previous studies, we used DNA oligomers and PCR to create the appropriate restriction sites in the foriegn genes (20,21). Once a suitable replicon has been constructed, the following procedures should be carried out to obtain stocks of encapsidated replicons: 3.2. Preparations of Template and In Vitro Transcription The preparation of the template replicon DNA required for in vitro transcription is a critical step, Single, well-isolated E. coli colonies transformed with replicon DNA are picked from the plate and inoculated directly mto 200 mL of LB plus 50 mg/mL of ampicillin. After overnight growth, the E. coli are collected by centrifugation and processed using the alkalme lysis procedure (25). The plasmid DNA is purified by ultracentrifugation in CsCl, gradients (25). After centrifugation, the band containing the DNA is visualized by UV
Poliovirus Vectors
109
and removed using a syringe. The ethidium is extracted from the DNA with water-saturated butanol and the DNA is dialyzed against TE buffer (10 mA4 Tris pH 7.85 mMEDTA) overnight. The DNA is then precipitated with 0.3M NaOAC and 3 vol of 100% ethanol at -7OOC overnight. The precipitate is collected by centrifugation, washed with 70% ETOH, dried, and resuspended in TE buffer; the DNA is quantitated by UV 260/280. The DNA (5 cl8>is linearized by Sal1 digestion, followed by successive phenol-chloroform (1: 1) and chloroform extractions. The DNA is precipitated with 0.3MNaOAC and 3 vol of 100% ethanol at-70°C for at least 2 h. The precipitate is collected by centrifugation, washed once with 70% ethanol, and dried. The in vitro transcription mixtures (100 pL) contain 5 ~18of linearized DNA template, 5X transcription buffer (100 mMTris pH 7.7,50 mMMgC12, 20 mM spermidine, 250 mA4 NaCl), 10 mM dithiothreitol, 2 mM each of GTP, UTP, ATP, and CTP, 40 U of recombinant RNasin (Promega, Madison, WI), and approx 5 m of purified T7 RNA polymerase per reaction mixture. After 90 min at 37OC,5% of the in vitro synthesized RNA was analyzed by native agarose gel electrophoresis. Under these conditions, the full-length RNA transcripts (approx 7.5 kb) comigrate with the 2.0-2.2 kb DNA markers obtained from Hind111 digest of phage h. We have found that the Megascript kit from Ambion works well for in vitro transcription of the replicon DNAs. The m vitro transcribed RNAs can be transfected immediately or frozen at -70°C overnight prior to use. The best results are obtained if the m vitro synthesized RNA is transfected without further processing (such as phenolchloroform extraction).
3.3. Transfecflon of rep/icon RNA The transfection of the replicon RNA into cells can be accomplished using a variety of established methods. In our laboratory, we have utilized DEAE-Dextran as a facilitator (mol wt 500 kDa, obtained from Sigma). For our transfections, HeLa cells were first infected with 10 plaque-forming units (PFU) per cell of VV-P 1. The amounts of virus and RNA used have been optimized for a single well of a six-well plate (Falcon, Los Angeles, CA, 3046). After 2 h of infection, the cells were transfected using a protocol adapted from the originally described method of Vaheri and Pagan0 (26). The cells are washed twice with phosphate buffered saline (PBS; calcium and magnesium-free). The cells are then incubated with approx 10 M of the in vitro synthesized RNA (approximately one-fifth of the in vitro reaction) with DEAE-Dextran at a concentration of 300 &mL in PBS at 37OC for approx 2 h. Although the cells have a tendency to round up from this procedure, they do not detach from the plate. Following two h incubation, cells are washed carefully with DMEM, followed by the addition of complete medium.
110 3.4. Serial Passage of Encapsidated
Morrow, Ansardi, and Porter Rep/icons
Transfection of the replmon RNA into cells previously infected with VV-P 1 results in expression of the appropriate poliovirus proteins (e.g., 3CD) for the proteolytic processing of the Pl protein expressed from VV-Pl (19-22). This results in the assembly of subviral particles and the subsequent encapsidation of the poliovirus repltcon RNA. The procedure for passaging the encapsidated replicons IS presented m Fig. 4. Twenty-four hours after transfection of the rephcon RNA into VV-Pl -infected cells, the cells are lysed on ice by the addttron of Trrton-X- 100 at a final concentration of 1%. The lysates are then treated with RNase A for 10 min at room temperature, and the extract IS clarified by low-speed centrifugation at 14,000g for 20 min. The supernatants are then adjusted to 0.25% SDS, and overlaid onto a 30% sucrose cushion (30% sucrose, 30 mMTris pH 8.0, 1% Triton X-100, and O.lMNaCl). Following centnfugation in a Beckman SW-55 TI rotor at 290,OOOgfor 1.5 h at 4”C, the supernatant is gently decanted and the pelleted virlons are washed with a buffer consrstmg of 30 mM Trrs pH 8.0,O. 1M NaCl. To avoid losses during the washing procedure, the pellet IS again recentrrfuged in the wash buffer for an additional 1.5 h using the Beckman SW 55 TI rotor at 290,OOOg.The pellets are then resuspended in complete medium and designated as passage one of the rephcons. For additional serial passage of the encapsidated replicons, BSC40 cells are first infected with 20 PFU/cell of VV-Pl. At 2 h postinfection, the cells are infected with passage one of the encapsidated replicons. At this stage, care IS needed to maintain the small volumes during the infection owing to the low titer of the encapsidated rephcons. After 24 h, the infection IS terminated by three successrve freeze-thaws, followed by sonication and clarification by a low-speed centrifugation at 14,000g for 20 min. Previous studies from this laboratory have determined that the low-speed centrifugation results in the removal of approx 90-95% of the vaccima virus from the cell lysate (19-221. The passage procedure is repeated by first infecting BSC40 cells with VVPl, followed by reinfection wrth the cell supernatant. After 5-6 serial passages, the cell monolayer takes on a cytopathic effect consistent with that of a poliovirus infection rather than a vaccima virus infection. This is easily visualized because pohovirus results in cellular destruction leading to the detachment of the cell monolayer from the culture flask, whereas vaccinia virus-infected cells do not readily detach. Once this type of cytopathrc effect IS observed, the stock of the encapsidated replicons can be increased by infecting larger amounts of BSC40 cells. Generally, this procedure is mitiated by increasing the size from one well of a 24-well plate to two and then four wells of a 24-well plate, followed by one well of a 6-well plate, leading eventually to the serial passage of stock m large tissue culture flasks.
Poliovirus Vectors Infect cells with W-P1 (Pl capsid protein expression)
24 hours later Detergent
lyse cells: Triton X-100 (1%) and SDS (0.25%) RNase A treatment Pellet virions: Sucrose cushion
Infect cells with W-P1 Infect cells with pelleted
replicons
24 hours later Freeze-thaw 3 times Pellet 20 minutes at 14,000 x g Remove lysate, use for passages
Infect cells with VV-Pl Infect cells with replicon
stocks
Metabolically label proteins lmmunoprecipitate viral proteins using specific antisera
Fig. 4. Replicon encapsidation and passaging protocol: The procedure for the serial passage and development of stocks of the encapsidated replicon is presented. The important feature of this system is that, once the replicon has been successfully encapsidated, it can be passaged in a manner similar to poliovirus as long as coinfecting VV-Pl is present. Once sufficient stocks of the replicon have been obtained, removal of VV-Pl by either detergent treatment or immunoadsorption results in a pure stock of the encapsidated replicons.
3.5. Detection of Encapsidated Rep/icons The detection of the encapsidated replicons can be accomplished by several methods. One method is to utilize radioactive probes specific for the inserted foreign gene sequence in the poliovirus genome. A simple dot blot procedure
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can be used to detect the presence of the replicons (see Section 3.7.). A second method is to use metabolic labeling followed by immunoprecipitation to assay for protein expression from the encapsidated replicon. In this case, we use antibodies specific for the foreign protem to be expressed or antibodies to the poliovirus 3D polymerase, if antibodies to the foreign protein are not available. It is important to note that because the poliovirus RNA is translated mto a single polyprotem that is then processed, a feature of the expression of foreign proteins using the replicons is that equivalent amounts of the foreign protein and the poliovirus rephcative proteins, such as the 3Dp0’ (or 3CD) protein, should be expressed. To metabolically label cells, the cells are infected with the stocks of encapsidated rephcons. At 6 h postmfection, the cells are starved for methiomne-cysteine by incubation in DMEM mmus methionine-cysteine for 30 mm. After starvation, 100 pCi of 35S-Translabel is added to these cultures for an additional 1 h. Cultures are then processed for mununoprecipitation of viral proteins by lysing the cells with a buffer consistmg of 150 mM NaCl, 10 mM Tris-pH 8.0, 1% Triton X-100, 1% sodium deoxychlate, and 0.2% sodium dodecyl sulfate (SDS, RIPA buffer). Upon lysis of the cells, the extract is centrifuged at 14,000g for 10 min to pellet any residual debris and the designated antibodies are added to the supernatants followed by incubation at 4OC for 24 h rocking. The immunoprecipitates are collected by addition of 100 &L of protein A Sepharose (10% wt/vol in RIPA buffer). After I h rocking at room temperature, the protein A Sepharose beads are collected by a brief centrifugation, washed four times with RIPA buffer, and the bound material is diluted by boiling for 5 min in gel sample buffer. The proteins can then be analyzed by SDS-PAGE; after fluorography the proteins can be visualized by autoradiography. In all casesthus far examined, we have found that the expression of the viral protems correlates with the capacity of the RNA to replicate m the infected cells. 3.6. Isolation of Encapsidated Poliovirus Rep/icons Devoid of Vaccinia Virus Once a sufficient stock of the encapsidated replicons IS obtained, it is important to concentrate these replicons and remove any residual vaccinia virus. This can be accomplished by adjusting the concentration of the supernatant to 1% Triton X- 100 and 0.25% SDS. The virus stock is then overlaid on a 30% sucrose cushion (30% sucrose, 30 mMTris pH 8.0, 1% Tnton X-100, and O.lMNaCl), and the encapsidated replicons are pelleted through the cushion in an SW 41 rotor at 160,OOOgfor 3 h at 4°C. Although the majority of the vaccima virus has been inactivated by the detergents, a residual amount of vaccinia virus, generally less than 0.1% of the input amount, is still detectable in the sample. To remove the residual vaccinia virus, the pelleted virions are resuspended in PBS (phos-
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phate-buffered saline) KI the presence of antivaccinia antibodres (Lee Biomolecular, San Diego, CA). The sample is incubated at 4°C for 24 h, followed by the addition of protein A Sepharose. The protein A Sepharose is collected by lowspeed centrifugatlon and the supernatant containing the encapsidated replicon is then used for further characterization. We have found that this procedure virtually eliminates any remaining vaccima virus from the preparations. 3.7. Estimation of the Titer of Encapsidated Poliovirus Replicons Since these repbcons have the capacity to infect a cell but lack capsid proteins, the replicons cannot form plaques, and therefore virus titers cannot be quantitated by traditional assay.To overcome this problem, we have devised a method to estimate the titer of the encapsidated rephcons by comparison with wild-type poliovirus of known titer. For these studies, RNA from a titered stock of poliovuus is isolated according to previously described procedures (27). The virion RNA is then precipitated in 2.5 vol of ethanol using 0.2M LiC12. The encapsldated replicon RNA is extracted using the same procedure. Following precipitation, the RNAs are washed consecutively wrth 80% ethanol and 100% ethanol, dried, resuspended in DEPC-treated H20, and serially diluted and spotted onto nitrocellulose paper (dot blot apparatus from Bio-Rad, Hercules, CA). For detection of poliovirus-specific RNA, a 503-bp riboprobe was generated complementary to nucleotrdes 67 l-l 174 of the poltovnus genome. The details for the construction of the plasmid to generate this riboprobe can be found in Choi et al. (I 7). The m vitro transcription of this plasmid results m a complementary RNA that will hybridize to the plus strand RNA of both wildtype poliovirus and the encapsidated chimeric RNA genomes. The in vitro transcriptions are performed under standard conditions using 5X transcription buffer (100 rmI4 Tris pH 7.7, 50 m&f MgC&, 20 mA4 spermidine, 250 rmI4 NaCl), 10 mM dithlothreitol, 2 miI4 each of ATP, GTP, and CTP, 32[P]-UTP, 40 U recombinant RNasin (Promega), and approx 5 pg of purified T7 RNA polymerase per reaction. The conditions for hybridization can be found in Choi et al. (I 7). To determine the relative titer of the encapsidated replicons, the radioactivity associated with each dilution was quantitated using a phosphorimager (or similar radiation scanner). A standard curve was generated in which the radioactive intensity is plotted vs the plaque-forming units of the wild-type poliovirus genome. Extrapolation of the radioactivity of the encapsidated replicon samples to the curve gives an estlmatlon of the approxtmate titer of the stocks of encapsidated replicons. The resulting titer is expressed in infectious units of replicons, since the infection of cells with the replicons does not lead to plaque formation owing to the absence of the Pl capsid gene, We have determined experimentally that the infectivity of equal
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amounts of infectious units of the encapsidated replicons correlates with similar amounts of plaque-forming units of wild-type pohov~rus. 3.8. Future Directions We have effectively encapsldated genomes that express a foreign gene of approx 3.0 kb (e.g., j3-galactosldase). We have grown such rephcons to titers of lo7 infectious U/mL using standard laboratory condltlons. Recent studies from other laboratories have described the use of internal ribosome entry sites (IRES elements) positioned within the poliovirus genome to construct polyclstromc replicons (28). This technology could easily be applied to these rephcons to produce two different proteins from the same replicon. The design of these constructs would be limited only by the upper limit for the encapsidation of the poliovirus RNA genome, which is probably around 8000 bp (40s 500 bp larger than the wild-type genome). Finally, one of the unique features of these encapsldated replicons is that it is now possible to use these replicons as a means to deliver foreign antigens or biologically active proteins to cells in the absence of an infectious virus. Once suitable stocks of the encapsldated replicons have been generated, the complementing vaccinia virus, VV-PI, can be effectively removed, resulting in a stock of encapsidated replicons that have the capacity to infect a cell and undergo one round of replication. Since these replicons do not encode a functional capsid protein, they can be viewed as noninfectious, thus greatly reducing concerns of disease caused by the vector used in delivery of the foreign gene. 4. Notes One of the critical features for the initiation and maintenance of the encapsidated genomes is to accurately titer the recombinant vaccima virus expressing the poliovirus capsid precursor protein (VV-Pl). It is important to titer on the particular cell type that will be used for the transfection and encapsidation process (i.e., HeLa or BSC40 cells). We have found that it 1s critical to maintain a multiplicity of infection of approx 20 PFU/cell so that every cell is infected. Great care has to be maintained during the passaging of the encapsidated genomes. In the early stagesof passaging, there is a low level of encapsldated genomes and it 1s imperative that low culture volumes are maintained to increase the multiplicity of infection of the encapsidated genomes. Since the inherent infectivity of poliovirus is somewhat low (approx 40-1000 particles per infectious units), it is important at early stages to optimize the conditions for infectivity. As the number of serial passagesincreases, the levels of encapsidated replicons increase, allowing for the expansion of the stock of the encapsidated replicons. Again, during the expansion, care has to be taken to avoid overdilution of the encapsidated replicons.
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Acknowledgments We thank Dee Martin for preparation of the manuscript, This work is supported in part by NIH grants AI25005, AI 15 128, AI28 147, DAMD I 7-94-J4403 (US Army), and a grant from the American Foundation for AIDS Research (AmFAR 50449-l 5-PG) to CDM.
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