1 Int.J.Curr.Biotechnol. Volume 3; Issue 1; Jan, 2015 Mouttou Vivek Srinivas, Suresh H. Basagoudanavar, R.P. Tamil Selvan and Madhusudan Hosamani, Modulating the level of expression of 3C protease in relation to capsid precursor (P1) of FMD virus enhances the yield of empty capsids, Int.J.Curr.Biotechnol., 2015, 3(1):1-7. Modulating the level of expression of 3C protease in relation to capsid precursor (P1) of FMD virus enhances the yield of empty capsids Mouttou Vivek Srinivas, Suresh H. Basagoudanavar, R.P. Tamil Selvan and Madhusudan Hosamani* FMD research centre, Indian Veterinary Research Institute, Hebbal, Bangalore –560024, India. ARTICLE INFO ABSTRACT Article History: Received 02 December 2014 Received in revised form 15 December 2014 Accepted 25 December 2014 Available online 08 January 2015 Key words: Foot and Mouth Disease Virus, Virus- like particle, Baculovirus expression. Empty capsid particles of FMDV are being pursued as the promising non-infectious safe alterna- tive to conventional vaccine. Successful efforts have been made in producing FMDV empty capsid particles using different systems. However, increasing the yield of the empty capsids still remains a challenge. Given that capsid precursor P1 polyprotein is cleaved by protease to produce structural proteins for assembly into virus particle, regulating the stoichiometry of the 3C and P1 might improve the yields of VP0, VP1 and VP3 inturn increasing the yield of empty capsids. In the present study, using baculovirus system, we compared three recombinant viruses expressing P1 and 3C sequences. One that had one each copy of P1 and 3C under the control of polyhedrin (Ph) and P10 promoters respectively. Another construct contained two copies of P1 in combination with one copy of 3C to have lower expression of 3C relative to P1 precursor protein. These two were compared with the one that expressed P1 and 3C as a single open reading frame under polyhedrin promoter. The expressed recombinant antigens were analyzed by western blotting and sandwich ELISA using antisera produced against the native FMDV particle (146S). When we compared these constructs it was observed that expression of P1 and 3C proteins under two independent promoters showed poor accumulation of the processed capsid subunits, while the construct carrying P1-2A under Ph promoter and P1-2A-3C gene under P10 promoter, that expressed two copies of P1-2A and single copy of 3C showed higher yields of processed structural proteins showing accumulation of the cleaved ~26 kDa VP1/VP3 and ~36 kDa VP0 proteins. This construct showed an efficient processing in insect cells to produce VLPs even when com- pared with the construct that expressed P1-2A-3C as single ORF under Ph promoter. Introduction Foot-and-mouth disease (FMD) is a highly contagious and economically important disease affecting cattle, buffaloes, sheep, goats, pigs and other cloven-hoofed livestock. The etiological agent, foot-and-mouth disease virus (FMDV), is classified within the Aphthovirus genus of the Picornaviridae family. Seven serotypes (A, O, C, Asia 1, and South African Territories 1, 2, and 3) of FMDV have been identified serologically, and multiple subtypes occur within each serotype (Bachrach et al., 1968). The virus consists of non-enveloped particles that contain a positive-sense, single-stranded RNA of approximately 8.5 kb genome size. Its translation yields a polyprotein which upon subsequent processing by virus-encoded proteases produces the structural and non-structural proteins required for virus assembly and replication. In the mature virus, the genome is assembled into an icosahedral structure that is composed of 60 copies each of four structural proteins (VP4, VP2, VP3, VP1). Viral polyprotein (P1) of 95 kDa is cleaved by the viral 3C protease to yield VP0, VP1 and VP3 which self assemble to form the capsid. Autocleavage of VP0 into VP2 and VP4 occurs during encapsidation of the viral genome to produce the mature virus (Curry et al., 1997). Currently, to control the disease, tissue culture-adapted field strains are used to produce serotype specific inactivated FMD vaccines. This requires cost intensive high-containment facility, which however poses the risk of virus escape from such facility. Sometimes there is possibility of incomplete virus inactivation leading to disease outbreaks in field. To address these concerns, research is ongoing worldwide to develop recombinant vaccines for FMD that are safe to produce, yet effective at eliciting protective immunity in animals. In this context, expression from the baculovirus system offers the potential for large-scale production of non-infectious FMDV capsids. Virus-like particles (VLPs), a result of recombinant DNA technology, are formed by viral structural proteins that can inherently self-assemble and mimic the morphology of the virus, without being infective or replicating (Brun et al., 2011). FMDV subunit vaccine based on empty capsid-like particles has been developed as one of the most promising alternatives to conventional vaccines (Li et al., 2008). These recombinant non-infectious FMDV empty capsid-like particles are potentially useful for the development of diagnostic assays and as vaccine antigens. Though studies so far have shown that co- expression of 3C is essential for processing P1 International Journal of Current Biotechnology Journal Homepage : http://ijcb.mainspringer.com *Corresponding author. Email address: [email protected]Mobile no: +91-80-23410729. ISSN: 2321 - 8371
Mouttou Vivek Srinivas, Suresh H. Basagoudanavar, R.P. Tamil Selvan and Madhusudan Hosamani, Modulating the level of expression of 3C protease inrelation to capsid precursor (P1) of FMD virus enhances the yield of empty capsids, Int.J.Curr.Biotechnol., 2015, 3(1):1-7.
Modulating the level of expression of 3C protease in relation to capsid precursor (P1) ofFMD virus enhances the yield of empty capsids
Mouttou Vivek Srinivas, Suresh H. Basagoudanavar, R.P. Tamil Selvan and MadhusudanHosamani*
FMD research centre, Indian Veterinary Research Institute, Hebbal, Bangalore –560024, India.
A R T I C L E I N F O A B S T R A C T
Article History:Received 02 December 2014Received in revised form 15 December 2014Accepted 25 December 2014Available online 08 January 2015
Key words:Foot and Mouth Disease Virus, Virus-like particle, Baculovirus expression.
Empty capsid particles of FMDV are being pursued as the promising non-infectious safe alterna-tive to conventional vaccine. Successful efforts have been made in producing FMDV emptycapsid particles using different systems. However, increasing the yield of the empty capsids stillremains a challenge. Given that capsid precursor P1 polyprotein is cleaved by protease toproduce structural proteins for assembly into virus particle, regulating the stoichiometry of the3C and P1 might improve the yields of VP0, VP1 and VP3 inturn increasing the yield of emptycapsids. In the present study, using baculovirus system, we compared three recombinant virusesexpressing P1 and 3C sequences. One that had one each copy of P1 and 3C under the control ofpolyhedrin (Ph) and P10 promoters respectively. Another construct contained two copies of P1in combination with one copy of 3C to have lower expression of 3C relative to P1 precursorprotein. These two were compared with the one that expressed P1 and 3C as a single open readingframe under polyhedrin promoter. The expressed recombinant antigens were analyzed by westernblotting and sandwich ELISA using antisera produced against the native FMDV particle (146S).When we compared these constructs it was observed that expression of P1 and 3C proteins undertwo independent promoters showed poor accumulation of the processed capsid subunits, while theconstruct carrying P1-2A under Ph promoter and P1-2A-3C gene under P10 promoter, thatexpressed two copies of P1-2A and single copy of 3C showed higher yields of processed structuralproteins showing accumulation of the cleaved ~26 kDa VP1/VP3 and ~36 kDa VP0 proteins.This construct showed an efficient processing in insect cells to produce VLPs even when com-pared with the construct that expressed P1-2A-3C as single ORF under Ph promoter.
IntroductionFoot-and-mouth disease (FMD) is a highly contagiousand economically important disease affecting cattle,buffaloes, sheep, goats, pigs and other cloven-hoofedlivestock. The etiological agent, foot-and-mouth diseasevirus (FMDV), is classified within the Aphthovirus genusof the Picornaviridae family. Seven serotypes (A, O, C,Asia 1, and South African Territories 1, 2, and 3) of FMDVhave been identified serologically, and multiple subtypesoccur within each serotype (Bachrach et al., 1968). Thevirus consists of non-enveloped particles that contain apositive-sense, single-stranded RNA of approximately8.5 kb genome size. Its translation yields a polyproteinwhich upon subsequent processing by virus-encodedproteases produces the structural and non-structuralproteins required for virus assembly and replication. Inthe mature virus, the genome is assembled into anicosahedral structure that is composed of 60 copies eachof four structural proteins (VP4, VP2, VP3, VP1). Viralpolyprotein (P1) of 95 kDa is cleaved by the viral 3Cprotease to yield VP0, VP1 and VP3 which self assembleto form the capsid. Autocleavage of VP0 into VP2 andVP4 occurs during encapsidation of the viral genome toproduce the mature virus (Curry et al., 1997).
Currently, to control the disease, tissue culture-adaptedfield strains are used to produce serotype specificinactivated FMD vaccines. This requires cost intensivehigh-containment facility, which however poses the riskof virus escape from such facility. Sometimes there ispossibility of incomplete virus inactivation leading todisease outbreaks in field. To address these concerns,research is ongoing worldwide to develop recombinantvaccines for FMD that are safe to produce, yet effectiveat eliciting protective immunity in animals. In this context,expression from the baculovirus system offers thepotential for large-scale production of non-infectiousFMDV capsids. Virus-like particles (VLPs), a result ofrecombinant DNA technology, are formed by viralstructural proteins that can inherently self-assemble andmimic the morphology of the virus, without beinginfective or replicating (Brun et al., 2011).
FMDV subunit vaccine based on empty capsid-likeparticles has been developed as one of the mostpromising alternatives to conventional vaccines (Li etal., 2008). These recombinant non-infectious FMDVempty capsid-like particles are potentially useful for thedevelopment of diagnostic assays and as vaccineantigens. Though studies so far have shown that co-expression of 3C is essential for processing P1
polyprotein into individual subunits (Fig. 1), it is crucialto have controlled expression of 3C protease. Theprotease has inherent adverse effect on the host cells byone of several mechanisms as reported previously(Belsham et al., 2000; Armer et al., 2008; Falk et al., 1990).For expression of empty capsids, the uncontrolledexpression of 3Cpro has proved to have adverse effectson the yields of processed capsids (Gullberg et al., 2013).Therefore, a reduced level of 3Cpro in relation to the P1-2A precursor would allow efficient capsid proteinprocessing (Polacek et al., 2013). One strategy to reducethe level of 3Cpro activity is to use mutant forms of thisprotein with reduced enzymatic activity (Sweeney et al.,2007). An alternative system is based on reducing theamount of 3Cpro expression relative to the P1-2A. Thiscan be achieved by expressing differential levels oftranscripts, e.g. employing separate promoters to drivethe expression of two different constructs as used withenterovirus 71 and FMD Asia 1 (Chung et al., 2010, Caoet al., 2009). Alternatively, a single cDNA cassettecontaining the coding sequences for FMDV P1-2A and3Cpro can been used with the two coding sequencesbeing separated by a translational frame shift signalsequence (Porta et al., 2013b). In addition, two separateopen reading frames (ORFs) can be expressed from asingle bi-cistronic mRNA with an inefficient (mutant)internal ribosome entry site (IRES) located between them(Gullberg et al., 2013). Thus it is critical to obtainoptimized expression of 3Cpro for efficient P1 structuralprotein processing.
Keeping in view the above, the present study was aimedat modulating the level of 3C protease co-expressed withP1 precursor protein so as to improve the yields ofprocessed capsids. Such studies are expected tocontribute to improving the yields of empty capsid ofFMDV, which could be potentially applied for developingnewer diagnostics tools and/ or vaccines.
Materials and MethodsCells and virusesSpodoptera frugiperda (Sf-21) and Trichoplusia ni (Tn5)insect cells were maintained at 27°C in SF-900 II SFM(Invitrogen) and BHK-21 cells maintained in Glasgow’sModified Eagles Medium (GMEM) were used in thestudy. FMD vaccine virus serotype O, (Ind-R2/75 strain)was used for viral RNA extraction and to prepare cDNAfor amplification of genomic sequences.
Primer design and gene synthesisBased on the genomic sequence of FMDV serotype O,(IND/R2/75 strain, GenBank accession: AF204276) primers
were designed using the SnapGene software (version2.5) (Table 1).
Construction of recombinant shuttle plasmids andgeneration of recombinant baculovirus clonesThe polyprotein coding region of P1-2A (2.3 kb) and 3Cviral protease (639 bp) were amplified from cDNA ofFMDV serotype O using specific primer pairs (Table 1).The following vectors containing capsid region and 3Cprotease of FMDV serotype O were constructed.Construct 1: P1-2A amplified using primers VP4-F and2B29R-NOTI-R was cloned under polyhedrin promoter(MCS I) while 3C amplified using primer pair 3C-F and3C-R was cloned under P10 promoter (MCS II) intopFastDual vector (Invitrogen) to generate a bidirectional-bicistronic cassette (Fig. 2A). Construct 2: P1-2A regioncloned under polyhedrin promoter (MCS I) while, P1-2Afused with 3C gene as a single transcriptional unit underP10 promoter. For this, amplicons using VP4-F and2B29R-SPEI-R and 3B3C-F and 3C-R were fused in SpeIenzyme site without any transcriptional stop betweenthese two sequences (no stop codon) and then clonedunder P10 promoter (MCS II) in pFastDual vector (Fig.2B). Construct 3: P1-2A-3C sequence as singletranscriptional unit, but cloned under polyhedrinpromoter into pFastBac 1 vector (Invitrogen) to generatea unidirectional-monocistronic expression (Fig. 2C)
All the constructs cloned into the transfer vectorpFastBac dual (pFD) or pFastBac 1 (pFB) vector wereanalyzed by restriction enzyme digestion and confirmedby nucleotide sequencing to verify the direction andcorrectness of their reading frame prior before expressionstudies. These recombinant transfer vectors weretransformed into DH10Bac E.coli cells (Invitrogen) toproduce the recombinant bacmid by site-specifictransposition based on blue-white colony screening andantibiotic selections. Representative bacmid DNAconstructs were isolated and transfected into Sf-21 insectcells to produce recombinant baculoviruses. TheseRecombinant baculoviruses were propagated 2-3 roundsto amplify the viral titers in Sf-21 insect cells and wereanalyzed for expression of the recombinant proteins uponinfection of Tn5 insect cells.
Expression of FMDV capsid proteins in Tn5 cells andtheir biochemical characterizationTn5 insect cells, infected with recombinant baculovirusesat multiplicity of infection 5 were incubated at 27°C for 3days. Infected cells were then recovered by pelleting (192x g for 10min) and lysed in lysis buffer (50mM Tris, 100mMNaCl, containing 0.1% Triton X 100 and proteaseinhibitors) (Porta et al., 2013b). The clear lysate was
Figure - 1: Schematic representation of recombi-nant FMD virus like particle synthesis. ProteolyticCleavage of expressed FMDV P1-2A-3Cpolyprotein subsequently into VP0, VP3 and VP1structural proteins would allow their self assem-bly into empty virus like particles. In P1-2A-3C,targets of 3C are indicated by upright arrow whileasterisk (*) indicates self cleavage site of 2A.
Figure - 2: Schematic representation of the cDNAcassettes constructed in this study. Ph, Polyhedrinpromoter; P10, P10 promoter; P1-2A, Capsid pre-cursor protein (FMDV serotype O); 3C, 3Cpro; ATG,start codon; TAG, stop codon.
Figure – 3: Expression screening of various FMDVP1-2A cDNA cassettes by immunoblotting usinganti-146S FMDV serum. The migration positionsof P1-2A, VP0 and VP1/VP3 are indicated. Num-bers to the left are the migration positions of pro-tein markers (in kDa). Lane 1-Protein Marker, Lane2- pFB-Ph-P12A3C, Lane 3- pFD-Ph-P12A/P10-3C,Lane 4- pFD-Ph-P12A/P10-P12A3C, Lane 5- pFB-P12A.
Figure - 4: Binding of expressed FMDVcapsid proteins obtained from variousFMDV P1-2A cDNA cassettes in thisstudy to O serotype-specific anti-FMDVantibodies.
Figure - 5a: Western blot analy-sis of sucrose gradient fractionsof FMDV capsid probed with rab-bit sera raised against FMDVtype O (Ind-R2/75) 146S. Lane 1-Protein Marker, Lane 2- BHK-21grown FMDV antigen, Lane 3 to6- Fractions collected from 60%,50%, 40% and 30% sucrosefractions.
Figure - 5b: Binding of purifiedFMD Virus like particles fromvarious sucrose gradient frac-tions indicated to anti-146SFMDV serum.
Figure – 6: VLP quantification of the constructs by sandwich ELISA. The cells (Tn-5) were infected with the recom-binant baculovirus (MOI 5), and the supernatant was harvested at 3 dpi for sELISA analysis using rabbit sera raisedagainst FMDV type O (Ind-R2/75) 146S.
obtained by homogenizing with syringe and supernatantcollected. The lysates were separated by 13% sodiumdodecyl sulphate polyacrylamide gel electrophoresis(SDS–PAGE) and blotted on PVDF membrane. Electrotransferred proteins were probed with rabbit antiserumraised against FMDV type O (Ind-R2/75 strain) 146S, bywestern blotting.
Sandwich ELISA (s-ELISA) for immunoreactivitydetection of insect cell expressed FMDV proteinsThe clear lysates were tested in s-ELISA. Briefly, the 96-well ELISA plate (Nunc, Maxisorp) was coated with FMDVanti-146S serum raised in rabbit at 1 in 1000 dilution incarbonate-bicarbonate buffer (pH 9.6). The plate wasincubated at 37p C for 1 hr and washed 3 times withPBST [Phosphate buffered saline (PBS) containing 0.05%Tween-20]. Samples were added in duplicates, with 4 wellseach for positive (BHK cell culture viral antigen) andnegative (Sf-21 cellular antigen) controls. The plate wasincubated and washed. The anti-146S guinea pig tracingantibody in blocking buffer (PBST + 5% skimmed milkpowder) was then added to each well. The plate waswashed after incubation at 37p C for 1 hr and anti-guineapig IgG conjugated to HRPO (Dako, Germany) at 1:3000dilution in blocking buffer was added and incubated. Afterwashing, freshly prepared orthophenylene diamine/hydrogen peroxide substrate was added and incubatedat 37p C for 15 min for colour to develop. Then the reactionwas stopped using 1 M H2SO4. The absorbance in theplates was read at 492 nm using ELISA plate reader (TecanInfinite50). Positive–negative cut off value was calculatedas twice the mean ± standard deviation of four blankwells.
Purification and quantification of baculovirus expressedFMD type O VLPsInfected Tn5 cells were collected by centrifugation on 3days post infection and the pellet lysed by re-suspendingin lysis buffer (50mM Tris, 100mM NaCl, containing 0.1%Triton X 100 and containing 1mM each of PMSF andleupeptin) (Porta et al., 2013b) and sonicated on ice(amplitude of 27% for 1 minute with pulse of 10 secondswith pause of 20 seconds). Unbroken cells and nucleiwere removed by centrifugation (4500 rpm, 15min) andthe clarified supernatant was layered onto a 30% sucrose(w/v in 50mM Tris, 100mM NaCl) cushion. Aftercentrifugation at 100,000g for 100 min the supernatantwas discarded and the pellet suspended in Tris-NaCl andstored at 4p C overnight. Re-suspended pellet was thenapplied to a preformed discontinuous 30–60% sucrosegradient with the increment of 10% of four gradient (w/vin Tris-NaCl) (2 ml of each sucrose gradient and remainingwith sample) in 17ml polyallomer tubes. Afterultracentrifugation at 100,000g for 16hr, 4 fractions of 2ml each were collected from bottom to top and tested insandwich ELISA, spectrophotometry and westernblotting.
To quantify the VLPs by sandwich ELISA(Basagoudnavar et al., 2015 in Press), each well in the 96-well plate was coated with 50µl of FMDV anti-146S serumraised in rabbit at 1 in 1000 dilution in carbonate-bicarbonate buffer (pH 9.6) and incubated overnight.After 3 washes with PBST buffer (0.05% Tween 20 inPBS), the wells were blocked with PBST containing 1%bovine serum albumin (BSA) for 1 h. After washes, seriallydiluted samples (expressed VLPs from construct 2 and 3)were added (50µl/well). In parallel, known amount ofpurified FMDV O 146s inactivated antigens were 2-foldserially diluted and added to the wells for standard curve
generation. After 2 h incubation and 4 washes, The anti-146S guinea pig tracing antibody in blocking buffer (PBST+ 5% skimmed milk powder) was then added to each well.The plate was washed after incubation at 37p C for 1 hrand anti-guinea pig IgG conjugated to HRPO (Dako,Germany) at 1:3000 dilution in blocking buffer was addedand incubated. After washing, freshly preparedorthophenylene diamine/hydrogen peroxide substratewas added and incubated at 37p C for 15 min for colourto develop. Then the reaction was stopped using 1 MH2SO4. The absorbance in the plates was read at 492 nmusing ELISA plate reader (Tecan Infinite50).
ResultsConstruction and production of recombinant baculovirusclonesAmplicons generated by PCR were purified and clonedinto pFastDual as well as pFastBac 1 using the restrictionenzymes. Clones were analyzed for gene of interest byrestriction enzyme analysis and then sequenced. Aftertransformation of the recombinant transfer vector cloneinto E. coli DH10Bac cell, the antibiotic resistance(kanamycin, gentamicin, tetracycline) selected bacmidDNA were transfected in insect cells (Sf-21) to recoverrecombinant baculoviruses. Recombinant viruses wereamplified to high titers (1 x 108 pfu/ml) by passaging inSf-21 cells.
Analysis of FMD capsid proteins in insect cellsTo analyze the expression of P1 precursor protein and itssubsequent processing these three recombinantbaculovirus constructs were compared. Viruses were usedto infect Tn5 cells at a MOI of 5 in T175 culture flasksunder stationary culture condition. Cells were harvestedwhen they showed cytopathic changes and subjected toexpression analysis by western blot and ELISA.Schematic representations of viral genes cloned underthe control of baculovirus promoters, are provided inFig. 2. Expression of capsid proteins by three recombinantviruses were constructed as follows. Construct 1:Expressing of P1-2A under Ph promoter and 3Cpro underP10 promoter. Construct 2: P1-2A under Ph promoter andP1-2A-3Cpro as a single transcriptional unit underP10 promoter. Construct 3: P1-2A-3Cpro under Phpromoter as single ORF.
Western blotRecombinant virus that contained only P1-2A but no 3Cshowed intact band of ~95 kDa polyprotein when reactedwith polyclonal antisera raised against whole FMDV typeO 146S virus particle (Fig. 3). Expression of processedviral capsids having 3C along with P1 showed interestingresults. It was observed that expression of P1-2A and 3Cas two ORFs under the control of two independentpromoters (construct 1) did not gave the desired results.In the lysate from construct 1, no processed capsids wereseen in the western blot, while expected bands ofprocessed capsids were obtained in the other twoconstructs (Fig 3). Constructs having dual promotersexpressing P1-2A under Ph promoter and P1-2A-3C underP10 promoter reacted strongly than the P1-2A-3Cconstruct expressing both genes as single transcriptionalunit.
ELISAResults of sandwich ELISA and western blot werecomparable (Fig. 4). Construct 1 showed lower reactivitywith anti-serum as compared to other two constructs.Construct 2 showed a higher OD value in ELISA ascompared to construct 3. (Fig. 4).
Fractionation of expressed protein and theirimmunoreactivityInfected cell lysate supernatant derived from theconstruct 2 was subsequently subjected to fractionationby ultracentrifugation, and reactivity of each fractionwere checked for antigenicity by comparing withinactivated BHK-21 grown FMDV antigen, in westernblot and sandwich ELISA. The results shows that emptycapsid accumulate in the sucrose density of 50% asevident from the western blot and s-ELISA (Fig. 5a &Fig. 5b). The fractions from the middle of the gradient (at50% sucrose level), had good immunoreactivity signalas seen in s-ELISA (Fig. 5b) and they showed bandscorresponding to VP1/VP3 subunit in immunoblot assay(Fig. 5a). In lanes 4 the pattern of VP0, VP1 and VP3typical of procapsid proteins was observed which werecomparable with the native FMDV infected in BHK cells.
To test for the expression of empty capsids, extracts ofinfected insect cells harvested at 3 days post infectionwere clarified by low speed centrifugation and theparticulate material present in the supernatant wascollected by sedimentation through a 30% sucrosecushion and then on a sucrose velocity gradient. Thegradient was developed by ultracentrifugation at 100,000gfor 16h and the 6 fractions collected in eppendorf frombottom to top were tested for the spectrophotometryfollowed by estimation of protein concentration (mg/ml)as per the formula [1.55 x A280]–[0.76 x A260].
Yield of VLPs with each construct using infected celllysate at 3 dpi was assessed by sELISA (Fig. 6). In Tn-5cells, P12A-P12A-3C Construct showed ~64-fold betterVLP yield than P12A3C construct.
DiscussionCleavage of precursor polyprotein (P1) of FMDV intostructural proteins is mediated mainly by virus coded 3Cprotease, a non-structural protein. Co-expression of P1and 3C has been shown to result in processing of P1precursor capsid into subunits VP0, VP1 and VP3 thatself assemble into empty capsids as demonstrated byseveral groups (Porta et al., 2013b; Lewis et al., 1992;Roosien et al., 1990). Assembled capsids behaveantigenically similar to that of FMDV virus particles andtherefore can be used as candidate immunogens (Lewiset al., 1992) or as reagent to detect FMDV-specificantibodies (Basagoudanavar et al., 2013). For expressionof empty capsids, the 3Cpro has proved to have adverseeffects on the yields of processed capsids (Polacek etal., 2013). This may result from the fact that the FMDV3Cpro has a number of cellular targets including certaintranslation initiation factors (eIF4G and eIF4A) (Belshamet al., 2000), cytoskeleton components (Armer et al., 2008)and histone H3 (Falk et al., 1990). As a result, increasedlevel of the FMDV 3C protease has an adverse effect onFMDV capsid protein production within cells.Subsequently attempts were made to decrease the levelof 3Cpro co-expressed with the P1-2A precursor. It hasbeen reported that equimolar amounts of the 3Cpro arenot required to achieve processing of the P1-2A, by co-transfecting plasmid containing structural gene anddifferent concentration of 3C plasmid within both insectand mammalian cells (Polacek et al., 2013; Porta et al.,2013a; Porta et al., 2013b). Several different activities mayunderlie this effect including effects on translation,transcription and other cellular processes in host cells.Thus regulating the expression of 3C is critical to enhancethe yields of processed capsids.
The present study was aimed at increasing the yields ofVLPs of FMDV type O, by limiting the expression levelsof 3C protease in tandem with P1-2A polypeptide. Threeconstructs of recombinant baculoviruses, intended atlowering the levels of 3C were evaluated. One of theconstructs had P1-2A and 3C under separate promoters.Use of dual expression system was intended to havereduced levels of 3C based on the fact that polyhedrinpromoter is relatively stronger as compared to P10promoter (Chung et al., 2010; Cao et al., 2009). Howeverthis failed to give processed capsids in our study, basedon western blot and ELISA analysis. This result was incontrast to earlier report where in, expression of P1-2Aand 3C under separate promoters with serotype Asia 1lead to formation of empty capsids (Cao et al., 2009).Another construct expressed fusion protein of P1-2A and3C (designated as P1-2A-3C) in insect cells usingbaculovirus system as shown previously in our lab(Basagoudanavar et al., 2013; Bhat et al., 2013). Howevercompared to the above two constructs, higher yield ofprocessed FMDV capsid proteins could be achieved, byexpressing two copies of polyprotein (P1-2A) relative toone copy of the 3C. This was demonstrable throughhigher signals obtained both in western blot and ELISAas compared to other two constructs. This constructshowed ~64-folds higher reactivity in ELISA as comparedto P12A3C construct.
Western blot of the sucrose gradient purified fractionsof the expressed antigens showed higher antigenicityfor fractions collected in 45-50% sucrose gradients. Theobservations are consistent with a recent studiesinvolving production of empty capsids either in insectcells (Porta et al., 2013b) or mammalian cells (Polacek etal., 2013). The results of western blot are comparable tothe antigenicity observed in sELISA. There wasdifference between the signals detected using the intactprecursor (P1-2A) or the processed one. UnprocessedP1-2A protein in our study reacted well in diagnosticserotype O specific antigen ELISA as evident from higherOD value observed in sELISA. These results areconsistent with earlier results, using a P1-2A of FMDVserotype C and serotype O (Sa’iz et al., 1994; Polacek etal., 2013).
Empty capsids are an attractive vaccine candidate forFMDV as they do not require high biological containmentfacilities to produce them and importantly they allowmodification to capsid sequences to accommodate theantigenic drift. Capsid engineering, for improvedstability, broadened immune response (Tobin et al., 2008),with DIVA (Differentiating Infected from VaccinatedAnimals) compliance is an important area for improvingthe current diagnostic tools and vaccines for FMD. Thestrategy shown here will be useful to circumvent the lowercapsid yields associated with expression of FMDV emptycapsids in different systems such as baculovirus oradenovirus vector platforms.
AcknowledgementAuthors acknowledge the Director, IVRI and JointDirector, IVRI Bangalore for providing the facilities.VMVS acknowledges the financial assistance of IVRIfellowship.
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