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Journal of Biotechnology 128 (2007) 875–894
Modeling rotavirus-like particles production in a baculovirusexpression vector system: Infection kinetics, baculovirus DNA
replication, mRNA synthesis and protein production
Antonio Roldao a, Helena L.A. Vieira a, Annie Charpilienne b, Didier Poncet b,Polly Roy c, Manuel J.T. Carrondo a,d, Paula M. Alves a, R. Oliveira d,∗
a IBET/ITQB, Apartado 12, P-2781-901 Oeiras, Portugalb Virologie Moleculaire et Structurale, CNRS-INRA, 1 Avenue de la Terrasse, 91198 Gif-Sur-Yvette Cedex, France
c London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdomd FCT/UNL, Laboratorio de Engenharia Bioquımica, P-2829-516 Caparica, Portugal
Received 10 November 2006; received in revised form 21 December 2006; accepted 2 January 2007
bstract
Rotavirus is the most common cause of severe diarrhoea in children worldwide, responsible for more than half a millioneaths in children per year. Rotavirus-like particles (Rota VLPs) are excellent vaccine candidates against rotavirus infection,ince they are non-infectious, highly immunogenic, amenable to large-scale production and safer to produce than those basedn attenuated viruses. This work focuses on the analysis and modeling of the major events taking place inside Spodopterarugiperda (Sf-9) cells infected by recombinant baculovirus that may be critical for the expression of rotavirus viral proteinsVPs). For model validation, experiments were performed adopting either a co-infection strategy, using three monocistronicecombinant baculovirus each one coding for viral proteins VP2, VP6 and VP7, or single-infection strategies using a multigeneaculovirus coding for the three proteins of interest. A characteristic viral DNA (vDNA) replication rate of 0.19 ± 0.01 h−1
as obtained irrespective of the monocistronic or multigene vector employed, and synthesis of progeny virus was found to beegligible in comparison to intracellular vDNA concentrations. The timeframe for vDNA, mRNA and VP synthesis tends toecrease with increasing multiplicity of infection (MOI) due to the metabolic burden effect. The protein synthesis rates coulde ranked according to the gene size in the multigene experiments but not in the co-infection experiments. The model exhibits
Abbreviations: Rota VLPs, rotavirus-like particles; Sf-9, Spodoptera frugiperda Sf-9 cells; VPs, viral proteins; vDNA, viral DNA; MOI,multiplicity of infection; BEVS/IC, baculovirus expression vector system/insect cells; TOI, time of infection; IBDV, infectious bursal dis-ease virus; AcMNPV, autographa californica nucleopolyhedrovirus; Sf-21, Spodoptera frugiperda Sf-21 cells; ATCC, American type culturecollection; SF900II, serum free media; PBS, phosphate buffer solution; Q-PCR, quantitative polymerase chain reaction; PIPES, piperazine-1,4-bis(2-ethanesulphonic acid); Vertel, 2,3-dihydroperfluoropentane; UTRs, untranslated regions
∗ Corresponding author at: Departamento de Quımica, Laboratorio de Engenharia Bioquımica, Faculdade de Ciencias e Tecnologia,niversidade Nova de Lisboa, P-2825-516 Caparica, Portugal. Tel.: +351 21 2948303; fax: +351 21 2948385.
E-mail address: [email protected] (R. Oliveira).
168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2007.01.003
876 A. Roldao et al. / Journal of Biotechnology 128 (2007) 875–894
acceptable prediction power of the dynamics of intracellular vDNA replication, mRNA synthesis and VP production for thethree proteins involved. This model is intended to be the basis for future Rota VLPs process optimisation and also a means to
evaluating different baculovirus constructs for Rota VLPs production.© 2007 Elsevier B.V. All rights reserved.K s assem
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eywords: Rotavirus; Virus-like particles; Protein expression; VLP
. Introduction
Rotavirus disease is responsible for more than halfillion deaths per annum in the young due to severe
iarrhoea. Only in the US, 3.1 million of cases ofotavirus disease per annum are reported, resulting in0 deaths and more than 1 billion dollars spent in treat-
ents (Glass et al., 1996; Smith et al., 1995).Rotavirus, a member of the family Reoviridae, is aon-enveloped virus that can be mimicked, for vaccineurposes, by a triple-layered concentric VLP protein
Nomenclature
DNAtotal total number of intracellular vDNA copies (inteDNAT
j total number of vDNA j copies inside the cell (D
DNAnucj intracellular vDNA j concentration (DNA cell−1
j subscript index 2, 6, 7 refers to recombinant bacuka attachment rate constant (ml cell−1 h−1)kd cell death rate (h−1)kd1 intrinsic cell death rate (h−1)kd2 increase in cell death rate due to infection (h−1)kRDNA first-order vDNA replication constant (h−1)kDRNA,j first-order mRNA j degradation rate (h−1)KRNA mRNA half-saturation constant for protein synthkSRNA,j the first order transcription rate (h−1)kVP,j maximum VP synthesis rate (�g cell−1 h−1)k* increase in cell death rate corresponding to 10 ink∗
VP,j maximum VPj synthesis rate corresponding to 10
Ni concentration of infected cells (cell ml−1)Nu concentration of uninfected cells (cell ml−1)RNAj intracellular mRNA j concentration (RNA cell−1
t time post-infection (hpi)Vj concentration of extracellular recombinant baculVPj total (intracellular plus extracellular) VP concentVPint
j intracellular VP concentration (�g cell−1)Y6/2 stoichiometric ratio of proteins VP6/VP2 in correY7/2 stoichiometric ratio of proteins VP7/VP2 in corre
b(lp
bly; Mathematical modelling; Process optimisation
tructure (Mattion et al., 1994). The innermost layer isomposed by 60 dimers of VP2 (102.7 kDa) (Labbe etl., 1991); the middle shell is formed by 260 trimers ofP6 (44.9 kDa) (Prasad et al., 1988) and the third, outer
ayer is composed by 780 monomers of glycoproteinP7 (37.2 kDa) (Prasad et al., 1988).Since its development in the early 1980s, the
rnalized + newly formed viruses)NA cell−1)
)lovirus coding for VP2, VP6 and VP7 respectively
esis (RNA cell−1)
tracellular vDNA copies (h−1)intracellular vDNA copies (�g cell−1 h−1)
)
ovirus carrying gene j (DNA ml−1)ration (�g ml−1)
ctly assembled particlesctly assembled particles
aculovirus expression vector system/insect cellsBEVS/IC) has demonstrated its capacity to expressarge quantities of proteins that mimic authentic viralroteins. Indeed, this system has been successfully
A. Roldao et al. / Journal of Biotechnology 128 (2007) 875–894 877
Greek lettersδD time instant when cell death rate increases (hpi)δDNA,high time post-infection for the halt in vDNA replication (hpi)δDNA,low onset of vDNA replication (hpi)δVP,high time post-infection for the halt in VP production (hpi)δVP,low onset of VP production (hpi)δ* critical time instant corresponding to 10 intracellular vDNA copies (hpi)
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ηtraf genes’ trafficking efficiency from the memτtraf time constant for virus trafficking (hpi)
sed to express capsid virus proteins with the ability toorm VLPs. These include, amongst others, rotavirusCrawford et al., 1994; Sabara et al., 1991). RotaLPs are excellent candidates for vaccination against
otavirus since these multi-protein structures mimic therganization and conformation of authentic native par-icles but lack the viral genome, potentially yielding aafer and cheaper vaccine than those based on attenu-ted viruses. Moreover, Rota VLPs can be engineeredo contain structural proteins corresponding to differentotavirus serotypes, thus providing broader protectionCrawford et al., 1999).
Due to the complexity of Rota VLP structure, thechievement of economical volumetric productivity oforrectly assembled Rota VLPs is not an easy task.revious studies (Cruz et al., 1998; Jiang et al., 1999;aranga et al., 2003; Palomares et al., 2002; Roldao
t al., 2006; Vieira et al., 2005) reported wastefulccumulation of unassembled proteins or formationf incomplete particles, thereby reducing the processroductivity. According to Vieira et al. (2005) lesshan 15% of expressed proteins find their way intoorrectly assembled Rota VLPs probably due to incor-ect stoichiometric ratios or inadequate thermodynamicggregation conditions. To improve the overall volu-etric productivity, different infection strategies are
nder analysis, namely co-infection with three mono-istronic baculovirus, each one coding for VP2, VP6nd VP7, and single-infection strategies using a multi-ene baculovirus coding for the three proteins ofnterest simultaneously. The single-infection strategy
ith a multigene baculovirus appears to be advanta-eous over co-infection (Vieira et al., 2005), suggestinghat process optimisation can be achieved through theedesign of multigene baculovirus vectors, namely atii(s
to the cell nucleus
he promoters’ level since different promoters maynduce different levels of expression at more convenientimes for correct particle formation.
The final Rota VLPs composition and titer washown to be highly dependent on MOI and on the timef infection (TOI) in many systems (Hu and Bentley,001; Maranga et al., 2003). In this respect, the co-nfection strategy offers additional degrees of freedomnd has thus gained more attention in recent years (Hund Bentley, 2001). Theoretically, the time evolution ofP2:VP6:VP7 stoichiometric ratio can be manipulatedy the MOI of the respective baculovirus coding forspecific VP and, to a less extent, by scheduling theOI of individual monocistronic baculoviruses. Thisoses a challenging optimal control problem requir-ng an accurate dynamical model of the critical eventsnvolved in Rota VLPs synthesis.
The modeling of baculovirus infection in dif-erent culture systems was studied by Licari andailey (1992), Rosinski et al. (2002), Dee and Shuler
1997a,b), Power and Nielsen (1996), Hu and Bentley2000, 2001), Power et al. (1992, 1994) and Roldao etl. (2006). These papers provide an extensive analy-is of the infection process and virus trafficking butrotein expression is treated more empirically. Theain objective in the present study was to develop a
etailed mathematical model of Rota VLPs synthesisor process optimisation. The model integrates somef previous model formulations but addresses moreeeply gene expression and protein synthesis. In partic-lar, the following steps are addressed: (i) infection of
nsect cells by recombinant baculovirus; (ii) traffick-ng of vDNA in the cytoplasm into the cell nucleus;iii) vDNA replication; (iv) mRNA synthesis; (v) VPynthesis in the cytoplasm (VP2, VP6 and VP7).
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. Model formulation
.1. Process description
The process main steps are represented schemat-cally in Fig. 1A and B for single (M2/6/7) ando-infection (Co2 + 6 + 7) strategies, respectively. Theriggering event is the infection of insect cells by theaculovirus (step 1). The extracellular baculovirus con-aining the viral genes coding for VP2, VP6 and VP7ind to the plasmatic membrane and enter the celly adsorptive endocytosis (Volkman and Goldsmith,
985). Then follows the virus trafficking into the cellucleus (step 2). The viruses are first enclosed withinndosomes in the cytoplasmic space. As the pH insidehe endosomes drops, the viruses’ nucleocapsids, con-pwew
ig. 1. Schematic representation of the Rota VLPs production process for sindsorptive endocytosis, (2) virus trafficking, (3) vDNA replication, (4) vDN6) Rota VLPs assembly and (7) Rota VLPs release to the extracellular med
nology 128 (2007) 875–894
aining the viral genes, are released to the cytoplasmnd migrate to the cell nucleus. Then, the nucleocap-ids bind to the nucleus membrane, thereby deliveringhe genes into the cell nucleus. Once inside the nucleus,iral genes are expressed in a well orchestrated cas-ade that is normally divided in three stages: early,ate and very late gene expression (Possee and King,992). The early genes, which are expressed within therst 1–6 hpi, prior to vDNA replication, encode severalegulatory proteins that are essential for vDNA repli-ation initiation and also for the late gene transcription.he early gene transcription is insured by a host RNA
olymerase, which is present in uninfected host cells,hile the late (6–15 hpi) and very late (>15 hpi) genexpression requires a viral specific RNA polymerase,hose synthesis is induced by viral infection. Viral
gle M2/6/7 (A) and co-infection Co2 + 6 + 7 (B) strategies: (1) virusA transcription into the corresponding mRNA, (5) VPs synthesis,
ium.
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NA replication (step 3) commences at the beginningf the late phase. The dnapol, dnahel and several otherarly and late genes are known to be involved in vDNAeplication (Blissard and Rohrmann, 1990; Possee anding, 1992; Ramachandran et al., 2001; Williams andaulkner, 1996). During the late and very late phasesf gene expression, some of the vDNA replicates arencapsidated leading to the formation of new virionarticles that eventually bud from the host cell; thiss known to occur between 12 and 48 hpi (Blissardnd Rohrmann, 1990; Hu and Bentley, 2000; Hu andentley, 2001; Possee and King, 1992). The vDNA is
hen transcribed into the corresponding mRNA dur-ng the very late phase, under the control of the polhromoter or under the control of the p10 promoterepending on the construct used (step 4). SynthesizedRNA molecules leave the cell nucleus and migrate to
ibosomes where the VPs are synthesized (step 5). Theynthesized proteins, VP2, VP6 and VP7, assemble intoriple layered Rota VLPs (step 6) according to mech-nisms that are not yet fully known. Finally, the RotaLPs are released to the extracellular medium (step 7).Of all the steps involved in this process, steps 1–2
infection and virus trafficking) are the most studied inhe literature, while steps 6–7 were not characterizedt all for Rota VLPs. The remaining intracellular steps,elated to vDNA replication and VP expression (steps–5), will be the main focus of this work.
.2. Modeling assumptions
A set of simplifying hypothesis were assumed basedither on the literature or on experimental evidence:
. Excess of nutrients: the extracellular nutrients donot limit the synthesis of virus related products.
. Synchronous infection for high MOIs: in a batchculture of insect cells with MOI greater than five,the ensuing infection process will be essentially syn-chronous, i.e., all cells will go through the infectioncycle simultaneously (Power and Nielsen, 1996).Such high MOIs ensure that all cells are infected,preventing cell division from competing for nutri-ent resources needed for virus and protein synthesis
(Dee and Shuler, 1997b).. Infection kinetics is independent of the viral genein the recombinant baculovirus. Power and Nielsen(1996) showed that recombinant baculovirus carry-
bmt1
nology 128 (2007) 875–894 879
ing different foreign genes present similar infectionkinetics in Sf-9 suspension cultures.
. Virus trafficking, encompassing attachment, endo-cytosis, uncoating and transport to the nucleus areindependent of intracellular state.
. One-half of internalized virus are routed to lyso-somes and degraded (Dee and Shuler, 1997a).
. The viral genes coding for each VP here studied donot affect vDNA replication. The size of the mono-cistronic and multigene constructs is approximatelythe same, thus the vDNA replication kinetics isassumed to be the same for the different constructs.
. Negligible virus budding and release. Our data sug-gests that the rate of virus budding is much lowerthan the DNA replication rate. This hypothesis hasalso been defended by Rosinski et al. (2002).
. Negligible VP2–VP6–VP7 interaction: the interac-tion between the different proteins during synthesisis unknown.
. Stoichiometric Rota VLPs assembly. The final RotaVLPs titer is determined by the limiting protein.
.3. Baculovirus adsorption
The baculovirus enters the cell by receptor-mediatedndocytosis (Volkman and Goldsmith, 1985). Accord-ng to Dee et al. (1995) and Dee and Shuler (1997a),he depletion of extracellular virus due to binding toell surface is:
dVj
dt= −ka(Ni + Nu)Vj (1)
ith Vj concentration of extracellular recombinant bac-lovirus coding for VPj (DNAj ml−1), with subscriptndex j = 2, 6, 7 representing VP2, VP6 and VP7,espectively, ka the attachment rate (ml cell−1 h−1),onsidered here to be independent of the gene cod-ng for the VPs, Ni the concentration of infected cellscell ml−1), Nu the concentration of uninfected cellscell ml−1) and t is time post-infection (hpi). Thettachment rate is defined as follows (Dee et al., 1995):
a = kf(αR) (2)
ith k the intrinsic forward rate constant for the
finding of a single viral attachment protein to a cellembrane receptor, α the number of attachment pro-eins per virus, ∼1000 for baculovirus (Wickham et al.,990, 1992) and R is the number of free surface recep-
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ors per cell. In typical infection conditions (MOI < 50),he number of surface receptors per cell is normallyn great excess, R ∼ 11,000 for Sf-21 cells (Dee andhuler, 1997b), thus the attachment rate ka may bessumed to be time-invariant. According to Dee andhuler (1997b), the attachment rate constant variationetween baculovirus infection for Sf-21 and Sf-9 cellsn suspended cultures is meaningless; thus, a ka of.8 × 10−8 ml cell−1 h−1 was adopted for this studyDee and Shuler, 1997b). Moreover, the adsorption ofaculovirus to Sf-9 cells was studied in detail by Powert al. (1994) for MOI < 10 in suspended cultures, inespect to the effect of cell density, MOI and composi-ion of cell growth medium. It was verified that thettachment rate is independent of MOI, but slightlyependent of the cell density, as it is reported in theresent study.
For the co-infection strategy Co2 + 6 + 7 (seeable 1, experiment 5), the total extracellular virus con-entration at a given instant, Vt (DNAj ml−1), is giveny the sum of individual extracellular virus concentra-ion:
t =∑
j
Vj (3)
hile for the single-infection strategies (Table 1, exper-ments 1–4), the total extracellular virus concentration
s equal to the concentration of any extracellular virusarrying viral gene j:t = Vj, ∀j (4)
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easured times
alibration experiments
alidation experiments
a mRNA data not available.
nology 128 (2007) 875–894
.4. Infected cells population
The material balance of infected cells has two terms.he first term accounts for the increase in infected cellsoncentration due to binding of baculoviruses to unin-ected cells, Nu (cell ml−1). The second term representshe cells death rate:
dNi
dt= kaNuVt
(1
MOI
)− kdNi (5)
The viability of infected Sf-21 cells exhibits twohases (Dalal and Bentley, 1999). The first phase isharacterized by a slight decrease in infected cells con-entration related to the intrinsic cell death rate. In theecond phase, cell lysis is considerably faster due toiral infection. Accordingly, the cell death rate, kd (h−1)as two terms:
d ={
kd1, t < δD
kd1 + kd2, t ≥ δD(6)
ith kd1 the intrinsic cell death rate, which is equal to.0008 h−1 (Dalal and Bentley, 1999; Hu and Bentley,000), kd2 the increase in cell death rate due to infec-ion (h−1) and δD (hpi) the time instant when the celleath rate increases. Both kd2 and δD (hpi) show a rela-ionship with the extent of host cells infection; namely,he parameter kd2 increases with the number of virions
nfecting the cells:d2 ={
k∗, DNAtotal ≤ 10
k∗ log(DNAtotal), DNAtotal > 10(7)
250 ml Spinner @ CCI ∼ 1 × 106 cell ml−1
0/5/10/15/20/25/30/35/40/45/57/72/84/96/120/144 hpi
(1) S2 − BacRF2A − MOI5
(2) S6 − BacVP6C − MOI5
(3) S7 − BacRF7 − MOI5
(4) M2/6/7 − BRV RF VLP2/6/7 − MOI5
(5) Co2 + 6 + 7 − BacRF2A + BacVP6C + BacRF7 − MOI5+5+5
(6) Co2 + 6 − BacRF2A + BacVP6C − MOI5+5a
(7) M2/6 − BRV RF VLP2/6 − MOI5a
(8) Co2/6 + 7 − BRV RF VLP2/6 + BacRF7 − MOI5+5a
(9) M2/6/7 − BRV RF VLP2/6/7 − MOI15a
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ith k* (h−1) the increase in cell death rate correspon-ent to 10 intracellular vDNA copies and DNAtotal theotal number of intracellular vDNA copies (see Eqs.11) and (12)). The calculation of δD is discussed at thend of this section.
.5. Healthy cells population
The material balance of uninfected cells, Nucell ml−1), comprises two terms. The first term repre-ents the “conversion” of uninfected cells into infectedells due to virus binding. The second term reports thentrinsic cell death rate:
dNu
dt= −kaNuVt
(1
MOI
)− kd1Nu (8)
.6. vDNA replication
The dynamics of vDNA in the cell nucleus, DNAnucj
DNAj cell−1) is given by the following material bal-nce equation:
dDNAnucj
dt= ηtrafkaVj(t − τtraf)
(1 + Nu
Ni
)
+kRDNADNAnucj
×fDNA,rep(t, δDNA,low, δDNA,high) (9)
he first term accounts for the transport of vDNA fromxtracellular virus into the cell nucleus. Parameter ηtrafs the trafficking efficiency, which is the fraction ofnternalized genes that manage to enter the cell nucleus.onsidering that about half of internalized baculovirus
s degraded, most likely in lysosomes which containhe hydrolytic enzymes able to degrade the virus rapidlyDee and Shuler, 1997a), then ηtraf is equal to 0.5. Sinceirus trafficking within the cell takes some time, thisransport term has a delay τtraf (h). According to Deend Shuler (1997a), the mean time for trafficking is asollows: 43 min for endocytosis, 40–60 min for uncoat-ng and 6–25 min for transport to nucleus, which sumo τtraf = 1.8 h.
The second term is the vDNA replication kinetics,
ith kRDNA (h−1) the first order replication con-tant. The time-dependent function fDNA,rep(t, δDNA,low,DNA,high) defines a time interval [δDNA,low, δDNA,high]or vDNA replication, accounting also for the effect
tn[δ
nology 128 (2007) 875–894 881
f the metabolic decay on vDNA replication (see Eq.17)).
The total number of vDNA copies inside the cell,NAT
j (DNAj cell−1), is the sum of vDNA within theucleus and vDNA in the cytoplasm:
dDNATj
dt= ηtrafkaVj
(1 + Nu
Ni
)
+kRDNADNAnucj fDNA,rep
×(t, δDNA,low, δDNA,high) (10)
he first term accounts for the internalization of extra-ellular virus while the second corresponds to theDNA replication kinetics.
The total number of vDNA copies inside the celln the co-infection experiments is given by the sum ofndividual intracellular vDNA copies:
NAtotal =∑
j
DNATj (11)
hile for multigene experiments, the total number ofDNA copies inside the cell corresponds to the indi-idual intracellular vDNA copies:
NAtotal = DNATj , ∀j (12)
.7. mRNA synthesis
The transcription of the genes coding for VP2, VP6r VP7 into the corresponding mRNA, under the controlf a very late promoter (either polh or p10 promoter),ollows first order kinetics on the corresponding vDNAemplates:
dRNAj
dt= kSRNA,jDNAnuc
j fVP(t, δVP,low, δVP,high)
−kDRNA,jRNAj (13)
ith RNAj (RNAj cell−1) the intracellular concen-ration of mRNA, kSRNA,j (h−1) the first orderranscription rate and kDRNA,j the first order mRNAegradation rate (h−1). The vDNA transcription and
ranslation processes obey to temporal control mecha-isms. Therefore, a time interval for mRNA synthesisδVP,low, δVP,high] is defined by the function fVP (t,VP,low, δVP,high) (see Eq. (18)).
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.8. Viral protein synthesis
The kinetics of structural VP synthesis, VPj
�g ml−1), was defined with Michaelis–Menten kinet-cs on RNAj:
dVPj
dt=kVP,j
RNAj
KRNA+RNAj
fVP(t, δVP,low, δVP,high)Ni
(14)
ith kVP,j (�g cell−1 h−1) the maximum VPj synthesisate and KRNA (RNA cell−1) the half-saturation con-tant for intracellular mRNA. The intracellular proteinontent, VP int
j (�g cell−1) is given by the followingquivalent equation:
dVPintj
dt=kVP,j
RNAj
KRNA+RNAj
fVP(t, δVP,low, δVP,high)
(15)
ote that both VPj and VPintj account for assembled
nd unassembled proteins altogether.The maximum VP synthesis rate, kVP,j, shows
n indirect relationship with the intracellular vDNAoncentration probably via the effect of translationnhancing factors coded by early and/or late genes (Lund Miller, 1995). Here, such effect has been expresseds follows:
VP,j ={
k∗VP,j, DNAtotal ≤ 10
k∗VP,j log(DNAtotal), DNAtotal > 10
(16)
ith k∗VP,j (�g cell−1 h−1) the maximum VPj synthesis
ate correspondent to 10 intracellular vDNA copies andNAtotal the total number of intracellular vDNA copies
Eqs. (11) and (12)).
.9. Temporal control
The expression of viral genes occurs in an orderedascade of events, whereby the early genes transac-ivate directly or indirectly the late genes, which inurn transactivate the very late genes. This sequencef events result in well defined time intervals for
DNA replication and VP expression that may bexpressed as functions of the intracellular viral loadHu and Bentley, 2000, 2001; Licari and Bailey,992).loiL
nology 128 (2007) 875–894
The vDNA replication, which commences in theate phase at δDNA,low = 6 hpi (Blissard and Rohrmann,990; Carstens et al., 1979; O’Reilly et al., 1994;ossee and King, 1992; Power et al., 1994; Rosinskit al., 2002; Tjia et al., 1979), is strongly affecteduring the overall process since infected cells grad-ally lose their ability to synthesize vDNA. Inheir segregated population framework, Licari andailey (1992) expressed this metabolic decay bylinear decay in time. The same concept was
dopted here to define the time window for vDNAeplication:
DNA,rep(t, δDNA,low, δDNA,high)
=
⎧⎪⎪⎨⎪⎪⎩
0, t < δDNA,low
1 − t − δDNA,low
δDNA,high − δDNA,low, δDNA,low ≤ t < δDNA,high
0, t ≥ δDNA,high(17)
The expression of VPs which are under the controlf very late promoters, polh or p10, starts approx-mately at δVP,low = 15 hpi (Hu and Bentley, 2000,001; Palomares et al., 2002; Possee and King,992). Hu and Bentley (2000, 2001) and Licari andailey (1992) considered a linear time decay func-
ion for VPs production. Here, a similar approach wasdopted:
VP(t, δVP,low, δVP,high)
=
⎧⎪⎪⎪⎨⎪⎪⎪⎩
0, t < δVP,low
1 − t − δVP,low
δVP,high − δVP,low, δVP,low ≤ t < δVP,high
0, t ≥ δVP,high(18)
Interestingly, our data suggests the existence ofhree critical events practically coincident in time:i) the increase in cell death rate; (ii) the halt inDNA replication; (iii) the halt in VPs expression.hese major events are somehow interconnected inhat appears to be an influence of intracellular viral
oad. This specific moment in time has been previ-usly expressed as a function of the number of virusesnfecting the host cell (Hu and Bentley, 2000, 2001;icari and Bailey, 1992). Here, this time instant is
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xpress as a dynamic function of the intracellularDNA copies:
DNA,high = δVP,high = δD
=⎧⎨⎩
δ∗, DNAtotal ≤ 10δ∗
log(DNAtotal), DNAtotal > 10
(19)
ith δ* (hpi) the critical time instant correspondent to0 intracellular vDNA copies.
.10. VLPs assembly
Most modeling studies overlook the VLP assemblyrocess. Hu and Bentley (2000, 2001) were the firsto propose a mathematical description of the assem-ly of infectious bursal disease virus (IBDV) VLPs.BVD VLPs are icosahedral particles composed of twotructural proteins only: VP2 and VP3. The authorsonsidered that VP2 and VP3 first form trimers andhen two different stable structural subunits, which con-titute the 20 triangular faces of an icosahedron. Thetructural subunits then aggregate resulting in a sta-le VLP. The assembly process was described usingthermodynamically based equilibrium model. RotaLPs are somewhat more complex to model since
hey involve three proteins: VP2, VP6 and VP7. Duringhe assembly process, many different stable subunitsre possible and there is yet limited knowledge ofhe details of rotavirus VLPs assembly in insect cellsMena et al., 2005). A simpler approach was thendopted, based on the VPs stoichiometric ratios (Y6/2nd Y7/2) and on their comparison with the compo-ition of correctly assembled particles, as a mean tovaluate the dynamics of Rota VLPs assembly for pos-ible process limitations analysis. The relative massomposition of VP6/VP2 and VP7/VP2 in correctlyssembled particles is Y6/2 = 2.8 (w/w) and Y7/2 = 2.4w/w), respectively.
. Materials and methods
.1. Cell culture and media
Spodoptera frugiperda, Sf-9 and Sf-21 cell linesere obtained from American Type Culture Collection
ATCC, US). Both cell types were cultured in serumhl
nology 128 (2007) 875–894 883
ree media SF900II (Gibco, Glasgow, UK) at 27 ◦Cn 250 ml (working volume) spinner flasks at 170 rpmr in 500 ml Erlenmeyers (50 ml working volume) at0 rpm. Cell concentration was determined by Fuchs-osenthal hemacytometer (Brandt, Wertheim/Main,ermany) and cell viability was evaluated by trypanlue exclusion dye (Merck, Darmstadt, Germany) usedt 0.4% in phosphate buffer solution (PBS).
.2. Baculovirus stocks and infection conditions
The recombinant baculovirus AcMNPV (Auto-rapha californica nucleopolyhedrovirus) expressesP2, VP6 and/or VP7 and were used to infect Sf-9
ells for virus propagation, using a MOI of 0.1 plaque-orming units (pfu) per cell, and VLPs production usingMOI of five pfu/cell. The recombinant baculovirusere also used to infect Sf-21 cells for virus titrationsing the assay described by Mena et al. (2003) basedn cell viability (Mena et al., 2003). The infectionsere performed when the cultures reached a cell con-
entration of approximately 1 × 106 cell ml−1 (CCI),hought some variability occurred due to differences inhe inoculum.
The monocistronic recombinant baculovirusesacRF2A (Labbe et al., 1991), BacVP6C (Tosser etl., 1992) and BacRF7 coding for VP2, VP6 andP7, respectively, and the multigene recombinantaculovirus VP2(RF)/VP6(RF) VLPs, all of bovineotavirus strain RF, were kindly provided by Dr.ean Cohen from Centre National pour la Recherchecientifique-Institut National Recherche AgronomiqueCNRS-INRA, Gif-sur-Yvette, France).
The multigene recombinant baculovirus BRV RFLP2/6/7 coding for three viral proteins, VP2, VP6
nd VP7, simultaneously is of bovine rotavirus strainF and was kindly provided by Prof. Polly Roy from
he London School of Hygiene & Tropical MedicineEngland). All genes were under the control of the polhromoter with the exception of the gene for VP7 in theecombinant multigene 2/6/7 baculovirus that is underhe control of p10 promoter.
.3. Nucleic acid quantification
The DNA and RNA extraction was performed usingigh pure viral nucleic acid kit and high pure RNA iso-ation kit (Roche Diagnostics, Mannheim, Germany),
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espectively. The total RNA was quantified by spectralbsorption (GeneQuant II, Pharmacia Biotech, UK).aculovirus DNA and mRNA levels were quantifiedy Quantitative Polymerase Chain Reaction (Q-PCR)nd reverse transcription Q-PCR, respectively, usingrimers specific for each gene coding for VP2, VP6nd VP7 (Vieira et al., 2005). To check RNA integrity,garose gel electrophoresis was performed.
.4. Western blotting analysis
Western blot techniques were applied for pro-ein quantification according to the method describedy Vieira et al. (2005). Depending on the infec-ion strategy, immunochemical staining was performedsing monoclonal or polyclonal antibodies. In theingle-infection case with the multigene baculovirusnd the co-infection experiment (Table 1, experi-ents 4 and 5, respectively), a mouse anti-VP2 IgGonoclonal antibody and a rabbit anti-rotavirus IgG
olyclonal antibody were used as primary antibodies.or the single-infection strategies (Table 1, experi-ents 1–3) different primary antibodies were used:
i) S2 strategy—mouse anti-VP2 IgG monoclonalntibody; (ii) S6 strategy—mouse anti-VP6 IgG mono-lonal antibody; (iii) S7 strategy—rabbit anti-rotavirusgG polyclonal antibody. All primary antibodies wereindly provided by Dr. Jean Cohen (CNRS-INRA, Gif-ur-Yvette, France). Blots were then developed afterncubation with anti-mouse and anti-rabbit IgG, bothonjugated with alkaline phosphatase using 1-stepTMBT/BCIP blotting detection reagents (Pierce). For2/6/7 and Co2/6 + 7 strategies (Table 1, experimentsand 5), as second antibody an anti-rabbit IgG antibodynd an anti-mouse IgG antibody (Sigma) were used. Inhe single strategies (Table 1, experiments 1–3), S2 and6 used an anti-mouse IgG antibody (Sigma) while for7 an anti-rabbit IgG antibody was used. The productield was estimated by densitometry analysis of thecanned images (ImageQuant® for Microsoft® Win-ows NT®, Molecular Dynamics, Inc., US, 1998).
.5. Rota VLPs purification
Cell culture bulks were harvested and cells removedy centrifugation at 500 × g for 10 min at 4 ◦C, atay 6. The supernatant was then ultracentrifuged at4,000 rpm (Beckman 45Ti rotor) for 1 h at 4 ◦C and
i(fr
nology 128 (2007) 875–894
he pellet resuspended in a small volume of 20 mMIPES buffer at 1 mM CaCl2 and pH 6.6 complementedith antiproteases (aprotinin and leupeptin). Follow-
ng, a lipid extraction was performed by adding threeolumes of Vertel to five volumes of pellet. A centrifu-ation step was then applied (low speed for 5 min); thepper phase containing the particles was transferredo a new centrifuge tube where four volumes of Vertelere added for a second extraction (the lower phase was
e-extracted by re-addition of buffer). At the end of theentrifugation steps, all aqueous phase containing RotaLPs was pooled together for product quantification.
.6. CsCl analysis for Rota VLPs assessment
To each 5 ml of aqueous phase resulting from pre-ious centrifugations, 2 g of CsCl was added for thereparation of a CsCl gradient (the refractive indexhould be 1.362). Following, an ultra-centrifugationtep was performed at 35,000 rpm for 18 h at 4 ◦CBeckman SWI55 rotor). Upon centrifugation, the bandontaining intact Rota VLPs was collected and particlesuantified. For that purpose the total protein contentn purified VLP’s was determined using BCA Proteinssay Kit from Pierce (Protein Assay Kit, Rockford,
L).
.7. Experimental planning
A series of infection strategies were performedn this study (see Table 1). For model calibration,ingle and co-infection experiments were used (exper-ments 1–5). In the single-infection case, experimentsere performed with recombinant baculovirus cod-
ng for VP2 or VP6 or VP7 individually (experiments–3, respectively) or simultaneously using a multi-ene baculovirus (experiment 4). In the co-infectionase, three monocistronic recombinant baculovirusoding for VP2, VP6 and VP7 were used to infect Sf-cells (experiment 5). As for model validation, four
dditional experiments (experiments 6–9) were used:i) co-infection using two monocistronic recombinantaculovirus coding for VP2 and VP6 (experiment 6),ii) single-infection with a multigene baculovirus cod-
ng for VP2 and VP6 simultaneously (experiment 7),iii) co-infection with a multigene baculovirus codingor VP2 and VP6 simultaneously plus a monocistronicecombinant baculovirus coding for VP7 (experiment
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), and (iv) single-infection with a multigene bac-lovirus coding for VP2, VP6 and VP7 simultaneouslyt a MOI of 15 pfu/cell (experiment 9).
.8. Kinetic parameters estimation
A kinetic parameters estimation program was devel-ped in MATLAB, The Mathworks, Inc., implement-ng the simultaneous parameter estimation strategy.he program minimizes the residuals in the sensef least squares employing the Levenberg-Marquardtlgorithm. The mathematical model equations (1)–(19)ere integrated using a fourth/fifth order Runge–Kutta
olver modified to handle ordinary differential equa-ions with time delays. The final residuals and Jacobian
atrix served to calculate an approximation to the Hes-ian matrix, thereby assuming that the final solution is aocal optimum. The Hessian matrix enabled to calculatehe parameters covariance matrix and parameters 95%
actp
able 2nfection and trafficking parameters
arameter Value
a 7.8 × 10−8 ml cell−1 h−1
d1 0.0008 h−1
traf 1.8 h
traf 0.5* 0.0017 ± 0.0003 h−1
* 233 ± 7 h
able 3iral protein expression: model parameters for vDNA replication, transcrip
VP2
RDNA (h−1)
DNA,low (hpi)
SRNA,j (h−1)polh 0.12 ± 0.01p10 –
DRNA,j (h−1) 0.019 ± 0.004
∗VP,j (�g cell−1 h−1) × 10−6
polh 0.19 ± 0.02p10 –
RNA (RNA cell−1)
VP,low (hpi)
a Fitted by trial and error.
nology 128 (2007) 875–894 885
onfidence intervals. The MATLAB ‘nlparci’ functionas used for this calculation.The determination and accuracy of precise virus
tocks titers is determined by the precision and repro-ucibility of the method employed. Unfortunately,ommonly applied methods (plaque assay and end-oint dilution assay) are known to be rather inaccurateMena et al., 2003), which inherently will affect the
OI determination for a specific experiment. Thexperimental error in the determination of the MOIs critical because it propagates exponentially in thealculation of the intracellular vDNA templates, thusaving a major impact on the overall virus dynamics.o overcome this problem, for the purpose of kineticarameters estimation, Eqs. (1)–(7) were “bypassed”
nd the data of the first 5 hpi was not taken for parameteralibration. This allowed to estimate more accuratelyhe vDNA replication, transcription and translationarameters.Reference
Dee and Shuler (1997b)Dalal and Bentley (1999) and Hu and Bentley (2000), this paperDee and Shuler (1997a)Dee and Shuler (1997a)This paperThis paper
tion and translation
VP6 VP7
0.19 ± 0.01
6
0.16 ± 0.02 0.11 ± 0.01– 0.46 ± 0.01
0.025 ± 0.005 0.013 ± 0.004
1.0 ± 0.1 0.058 ± 0.007– 0.751 ± 0.007
0.2 × 104a
15
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. Results and discussion
.1. Model calibration and validation
The calibration of model parameters was madesing the first five experiments specified in Table 1.s for model validation, four additional experiments
experiments 6–9) were used. The final results are pre-ented in Tables 2 and 3. Table 2 shows the parameters
elated to the infection and trafficking steps whileable 3 compiles the parameters related to viral proteinxpression. The parameters’ estimates show high sta-istical confidence (narrow 95% confidence intervals).4
k
ig. 2. Model predictions over measured variables: infected cells concentratoncentration (C) and total VP concentration (D). The full line (—) indicate5% confidence prediction errors. Open (�, � and ©) and closed (�, � anxperiments, respectively. Triangles, squares and circles represent data of g
nology 128 (2007) 875–894
ig. 2 shows model predictions against the corre-ponding measured variables for both the calibrationnd validation datasets. The 95% confidence predic-ion errors are as follows: Ni ± 0.10 × 106 cell ml−1,NAnuc
j ± 0.58 × 104 DNA cell−1, RNAj±0.32×104
NA cell−1 and VPj ± 1.19 × 101 �g ml−1. In theollowing sections, we analyze in detail the modelingesults.
.2. Effect of viral infection on cell death rate
The intrinsic cell death rate of Sf-9 cells,d1 = 0.0008 h−1 is in agreement with previous stud-
ion (A), intracellular vDNA concentration (B), intracellular mRNAs the average model prediction and the dashed lines (- - -) representd �) symbols denote data resulting from calibration and validationenes coding for VP2, VP6 and VP7, respectively.
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A. Roldao et al. / Journal of
es (Dalal and Bentley, 1999; Hu and Bentley, 2000).pon viral infection, the increase in cell death rate,
haracterized here by the kd2 parameter, exhibits someariability depending on the viral loading. For exam-le, the kd2 is reportedly higher in the co-infectionxperiment (Co2 + 6 + 7) than in the single-infectionxperiments (S2, S6, S7 and M2/6/7) in consequencef the higher MOI used (MOI5+5+5 versus MOI5),ringing about a more rapid decrease in cell viabil-ty (see for instance Licari and Bailey (1991)). Higher
OI leads to higher packing of baculoviruses insidehe cell promoting faster cell death through apop-osis. The baculovirus can prevent premature deathn infected Sf cells through the p35 gene, which ishought to block the apoptotic response to AcMNPVClem et al., 1991). However, according to Clemt al. (1991), such effect is overtaken by the apop-otic effect, inducing greater cell death rate in cellsnfected at higher MOI. The effect of virus packingnside the cells on the kd2 is well described by thempirical function defined by Eq. (7), as demonstrated
n Fig. 3.ig. 3. Infected cells population dynamics. The lines and symbolsenote model simulations and measurements, respectively: experi-ent S2 (full line (—) and open triangles (�)), experiment S6 (dashed
ine (- - -) and open squares (�)), experiment S7 (dotted line (· · · ·)nd open circles (©)), experiment Co2 + 6 + 7 (dash and dot line (--) and open diamonds (♦)) and experiment M2/6/7 (dash-dot-dotine (··-··-··) and closed triangles (�)).
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v(pbiawvg2artdeTrtwodMarv
nology 128 (2007) 875–894 887
.3. vDNA replication
Fig. 4A–C shows vDNA measurements and modelredictions for the single and co-infection experimentsS2, S6, S7, Co2 + 6 + 7 and M2/6/7). The vDNAeplication patterns are well described by first ordereplication kinetics, which is consistent with template-imited replication. The apparent vDNA replicationate is 0.19 ± 0.01 h−1 and appears to be independentf the construct used. Indeed, in the monocistronic andultigene vectors used in this work, the size of genes
oding for the three VPs of interest represents lesshan 5% of the baculovirus genome size (Summersnd Smith, 1978). Therefore, the genome size factors expected to have a minor effect on the replicationinetics. At least nine genes are known to be involvedn vDNA replication (Lu and Miller, 1995). The expres-ion of these genes, including the dnapol and dnahel,eems not to be a differentiation factor between theonocistronic and multigene vectors. The number of
rigins of vDNA replication is also expected to be theame in these vectors.
The concentration of intracellular vDNA con-erges asymptotically to a maximum intracellular loadFig. 4A–C). Rosinski et al. (2002) suggested that therogressive decrease in vDNA replication rates coulde caused by a virus shift from DNA polymerase tots own dedicated viral RNA polymerase, which onlycts on its unique promoter motifs. This hypothesisas supported by the observation that the maximumDNA content coincided with the onset of very lateene expression, at approximately 20 hpi for a MOI of0 pfu/cell. In our system, the maximum vDNA levelsre reached much later, between 48 and 60 hpi, thusefuting the aforementioned hypothesis. This suggestshat the halt of vDNA replication is practically coinci-ent in time, with few exceptions, with the halt of VPxpression and also with the increase in cell death rate.herefore, this behavior is here interpreted as being
elated with the intrinsic metabolic decay due to infec-ion. In our model, the effect of the metabolic decayas expressed by Eqs. (17) and (19) as a functionf intracellular vDNA. Fig. 4D illustrates this depen-ency. The MOI plays here an important role. High
OIs result in faster vDNA replication and eventu-lly in the reduction of the time interval for vDNAeplication and VP expression. This effect has been pre-iously observed in several studies (Hu and Bentley,
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88 A. Roldao et al. / Journal of
000, 2001; Licari and Bailey, 1992; Rosinski et al.,002).
.4. Virus budding
In theory, the apparently constant vDNA concentra-ion for t > δD (hpi) could result from vDNA releasento the extracellular medium through virus buddinghat would compensate for vDNA replication. Buddedirus release is known to commence around 17–20 hpior the BEVS/IC Sf-9 system (Power and Nielsen,996; Wong, 1997). However, according to Rosinski
t al. (2002), virus budding is considerably lower thanhe intracellular replication. This would suggest thathe virus makes much more vDNA than it is ableo encapsidate and release. To confirm this hypoth-p(ii
ig. 4. Dynamics of intracellular vDNA2 (A), vDNA6 (B) and vDNA7 (C).ata, respectively: experiments S2, S6 and S7 (full line (—) and closed triamonds (�)) and experiment M2/6/7 (dotted line (· · ·) and closed squareschedule for viral replication (metabolic decay) was also assessed based onnd closed diamonds (�) experimental data.
nology 128 (2007) 875–894
sis, the extracellular vDNA (originated from bothirus budding and cell lysis) was quantified for theo-infection experiment Co2 + 6 + 7. The maximumxtracellular vDNA obtained was 2.54 × 109 copies/mlDNA2 + DNA6 + DNA7) at time t = 80.5 hpi, whichepresents 11% of the intracellular vDNA, thus con-rming that virus budding is much lower than vDNAeplication.
.5. mRNA synthesis
Fig. 5A–C shows mRNA measurements and model
redictions for the single and co-infection experimentsS2, S6, S7, Co2 + 6 + 7 and M2/6/7). Typically,ntracellular mRNA increases fast at the onset of thenfection cycle up to a maximum level after which itThe lines and symbols denote model simulations and experimentaliangles (�)), experiment Co2 + 6 + 7 (dashed line (- - -) and closed(�)). The evaluation of total intracellular vDNA effect on the time-Eqs. (17)–(19) (D). The full line (—) represents model simulation
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tarts decaying. The increase in mRNA concentrationollows closely the increase in vDNA. The maximumarks the time instant when the mRNA transcription
ate becomes lower than the corresponding degrada-ion rate. The halt in mRNA synthesis is practicallyoincident in time with the halt in vDNA replication.n general, the higher the intracellular vDNA levels theigher are the corresponding mRNA levels. The onlyxception to this scenario was verified for the geneoding for VP7, whose transcription is controlled byifferent promoters: polh in the monocistronic vectorsexperiments 1, 2, 3, 5) and p10 in the multigeneectors (experiment 4).
The synthesis of mRNA is well described by first
rder transcription kinetics. The characteristic tran-criptional rates for the genes coding for VP2, VP6nd VP7 under the control of the polh promoter are.12 ± 0.01 h−1, 0.16 ± 0.02 h−1 and 0.11 ± 0.01 h−1bRrt
ig. 5. Intracellular dynamics of RNA2 (A), RNA6 (B) and RNA7 (C). Theespectively: experiments S2, S6 and S7 (full line (—) and closed triangles (�)) and experiment M2/6/7 (dotted line (· · ·) and closed squares (�)).
nology 128 (2007) 875–894 889
or mRNA2, mRNA6 and mRNA7, respectively. Thesealues fail to conform with the rule of the size of theenes (the smaller the gene the faster it is transcribed)ainly due to the low transcriptional rate observed for
he vDNA7. The sizes of the genes coding for VP2, VP6nd VP7 are 2690, 1356 and 1062 bp, respectively. Pre-uming a characteristic RNA polymerase elongationate profile, the resulting transcriptional rates shoulde such that kSRNA,7 > kSRNA,6 > kSRNA,2. Since theforementioned rates were consistent for all mono-istronic and co-infection experiments (S2, S6, S7 ando2 + 6 + 7), a possible justification for this ranking is
he use of different restriction sites for cloning. Theranscription of polh and p10 genes, starts within a
aculovirus late promoter motif TAAG (Blissard andohrmann, 1990). Modifications in the 5′ untranslatedegions (UTRs), between the TAAG motif and the ATGranslational start codon, and in the 3′ UTR of the bac-
lines and symbols denote model simulations and experimental data,�)), experiment Co2 + 6 + 7 (dashed line (- - -) and closed diamonds
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lovirus genome are known to have some influence inhe translation and transcription levels (Luckow andummers, 1988; Ooi et al., 1989; Possee and Howard,987; Qin et al., 1989; van Oers et al., 1999; Weyer andossee, 1988).
It should be noticed that the previous transcriptionates were the same for all the experiments except forhe transcription of the gene coding for VP7 (under theontrol of p10 promoter) in the M2/6/7 case (exper-ment 4), which was 0.46 ± 0.01 h−1, respectively. Aeculiar result is the exceptionally high kSRNA,7 inhe M2/6/7 experiment, which resulted in a muchigher mRNA concentration in M2/6/7 than in S7 and
7n Co2 + 6 + 7, even when the vDNA7 concentrationsere higher in the latter experiments (Fig. 5C). The jus-ification for this variation cannot be directly attributedo the strength of the promoter since the levels of polh
t((
ig. 6. Total (intracellular plus extracellular) protein dynamics of VP2 (A), Vnd experimental data, respectively: experiments S2, S6 and S7 (full line (- - -) and closed diamonds (�)) and experiment M2/6/7 (dotted line (· · ·) an
nology 128 (2007) 875–894
nd p10 driven expression are expected to be similarvan Oers et al., 1992). The interaction between polhnd p10 promoters are however known to influence theevel of gene expression (Chaabihi et al., 1993).
Another relevant point is that, for the M2/6/7 exper-ment, where the transcription of the viral genes occursrom the same DNA template, the ranking of the tran-criptional rates according to gene size is obeyed. Themaller gene for VP7 resulted in considerably higherranscription rates.
.6. Protein synthesis
Fig. 6A–C shows the dynamics of measuredotal (intracellular plus extracellular) protein, VPj
�g ml−1), and model predictions as given by Eq.14). Fig. 7A–C shows model predictions of intra-
P6 (B) and VP7 (C). The lines and symbols denote model simulations—) and closed triangles (�)), experiment Co2 + 6 + 7 (dashed lined closed squares (�)).
A. Roldao et al. / Journal of Biotechnology 128 (2007) 875–894 891
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ig. 7. Intracellular VP2 (A), VP6 (B) and VP7 (C) concentrations. T7 strategies, the dashed line (- - -) strategy Co2 + 6 + 7 and the dotte
ellular protein only (Eq. (15)). The dynamics ofntracellular protein and vDNA show similar patterns.ntracellular protein concentration VPint
j convergessymptotically to a maximum level that is highlyorrelated with the corresponding maximum vDNAevel. Indeed, higher intracellular vDNA concentra-ions induce higher mRNA transcription levels andltimately higher intracellular VP concentrations. Thisas verified whenever the gene expression was under
he control of the polh promoter. The unique exceptionas the gene coding for VP7 in single infection (under10 promoter), where significantly higher mRNA7oncentrations were obtained from significantly lowerNA templates (Figs. 4C and 5C).
7Protein synthesis seems to be reasonably wellescribed by Michaelis–Menten kinetics on mRNA.he maximum protein synthesis rate varies consider-bly (see Table 3). The rate of protein synthesis may
Tpse
s represent model simulations: the full line (—) denotes S2, S6 and· · ·) strategy M2/6/7.
ary due to codon usage and/or size of protein and alsohe time for maturation. The VP7 formation takes moreime, approximately 0.5 h (Lodish et al., 1986) since its a glycosylated protein.
.7. Analysis of protein dynamics
The following analysis will be centered onhe dynamics of VPs stoichiometric ratios and onheir comparison with the composition of correctlyssembled particles in the Co2 + 6 + 7 and M2/6/7xperiments. Fig. 8 shows the dynamics of the stoi-hiometric ratio Y6/2 and Y7/2 (VP6:VP2 and VP7:VP2,espectively) for the M2/6/7 and Co2 + 6 + 7 strategies.
he mass stoichiometric ratio of correctly assembledarticles VP2:VP6:VP7, is 1:2.8:2.4 (w/w). Fig. 8hows that in the M2/6/7 experiment there is anxcess of VP7, especially during the first 48 hpi. Dur-
892 A. Roldao et al. / Journal of Biotech
Fig. 8. Dynamics of VPs stoichiometric ratios. The doted linesrepresent the theoretical mass stoichiometric ratios Y6/2 = 2.8 andY7/2 = 2.4 (VP6:VP2 and VP7:VP2, respectively). Model simulationsare denoted by symbols: closed squares (�) indicate Y6/2 whereasopen squares (�) represents Y7/2 in Co2 + 6 + 7 strategy; closed trian-gi
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les (�) and open triangles (�) represents Y6/2 and Y7/2, respectivelyn M2/6/7 strategy.
ng the first 48 hpi approximately 80% (w/w) of thenal VP7 was already synthesized (see Fig. 6C). It is
ikely that the VP7 outer layer can only be formedfter the VP2 and VP6 layers are correctly assem-led. Therefore, such high VP7 levels at the beginningill probably favor the formation of wrong particles.urthermore, the experimental Y6/2 is systematicallybove the theoretical Y6/2 = 2.8 (w/w) value. Thus, it isikely that VP2 is the limiting protein for the VLPsssembly. Analyzing the case of Co2 + 6 + 7 strat-gy, the experimental values of Y7/2 are lower thanhe theoretical values. Thus, this strategy is prone toroduce particles composed of VP2 and VP6 layerssince the VP6 is not limiting the assembly process),ut with the outer VP7 shell only partially formedr even completely absent. The volumetric produc-ivity obtained for M2/6/7 and Co2 + 6 + 7 strategiesfter purification, based on total protein quantificationSection 3) reflects the observed strong underexpres-ion of VP7 in the Co2 + 6 + 7, leading to a lowerLP titer: 430 �g/ml in Co2 + 6 + 7 and 662 �g/ml in2/6/7. Nevertheless, the Co2 + 6 + 7 strategy can be
urther optimised in future studies through the MOInd TOI of the individual genes. The mathematicalodel proposed here incorporates both the effects of
OI and also the time constants for the infection andrafficking, thus having potential to support the optimi-ation of both the MOI and TOI in future co-infectionxperiments.
7
nology 128 (2007) 875–894
. Conclusions
In this work we propose a mathematical model ofhe baculovirus expression vector system/insect cellsor the production of Rota VLPs. The model addresseshe most critical steps of virus internalization and traf-cking, and gene expression mechanisms. The mainbjective of this modeling study was to understandnd to quantify the effect on VP synthesis of differentnfection strategies, and evaluate the effect of differ-nt promoters. Such modeling tool may allow to betteredesign baculovirus vectors. It is also a starting pointo optimise infection parameters such as TOI and/or
OI in co-infection experiments. The proposed modelepresents a good compromise between complexity andccuracy. A relatively small number of kinetic param-ters are involved, which were estimated with hightatistical confidence in most cases. From the analy-is of the modeling results, the following more specificonclusions may be outlined:
. A characteristic apparent vDNA replication rate of0.19 ± 0.01 h−1 was obtained for all monocistronicand multigene constructs used in this work.
. Virus budding is much lower than vDNA replica-tion.
. The vDNA translation and transcription is welldescribed by first order and Michaelis–Mentenkinetics, respectively. Reproducible kinetic param-eters for the polh and p10 promoters were obtained.
. The p10 promoter in the M2/6/7 construct leads con-sistently to a much higher VP7 expression than whenpolh controls VP7 expression (in the Co2 + 6 + 7 andS7 experiments).
. The time windows for effective vDNA, mRNA andVP synthesis are highly correlated. High MOIstend to increase vDNA replication, but, at the sametime, the metabolic burden is more severe, therebydecreasing the time window for effective proteinexpression.
. In terms of VLPs synthesis, the M2/6/7 experimentleads to both VP6 and VP7 overexpression. How-ever, the high VP7 expression at the beginning of theinfection cycle is undesirable. It is the expression of
VP2 that limits the Rota VLPs assembly.. In co-infection Co2 + 6 + 7, VP7 is largely under-expressed. Thus, probably particles of VP2 andVP6 with incomplete VP7 shell are formed. Opti-
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mized infection strategies are required to increasethe expression of VP7.
cknowledgements
The authors wish to thank Catarina Estevao andaria Candida Mellado for thoughtful discussions.
he authors are also grateful to Dr Pedro Cruz forxcellent advice, to Dr. John Aunins and Dr. Luisaranga from Merck & Co., West Point, USA,
nd Prof. Hansjorg Hauser, GBF, Germany for valu-ble comments on the manuscript. This work wasupported by European Commission project (QLRT-001-01249) and the Portuguese Fundacao para aiencia e Tecnologia (POCTI/BIO/55975/2004 andFRH/BD/21910/2005).
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