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Text S1: Supplementary Materials and Methods
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Construction of an infectious clone of FMDV C-S8c1 containing defective genomes and
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associated mutations from passage 260.
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Plasmids pMT∆417 and pMT∆999 were constructed by substituting the L- and the
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structural protein-coding regions (s region), spanning nucleotides 436 to 4201 of pMT28,
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with the corresponding region of the defective genomes ∆417 and ∆999, respectively, as
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previously described [1]. Plasmids pMT260∆417ns and pMT260∆999ns were constructed by
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replacing the nonstructural protein-coding region (ns region; see [2,3]), spanning nucleotides
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4201 to 7427 of pMT∆417 and pMT∆999, respectively, with the corresponding region from
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C-S8p260p3d. To this end, the ns region from C-S8p260p3d was amplified by using
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separately primer pairs: 5’-TTGGTGTCTGCTTTTGAGGAAC-3’ (sense; initial nucleotide, 3988
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according to [4]) / 5’-CATGACCATCTTTTGCAGGTCAG-3’ (antisense; initial nucleotide, 6009),
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5’-GCGGGCTCAGAGTTCACGTCATC-3’
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TGTGGAAGTGTCTTTTGAGGAAAG-3’ (antisense; initial nucleotide, 7783). Subsequently, the
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two resulting amplicons were shuffled using external primers. PCR amplifications were
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performed with Pfu polymerase (Stratagene), as specified by the manufacturer. The resulting
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DNA fragment was digested with BglII (position 4201) and Bam HI (position 7427), and
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ligated to pMT∆999 and pMT∆417, that were previously digested with the same restriction
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enzymes. Procedures for the purification of plasmids, transformation of competent
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Escherichia coli DH5α cells, and isolation of bacterial colonies, have been previously
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described [5,6].
(sense;
initial
nucleotide,
5704)
/
5’-
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The pMT260p3d infectious plasmid was constructed by replacing the genomic region
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spanning residues 638 to 2046 from pMT260Δ417ns, which includes the deletion in the L-
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coding region, by the same region from pMT260Δ999ns (with no deletion). FMDV genomic
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residues are numbered according to [4]. For this purpose, pMT260Δ417ns was digested with
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XbaI, that releases the indicated fragment, and the linear plasmid was separated by agarose
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gel electrophoresis and purified with the Wizard SV Gel and PCR Clean-Up System (Promega).
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Then, the cDNA that included genomic region 640-2068 was amplified with a forward primer
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spanning residues 628 to 651 of the FMDV genome, and a reverse primer spanning residues
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2065 to 2042 of the FMDV genome. Both reverse and forward primers are homologous to
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each terminus of the linear plasmid, permitting the cloning of the resulting PCR product by
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recombination. To this aim, we used the In-Fusion Dry and Down Mix kit (Clontech), as
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indicated by the manufacturer. The correctness of the constructions was verified by
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nucleotide sequencing. The nucleotide sequence of the primers employed during the
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cloning process is available upon request.
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Transcription of viral RNA and electroporation of BHK-21 cells
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Plasmid DNA was linearized by cleavage with Nde I, and purified using the Wizard PCR
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Preps DNA purification resin (Promega). Infectious FMDV RNA was transcribed from the
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linearized plasmids using the Riboprobe in vitro transcription system (Promega) as specified
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in [7]. The RNA concentration was estimated by agarose gel electrophoresis, with known
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amounts of rRNA as marker. Cells were electroporated with 12.5 μg of the corresponding
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viral RNA as previously described [7].
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Determination of viral titer of C-S8p260 and C-S8p260p3d
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The production of lytic plaques in a population of complementing viruses follows a
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two-hit kinetics. Following the model by Manrubia et al, 2006 [8], we call A and B the two
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defective, complementary populations, and define
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n0  (actual) number of infectious particles
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N  total number of cells
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PFU  (observed) number of lytic plaques in a plate
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n0
 number of particles of type A, in the approximation that there is an equal number of
2
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particles of types A and B
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The probability that k particles of either type infect a cell is given by a Poisson distribution of
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average λ=n0/(2N),
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P(k)=e- λ λ/k!
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The probability that a cell is infected at least by one particle of type A is (1- e- λ), and equals
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the probability that a cell is infected at least by one particle of type B. Thus, the number of
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cells simultaneously infected by both types, that is, the number of PFUs, is
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PFU=N (1- e- λ) (1- e- λ)
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To first order in λ,
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PFU=N λ2
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Substituting the value of λ, we can therefore derive the actual number of infectious particles
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from the observed PFU,
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n0  4 N  PFU
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Once the number of viral particles is known, the viral titre of the ST and complementing
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(A+B) population is given by,
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titre (in fectious particles / ml) 
n0  inoculum volume
dilution factor
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Estimate of RNA packaging density
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The parameter Vm describes the volume occupied per unit mass (dalton) of a biologic
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macromolecule in a molecular crystal [9], or in a container such as a virus capsid [10]. The
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approximate molecular mass of the full-length RNA of FMDV C-S8c1 (8415 nucleotides,
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including a 300-nucleotide-long polyC) is Mr=2.7x106 Da; the interior ratio r of the FMDV C-
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S8c1 capsid, approximated to a spherical shape, is about 108 Å. Thus, the internal volume
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Vi=(4/3)πr3= 5.27x106 Å3. Hence Vm= Vi/Mr=1.95 Å3/Da.
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The partial specific volume of dry RNA is 0.55 cm3/g, or 0.91 Å 3/Da [10]. Thus, the
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volume occupied by the full-length RNA of FMDV if no hydration waters were present would
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be VRNA=2.7x106x0.91 Å3/Da=2.46x106 Å3, and the fraction of Vi occupied by a fully
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dehydrated RNA molecule would be VRNA/ Vi=0.47, or 47%.
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Additional information on capsid stability and RNA packaging density
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The molecular basis for the higher thermal stability and fitness of the infectious C-S8p260
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population relative to the ST virus is unclear. However, here a working model is proposed
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based on the length of the genomic RNA molecule packed inside the FMDV virion. Thermal
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inactivation of FMDV is not due to dissociation of the capsid into pentameric subunits, as the
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latter process occurs much more slowly under the conditions used in the present study [11].
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However, several amino acid substitutions in the capsid alter the inactivation rate [11,12],
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which indicates that the inactivation process involves the viral capsid. In other viral models it
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has been established that by providing enough energy, heat may facilitate in vitro the same
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conformational rearrangements that are triggered by other agents in vivo (i.e. a
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conformational change of the poliovirus capsid upon receptor binding that can be triggered
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also by heat in vitro) [13,14,15]. Thus, heat could provide the extra energy needed to
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facilitate a conformational rearrangement of the FMDV capsid that, outside the cell, would
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lead to loss of infectivity. The observed effects of capsid mutations on the inactivation rate
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would be due to their lowering or rising of the energetic barrier that leads to the altered
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conformational state [11]. We suggest that the amount of RNA inside the virion may also
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influence the kinetic barrier of the inactivation process, because of packaging
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considerations, as justified next.
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The Vm (volume occupied per unit mass) value [9] of full-length RNA inside the FMDV
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virion is about 1.95 Å3/Da . This corresponds to a very high packing density, slightly higher
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than that of RNA in a molecular crystal (about 2.1 Å3/Da), and substantially higher than those
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reported for other icosahedral RNA viruses like cowpea chlorotic mosaic virus and satellite
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tobacco necrosis virus [10]. Because the partial specific volume of dried RNA corresponds to
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about 0.91 Å3/Da, RNA molecular crystals will be about 43% RNA and 57% hydration water in
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volume, while packaged material inside the FMDV virion will be about 47% RNA and,
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provided no other molecules are present, 53% hydration water. Thus, packaging a full-length
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RNA genome within the confined limits of the FMDV capsid may involve some energetically
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unfavourable dehydration of the RNA. Packaging a longer RNA would involve a more severe
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dehydration because of the limited volume inside the capsid. In contrast, packaging 5%-12%
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shorter RNAs, like those in the C-S8p260 virions, would lead to Vm values of about 2.05
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Å3/Da-2.20 Å3/Da, and thus may involve no dehydration at all. Based on these simplified
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estimates (and ignoring other energetic effects on RNA packaging, that are more difficult to
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predict), one could surmise that the C-S8p260 virions would be at an energetically lower
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state than the ST virion, and this in turn would be at a lower energetic state than virions
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harboring longer RNAs.
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If the above scenario is correct, the extra energy needed to trigger the proposed
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conformational rearrangement leading to FMDV inactivation could be higher for the C-
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S8p260 virions than for the ST virions, because the former could be at an energetically lower
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state. Thus, the C-S8p260 virions would be more resistant to thermal inactivation.
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Conversely, the extra energy needed to trigger that rearrangement could be lower for
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virions that package longer RNAs, because they could be at an energetically higher state.
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Estimation of the experimental value of the decay factor dS, the key parameter in the
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computational model
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According to Figure 5, the infective populations of the standard and of the segmented forms
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decay in time at different rates while not actively replicating. Let us call NS(t) the population
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of the standard type at time t, and NA(t) the population of one of the defective,
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complementary forms. It has been shown that
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N S (t )  N S (0)e 1.19t
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N A (t )  N A (0)e 0.91t
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where NS(0) and NA(0) are the initial populations of S and A types, respectively, and t is time
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in hours. The amount of S relative to A is a time-dependent quantity d (t ) defined as
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d (t ) 
N S (t ) / N S (0)
 e 0.28t
N A (t ) / N A (0)
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This expression holds in the extracellular medium and for the inactivation dynamics before
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replication starts, i.e. during the first hour of inoculation of the viruses to the cells.
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The value of the decay factor dS is obtained by substituting in the expression above the
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experimental value of t, that is, the time elapsed between the moment when viruses are
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released to the extracellular medium and the next initiation of replication. The infection of
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the viruses takes about 4h. Based in Figure 3D, we can estimate that cells start to seed virus
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to the extracellular medium at minute 110. Thus, the virus remains in the extracellular
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medium for 240min-110min=130min. Figure 3B shows that in about 30min the
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internalization of viruses reaches a maximum. The virus is therefore exposed to the
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extracellular medium for about 160min (=2.7h). This time yields the decay value
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d S  e 0.282.7  0.47.
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Note that, during the replicative period, both populations multiply the particle number in
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the same amount r. Hence, the amount of S relative to A remains unchanged during
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replication. As a consequence, the global dynamics turns out to be independent of r and is
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solely controlled by the value of the decay factor d s .
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