Genome Assembly and Particle Maturation of the Birnavirus Infectious Pancreatic Necrosis Virus

In this study, we have analyzed the morphogenesis of the birnavirusinfectious pancreatic necrosis virus throughout the infectivecycle in CHSE-214 cells by using a native agarose electrophoresissystem. Two types of viral particles (designated A and B) wereidentified, isolated, and characterized both molecularly andbiologically. Together, our results are consistent with a modelof morphogenesis in which the genomic double-stranded RNA isimmediately assembled, after synthesis, into a large (66-nmdiameter) and uninfectious particle A, where the capsid is composedof both mature and immature viral polypeptides. Upon maturation,particles A yield particles B through the proteolytic cleavageof most of the remaining viral precursors within the capsid,the compaction of the particle (60-nm diameter), and the acquisitionof infectivity. These studies will provide the foundation forfurther analyses of birnavirus particle assembly and RNA replication.

INTRODUCTION

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Introduction

The Birnaviridae family of viruses includes members with a bi-segmented,double-stranded RNA (dsRNA) genome encapsidated within unenveloped,single-shelled icosahedral particles of 60 to 70 nm in diameter.Birnaviruses do not infect mammals, and the family containsthree genera: (i) genus Aquabirnavirus, including infectiouspancreatic necrosis virus (IPNV), which spreads in salmonidsand trouts; (ii) genus Avibirnavirus, including infectious bursaldisease virus (IBDV), which infects young chickens; and (iii)genus Entomobirnavirus, including Drosophila X virus, whichinfects the fruit fly Drosophila melanogaster (14, 19). The5' ends of both segments of the dsRNA genome of the birnavirusesare bound to a genome-linked protein (VPg) (6, 51, 57). Amongthe multisegmented dsRNA viruses, these unique properties, amongothers, made it necessary to classify the birnaviruses in theirown family (5), apart from their reovirus relatives (54).

The IPNV particle presents a buoyant density of 1.33 g/cm³ inCsCl (17) and contains both segments of the dsRNA genome (14S)(named segments A and B) (15, 41). Genome segment A ( $~$ 3,100 bp)involves two partially overlapping open reading frames. Theshort one encodes a 17-kDa arginine-rich nonstructural protein(42), which, for IBDV, is not required for viral replicationbut plays an important role in pathogenesis (67). The long oneencodes a precursor of a 106-kDa protein (polyprotein [PP])(NH2-pVP2-VP4-VP3-COOH), which is cotranslationally cleavedby the viral VP4 protease (29 kDa), generating a precursor ofthe major capsid protein pVP2 (62 kDa) and VP3 (31 kDa) (20).pVP2 is further processed to generate VP2 (54 kDa) (17). VP3forms the trimeric subunits that wrap the inner face of thecapsid (4, 8). The external surface of IBDV particles is madeof trimeric subunits containing variable pVP2/VP2 ratios (9,40). It has been suggested that pVP2/VP2 conversion is associatedwith capsid maturation (9, 17, 48). The uncleaved PP is alsodetected, although in trace amounts, in highly purified IPNVpreparations (43). Genome segment B ( $~$ 2,900 bp) encodes a minorinternal polypeptide, VP1 (94 kDa), the putative viral RNA-dependentRNA polymerase (22). VP1 is found within the virion in two forms:as a free polypeptide, RNA-dependent RNA polymerase-associatedactivity, and as the genome-linked protein, VPg. As VPg, VP1is linked to the 5' end of each genome segment by a Ser-5'-GMPphosphodiester bond (6). This bond can be generated in vitroduring the guanylylation of VP1, yielding VP1pG (13). Interestingly,by electron microscopy, it has been observed that, in vitro,the binding of VPg to the viral RNA confers to the genome amarked propensity to aggregation, where the interactions werefound to take place at the end of the segments (6, 51).

The IPNV replication cycle has been mainly studied on continuouscell lines from teleost fishes in culture (66). In Chinook salmonembryo cells (CHSE-214 cells) (37), a single round of virusreplication takes approximately 24 h at 15°C. As a result,a characteristic cytopathic effect (CPE) is observed (44), inwhich apoptosis precedes the pathological changes of necrosis(30-32). Following penetration, viral particles are localizedwithin vesicular compartments in the cytoplasm (11). Early afterinfection, a putative transcription intermediate (14-16S) isdetected between 2 to 4 h postinfection (hpi) (62). After 4to 6 hpi, viral mRNA species can be identified (62) as wellas viral-specific polypeptides, with viral proteins synthesizedin the same relative proportions throughout the infective cycle(16). The synthesis of viral genomic RNA in infected cells reachesa maximum level between 8 to 10 hpi, correlating with the detectionof genomic dsRNA (62). After 14 to 16 hpi, viral RNA synthesisis diminished. Although the temporal virus-specific synthesisof macromolecules has been characterized, the structures associatedwith birnavirus replication and genome assembly in infectedcells are largely unknown.

In this study, we analyzed the morphogenesis of IPNV throughoutthe infective cycle in CHSE-214 cells by native agarose electrophoresis.Two types of particles (A and B) were identified, isolated,and characterized. Our results are summarized in a model ofIPNV morphogenesis during the replication cycle. These studieswill provide the foundation for the analysis of birnavirus particleassembly and RNA replication.

MATERIALS AND METHODS

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Materials and Methods

Cells and viruses. Virus stocks (IPNV, strain VR-299) were propagated by inoculatingChinook salmon embryo cells (CHSE-214 cells) (37) at a multiplicityof infection (MOI) of 0.1 to 1 PFU/cell in minimal essentialmedium (MEM) supplemented with 2% fetal bovine serum (FBS) andantibiotics. Infected cultures were incubated at 15°C untilthe CPE was evident, and the clarified supernatants were aliquotedand stored at –20°C. Virus fractions were titratedin a plaque formation assay as previously described (34).

Extraction, concentration, and partial purification of IPNV particles. Monolayers of CHSE-214 cells were infected with IPNV at a MOIof 1 to 5 PFU/cell. After 1 h of adsorption at 15°C, cellswere washed three times with phosphate-buffered saline, andthe inoculum was replaced with methionine- or phosphate-deficientMEM supplemented with 2% FBS and antibiotics, containing 50µCi of either [³⁵S]methionine or [³²P]HPO₄^2–/ml,respectively. The cells were then incubated at 15°C (thiswas defined as time zero of infection), and at 2 hpi, 0.5 µgof actinomycin D/ml was added.

Cultures showing complete CPE were subjected to three freeze-thawingcycles, and cell debris was discarded by centrifugation at 5,000x g for 10 min at 4°C. For cultures harvested at differenttime points during the infective cycle, cells were washed threetimes with phosphate-buffered saline and incubated for 10 minat 4°C with lysis buffer (3 mM Tris-HCl [pH 8.0], 0.5 mMMgCl₂, 3 mM NaCl), scraped with a rubber policeman, and homogenizedwith a Dounce at 4°C for best results. The homogenate wascentrifuged for 10 min at 5,000 x g at 4°C, and the pelletwas discarded. Nonidet P-40 (1%) was added, since viral progenyremained trapped within cellular debris (44), and the supernatantwas incubated for 60 min at 4°C with mild agitation. Viralparticles were concentrated by ultracentrifugation at 130,000x g for 2 h at 4°C, resuspended in ice-cold 50 mM Tris-HCl(pH 8.0), and then partially purified by ultracentrifugationthrough a 25% sucrose cushion at 145,000 x g for 4 h at 4°C.The pellet was resuspended in a buffer containing 10 mM HEPES-KOH(pH 8.0), 2 mM dithiothreitol, and 10% glycerol and stored at–20°C.

The progression of the IPNV infective cycle was monitored byviral dsRNA synthesis and the simultaneous inoculation of CHSE-214cells (4 to 5 x 10⁵ cells) with 50 µCi of [³²P]HPO₄^2–/ml.At several time points, the supernatant was discarded, the monolayerwas lysed, and the extract was subjected to digestion in bufferK (0.2 M Tris-HCl [pH 8.0], 1% sodium dodecyl sulfate [SDS],10 mM EDTA, 1.3% [vol/vol] ß-mercaptoethanol, 0.7M NaCl, 160 µg of proteinase K/ml) at 40°C for 12h. Total RNA was extracted with phenol-chloroform and ethanolprecipitated. Viral dsRNA was resolved by 7% polyacrylamidegel electrophoresis (PAGE) as previously described (27).

In pulse-chase experiments, after virus adsorption, the inoculumwas replaced with MEM containing 2% FBS and antibiotics. Duringthe pulse time (4 to 8 hpi), methionine- or phosphate-deficientMEM, containing 50 µCi of either [³⁵S]Met or [³²P]HPO₄^2–/ml,respectively, was added. The monolayers were then washed threetimes with MEM and placed in isotope-free MEM supplemented with2% FBS and antibiotics and incubated at 15°C until the indicatedharvest time.

Isopycnic CsCl gradient. Partially purified viral particles ([³⁵S]Met- or [³²P]HPO₄^2–-labeled)were isopycnically banded in a 38% (wt/vol) CsCl solution (in50 mM Tris-HCl [pH 8.0]) by ultracentrifugation at 195,000 xg for 18 h in an SW 55 Ti rotor. Two hundred-microliter fractionswere collected from the top to the bottom of the tube, and boththe radioactivity and the refractive index of each fractionwere determined. The buoyant densities were calculated fromthe refractive index measurements. Radioactive fractions werediluted, and the particles were concentrated by ultracentrifugationat 145,000 x g for 4 h at 4°C.

TGA electrophoresis. Aliquots of viral particles were mixed with Tris-glycine-agarose(TGA) loading buffer (3 mM Tris-HCl [pH 8.0], 6.6 mM NH₄Cl,3 mM magnesium acetate, 14 mM potassium acetate, 1 mM dithiothreitol,10% glycerol, 0.005% bromophenol blue) and subjected to electrophoresisin either 0.8 or 1% agarose gels containing 50 mM Tris and 200mM glycine at 8 V/cm for 3.5 h at 4°C (26). Gels were driedand exposed to autoradiography. Alternatively, after electrophoresis,viral particles were recovered from fresh gel slices by electroelutionat 100 V for 2 to 3 h at 4°C and then dialyzed for 1 h at4°C against TAE buffer (40 mM Tris-acetic acid, 2 mM EDTA).For the in vitro maturation assays, aliquots of dialyzed particlesA and B were subjected either immediately (t = 0) or after anincubation of 24 h at 4°C (t = 24) to a second round ofTGA electrophoresis. To analyze the polypeptide compositionof gel-recovered [³⁵S]Met-labeled particles, the proteins wereprecipitated with 15% trichloroacetic acid and resolved by SDS-12%PAGE. To analyze the [³²P]HPO₄^2–-labeled dsRNA content,electroeluates were incubated with buffer K and total RNA wasextracted with phenol-chloroform and ethanol precipitated. ViralRNA was resolved on a 7% polyacrylamide gel and autoradiographed(27).

Electron microscopy of IPNV particles recovered from TGA gels. Aliquots of concentrated electroeluates from TGA gels (bandsA and B) were fixed for 10 min on copper grids covered withFormvar membranes, previously shaded with carbon. The sampleswere stained for 10 min with 3% ammonium molybdate, pH 7.0.The particles were observed with a Zeiss electron microscope(Facultad de Ciencias, Universidad de Chile), amplified 50,000to 80,000 times, and then photographed. The diameters of bothparticles were estimated by comparison to rotavirus particles(data not shown).

Antiprotease assay. CHSE-214 cells were infected with IPNV in the presence of 50-µCi/ml[³⁵S]Met. At 8 hpi, the supernatant was discarded and the monolayerswere lysed as described above. Extracts were further incubatedfor 4 or 8 h at 15°C with 10 mM concentrations of the followingantiprotease compounds: iodoacetamide (IAA), phenylmethylsulfonylfluoride (PMSF), and EDTA. Particles were then extracted andpartially purified as described above in the presence of thesame concentration of each of the antiprotease compounds. Particleswere then identified by TGA electrophoresis.

RESULTS

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Results

Isolation and identification of IPNV particles. A native electrophoresis system with polyacrylamide gels inTris-glycine has been previously utilized to characterize smallnuclear particles involved in the splicing of cellular mRNAprecursors (35). Later, agarose replaced polyacrylamide foranalyzing the assembly of subviral particles of rotaviruses(26). Figure 1A, lane 1, shows the migration of [³⁵S]Met-rotavirusparticles in TGA electrophoresis, utilized as a migration controlfor IPNV particles. As previously reported, two bands were resolvedin the gel: double-shelled virions and single-shelled rotavirussubparticles, with corresponding diameters of 80 and 70 nm,respectively (26).

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FIG. 1. Isolation and identification of IPNV particles in TGA gels. (A) Radiolabeled and partially purified IPNV particles from culture supernatants were resolved by 0.8% TGA electrophoresis and visualized by autoradiography. Lane 1, [³⁵S]Met-labeled rotavirus particles (strain SA-11) used as migration control. Arrows on the left indicate double-shelled (ds) virions and single-shelled (ss) rotavirus subparticles, corresponding to diameters of approximately 80 and 70 nm, respectively (26). IPNV particles labeled with either [³⁵S]Met (lane 2) or [³²P]HPO₄^2– (lane 3) are resolved as two bands designated by arrows (particles A and B) on the right. IPNV A and B particles resolved by TGA electrophoresis were recovered from agarose and subjected to electron microscopy analyses. (B) Electron micrograph of structures visualized in particles A. (C) Electron micrograph of structures visualized in particles B. Bar, 100 nm.

In our initial experiments, we wanted to identify the differentviral particles produced during IPNV infection. A single roundof IPNV infection in monolayers of CHSE-214 takes 24 h at 15°C(14, 44). To avoid the production of defective interfering particlesinduced at high MOI (52), infections with virus stocks wereperformed at low MOI (0.1 to 1 PFU/cell) in the presence ofa radioactive precursor ([³⁵S]Met or [³²P]HPO₄^2–), andmonolayers were incubated until a visible CPE was observed (3to 4 days), indicative of complete virus spread. Viral particleswere concentrated, partially purified from cellular supernatants,and resolved by TGA electrophoresis. Figure 1A, lanes 2 and3, shows the migration of IPNV particles labeled with [³⁵S]Metand [³²P]HPO₄^2–, respectively. Two discrete bands, designatedparticles A and B, were identified in the presence of eitherradioactive precursor, suggesting that both are viral ribonucleoproteincomplexes. Such particles could not be detected when supernatantsfrom mock-infected cells were labeled and analyzed under similarconditions (data not shown). The migration of particles A issimilar to that of the single-shelled rotavirus particles, indicatingthat their diameters are comparable. Consistent with its migration,particles B correspond to a diameter of about 60 nm. We cannotdiscard the possibility that different electrostatic contributionsaffect the migration of both particles during their separationin the agarose gel. However, these contributions are currentlyuncertain.

Since the particles A and B were resolved under nondenaturingconditions in TGA electrophoresis, concentrated eluates correspondingto bands A and B were processed for electron microscopy (EM)analysis. As a control, a partially purified IPNV preparation(input for TGA gel) was prepared in parallel for negative staining.Figure 1B shows that eluates from band A contain species withthe general morphology of a viral particle. However, they areapparently open and permeable to ammonium molybdate staining.The careful observation of different individual particles suggesteddistinct degrees of openness but similar icosahedral architecture(data not shown). The average EM-estimated diameter for theseparticles is 66 ± 2 nm.

The viral particles recovered from the band B (Fig. 1C) showeda regular, icosahedral shape and were not permeable to ammoniummolybdate staining, which suggests a completely compacted structure.Particles B have an EM-estimated diameter of 60 nm. A partiallypurified IPNV preparation apparently showed a mixture of bothkinds of particles, A and B, but it was difficult to unequivocallydistinguish between them (data not shown). Consistent with ourobservations, populations of different particles have been previouslyobserved for purified birnaviruses (10, 50, 59). These resultsconfirm the different sizes of particles A and B observed byTGA electrophoresis. The appearance of both particles suggeststhey are late morphogenesis intermediates, in which one probablycorresponds to the particle precursor and the other correspondsto the virion.

Identification of IPNV particles obtained during the infective cycle. To identify the particle(s) produced at the different stagesof the IPNV infective cycle, infections were carried out inthe presence of a radioactive precursor ([³⁵S]Met or [³²P]HPO₄^2–)and then arrested at 8, 12, or 18 hpi. Since a single roundof the viral cycle may have slightly different extents betweendifferent infections (i.e., 18 to 24 h) (14, 44), we correlatedthe progression of each infection by monitoring viral dsRNAsynthesis at identical time points. Results from TGA electrophoresisshowed no particles detectable at 8 hpi for infections performedin the presence of either radioactive precursor (Fig. 2A and B,lanes 8 hpi). In control infections (in the presence of [³²P]HPO₄^2–),no viral genomic dsRNA was detected from infected cells at 8hpi. Viral RNA was detected at 12 and 18 hpi, as indicated by7% PAGE (data not shown) (27). At 12 hpi, particles A and Bwere both identified, and at 18 hpi, they appeared to be accumulatedin infected cells, suggesting a continuous assembly (Fig. 2A and B,lanes 12 and 18 hpi). These results indicate that thefirst IPNV particle is assembled at time points between 8 and12 hpi during the infective cycle.

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FIG. 2. Identification of IPNV particles produced during the infective cycle. CHSE-214 cells were infected with IPNV in the presence of [³⁵S]Met (A) or [³²P]HPO₄^2– (B). At 8, 12, or 18 hpi, viral particles were partially purified from cell extracts and resolved by TGA electrophoresis. To determine the time point at which the first viral particle is formed in the presence of [³⁵S]Met (C) or [³²P]HPO₄^2– (E), the cells were lysed every hour between 7 and 12 hpi and the viral particles were resolved by TGA electrophoresis. (D and F) To monitor the progression of the viral cycle in the experiments described for panels C and E, respectively, cells were simultaneously infected with IPNV in the presence of [³²P]HPO₄^2– and the monolayer was lysed at identical time points. Viral [³²P]HPO₄^2–-labeled dsRNA was extracted and analyzed by 7% PAGE (27). Control lanes correspond to RNA extracted from [³²P]HPO₄^2–-labeled IPNV particles obtained at 48 hpi. Arrows indicate positions of the segments of viral genomic dsRNA.

To determine the first viral particle produced during the IPNVinfection, a narrow period of the infective cycle, 7 to 12 hpi,was monitored. Results showed that particles A become detectablefirst, at 8 hpi (Fig. 2C and E, lanes 8 hpi), whereas particlesB were only identified 2 to 3 h later, at 10 to 11 hpi, witheither radioactive precursor (Fig. 2C and E). In both time courseexperiments, the viral genomic dsRNA was first detected at 8hpi from infected cells (Fig. 2D and F, lanes 8 hpi). This timepoint for viral dsRNA synthesis correlates with the identificationof particles A by TGA electrophoresis. These results show thatduring the infective cycle, particles A assemble first, simultaneouswith the detection of viral dsRNA. Then, 2 to 3 h later, particlesB become identified, probably after the intracellular accumulationof particles A. This sequence of events suggests that particlesA are the morphogenesis precursors of particles B. Since bothparticles A and B are likely constituted of genome and viralproteins, their molecular compositions were analyzed in somedetail.

Buoyant density and molecular composition of IPNV particles. To evaluate the buoyant density of the different IPNV particles,partially purified viral preparations were layered onto CsClgradients and ultracentrifuged to equilibrium. Consistent withprevious results (17, 18), a single peak of radioactivity wasobtained at densities of 1.33 g/cm³, as shown in Fig. 3A. Fractionscontaining the peak of radioactivity (fractions 9, 10, and 11)were further analyzed for their particle composition by TGAelectrophoresis (Fig. 3B). These fractions were shown to be,in fact, a mixture of both particles A and B. Fraction 9 (1.32g/cm³) contained a greater proportion of particles B, whereasfraction 11 (1.335 g/cm³) contained more particles A than B.Consistent with a symmetric peak, fraction 10 (1.33 g/cm³),with most of the radioactivity, is composed of nearly equivalentamounts of both particles, indicating their comigration in thegradient and implying the similar buoyant density of particlesA and B. The overall higher density of particles A may be explainedby a CsCl uptake during the experimental procedure. This uptakewould be consistent with our EM observations where particlesA were permeable to ammonium molybdate staining (Fig. 1B).

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FIG. 3. Molecular characterization of IPNV A and B particles. (A) Partially purified [³⁵S]Met-labeled IPNV particles were banded in isopycnic CsCl gradients, and both the density and radioactivity of each 200-µl fraction were determined and plotted. (B) Fractions containing the peak of radioactivity (fractions 9 to 11) were subjected to TGA electrophoresis. The migration of A and B particles is indicated by arrows on the left. Upon resolution on TGA gels, particles A and B recovered from the gel were analyzed for [³²P]HPO₄^2–-labeled dsRNA content by 7% PAGE (C) and for [³⁵S]Met-labeled protein composition by SDS-PAGE (D). The inset shows the densitometric analysis of viral proteins. The migration of viral components is indicated on the left of the panels, and the positions of protein markers are indicated on the right.

We next went on analyze the macromolecular composition of bothviral particles. As earlier expected, both viral particles Aand B contained the two segments of viral genomic dsRNA (Fig.3C). The profile of viral proteins is shown in Fig. 3D. Bothpopulations of particles contain all of the viral structuralpolypeptides, PP, VP1, pVP2, VP2, VP3, VP4, and the truncatedform of VP4 (VP4t) (42). However, the composition of the viralprotein precursors varied between them. Quantification of theprotein bands from SDS-PAGE (Fig. 3D, inset) indicated thatparticles A are composed approximately of 1.5% PP and 15% pVP2,whereas particles B contain nondetectable amounts of PP andpVP2. Importantly, particles A were shown to be composed of30% VP2 and 27% VP3, with a pVP2/VP2 ratio of 0.44, whereasparticles B were shown to be composed of up to 35% VP2 and 30%VP3. These values are different than those previously publishedfor birnavirus particles (4, 17, 18), and the differences probablyreflect distinct procedures for separation, purification, andanalyses. Since both populations of particles A and B containgenomic RNA and the viral structural proteins, these resultsagree with the notion that particles A are the precursors ofparticles B. Particle maturation has been associated with postassemblyproteolytic cleavage of capsid precursor proteins in other viralfamilies (see Discussion), and it was further investigated forIPNV.

Pulse-chase, maturation experiments, and effect of antiproteases. To test whether particles A are the precursors of particlesB, a series of pulse-chase experiments were performed. The viralparticles were analyzed by TGA electrophoresis (Fig. 4A), andthe bands corresponding to particles A and B were quantifiedfor each time point, as shown in Fig. 4B. With these particularkinetics, particles A became identified at 8 hpi, which correlatedwith the first detection of viral dsRNA in a control infection(data not shown). Similar to our previous results, the inputof radioactivity derived from particles A remained accumulatedin infected cells until 12 hpi (Fig. 4A). At 14 hpi, the radioactivityassociated with band A diminished 12.5% with respect to earliertime points and a proportional amount of label was detected,as incorporated in the form of particles B, as shown in Fig.4B. Analysis of later time points indicated that further chaseof radioactivity from particles A into particles B is a slowprocess with a maximum at 48 hpi, where more than 30% of radioactivitywas chased in the form of particles B (Fig. 4A and B). Theseresults are consistent with the interpretation that particlesA are the morphogenesis precursors of particles B.

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FIG. 4. Pulse-chase experiments, maturation, and effect of antiproteases. (A) CHSE-214 cells were infected with IPNV as described above. After a [³⁵S]Met pulse from 4 to 8 hpi, cells were further incubated for chase periods in the absence of label and then lysed at the indicated time points. The viral particles were partially purified and identified by TGA electrophoresis. (B) Radioactive bands from particles A and B, as shown in panel A, were quantified and plotted. Consistent results were obtained from two independent pulse-chase experiments. (C) Toperform an in vitro maturation assay, [³⁵S]Met-labeled IPNV particles A and particles B (lane 4, input) were recovered by electroelution. Lanes 1 and 2 correspond to particles recovered from bands A and B, respectively, and were run immediately after electroelution (t = 0). Lane 3 corresponds to the dialyzed electroeluate of particles A, incubated at 4°C for 24 h (t = 24). Arrows on the left of the figure indicate the migration of particles A and B. (D) The effect of antiproteases on particle maturation was tested in a cell-free assay. Cells were infected with IPNV in the presence of [³⁵S]Met. At 8 hpi, the cell extracts were incubated with 10 mM IAA, PMSF, or EDTA for 4 or 8 h. Partially purified particles were then resolved by TGA electrophoresis. Lanes 2 and 6 correspond to IAA treatment for 4 and 8 h, respectively; lanes 3 and 7 correspond to PMSF treatment for 4 and 8 h, respectively; lanes 4 and 8 correspond to EDTA treatment for 4 and 8 h, respectively; and lanes 1 and 5 correspond to incubation of extracts in the absence of antiproteases for 4 and 8 h, respectively. Arrows on the left of the figure indicate the migration of control particles.

To directly prove the relationship between both populationsof particles, a cell-free maturation assay was envisioned (Fig.4C). For this assay, [³⁵S]Met-labeled IPNV particles A and Bwere separated by TGA electrophoresis and then recovered fromthe gel by electroelution, defining this time point as timezero (t = 0). Recovered particles A were incubated at 4°Cfor 24 h (t = 24). We used incubation at 4°C to excludeparticle instability due to a higher temperature during theassay. Both t = 0 and t = 24 particles were then subjected toa second round of TGA electrophoresis. As shown in Fig. 4C,at t = 0, particles A (lane 1) or B (lane 2) were clearly identified,with no detectable levels of cross-contamination. Upon 24 hincubation at 4°C, particles A yielded particles B (lane3). Altogether, these results present strong evidence that particlesA are the precursors of particles B. Based on these data, allof the machinery required for viral maturation is probably containedwithin particles A.

Electroeluted fractions (t = 0) corresponding to either particlesA or B (Fig. 4B, lanes 1 and 2, respectively) were also testedfor infectivity in a plaque formation assay, as previously reported(34). For comparison, the results were normalized by the specificactivity of the [³⁵S]Met incorporated in each recovered band.Particles A gave a titer of 3.6 x 10⁷ PFU/µCi, whereasparticles B gave a titer of 9.1 x 10⁸ PFU/µCi, indicatingthat the maturation of particles A is accompanied by a gainin virus infectivity of particles B. Thus, the viral titer ofisolated particles A may correspond to the continuous maturationof virion precursors.

Previous results from this study have suggested the involvementof the proteolytic cleavage of the viral precursors on the maturationof particles A. The birnavirus-encoded protease VP4 has beenshown to be a Ser-Lys protease belonging to the family of Lonproteases (3, 56) which is incorporated into virions (4, 42).To test a role of VP4 in particle maturation, compounds thatcould have antiprotease activity were assayed as inhibitors:IAA, PMSF, and EDTA. IAA is an alkylating agent for Cys andHis residues, and PMSF is a specific inhibitor of serine proteases.Since VP4 is not a metalloprotease (14), EDTA is not expectedto produce inhibition. CHSE-214 cells were infected with IPNVin the presence of [³⁵S]Met, and the cells were harvested earlyat 8 hpi, a time corresponding to the assembly of particlesA in the absence of particles B (data not shown). Then cellularextracts were immediately treated for either 4 or 8 h with theantiprotease compounds, and viral particles were identifiedby TGA electrophoresis. Figure 4D shows that IAA (lanes 2 and6) and PMSF (lanes 3 and 7) treatments were able to eliminatevirion formation at 12 and 16 hpi, respectively, indicatingthat the maturation of particles A was arrested by proteaseinhibitors. In contrast, untreated samples incubated eitherfor 8 to 12 hpi (lane 1) or for 8 to 16 hpi (lane 5) yieldedparticles B from particles A. As predicted, particles A isolatedafter an 8-h treatment with either IAA (Fig. 4D, lane 6) orPMSF (Fig. 4D, lane 7) did not produce lysis plaques. Thus,they are not infectious. However, consistent with previous titrations,a partially purified IPNV preparation composed of a mixed populationof particles A and B gave an average titer of 1.0 x10⁷ PFU/µCiwhen purified with IAA and a titer of 1.9 x10⁷ PFU/µCiwhen purified in the presence of PMSF. Furthermore, a preparationof particles B purified in the presence of PMSF produced 1.2x10⁸ PFU/µCi, indicating that this antiprotease did notaffect the infectivity of virions. These results suggest theinvolvement of the associated VP4 protease in the proteolyticmaturation of particles A, since PMSF completely eliminatedthe production of particles B.

DISCUSSION

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Discussion

In this study, we have isolated, identified, and characterized,both molecularly and biologically, stable particles producedduring the infective cycle of IPNV in CHSE-214 cells. A summaryof our results in the context of the infective cycle is presentedin Fig. 5. The earliest particle, named particle A (provirion),was first detected simultaneously with the viral dsRNA in infectedcells, suggesting that viral assembly occurs as soon as dsRNAreplication has begun. No prior intermediates could be detected.Particles A showed appearances of an incomplete viral assembly,and although they are composed by the viral genome, their patternof viral precursor proteins suggested that they correspond toimmature intermediates of the morphogenesis. Upon provirionmaturation, infectious particles B (virion) are yielded, 3 to4 h later during the viral cycle. Particles B were shown toexhibit the icosahedral shape and to be constituted mostly bya pattern of mature capsid proteins as well as by viral dsRNA.Maturation of IPNV particles A not only involved the acquisitionof infectivity but also, and remarkably, a reduction in theparticle diameter, which may involve a coordinated series ofconformational changes visualized by a more-compact particlearrangement.

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FIG. 5. IPNV infective cycle and morphogenesis. Our model assumes that the viral events such as transcription, either of the positive or negative strand of RNA, translation, or intracellular accumulation of viral proteins have taken place to favor genome RNA replication. Once dsRNA synthesis has been initiated, it immediately triggers its assembly into immature particles A (provirions). As indicated, prior intermediates are unknown. Upon maturation, where both precursor cleavage and compaction of the capsid occur, the infectious virion (particles B) is yielded.

Postassembly maturation has been previously described for otherfamilies of unenveloped viruses, with particularities in eachcase. Unlike IPNV, the morphogenesis of poliovirus particles(a single-stranded RNA virus) takes place through a discreteset of capsid intermediates which assemble the viral genomicsingle-stranded RNA into an immature precursor provirion andthen triggers proteolytic processing of viral precursors (28,58). Furthermore, provirion maturation is also a requirementfor release of the RNA genome into a new host cell. On the otherhand, the morphogenesis of nodaviruses would proceed throughan uninterrupted set of intermediaries. Following flock housevirus infection of Drosophila cells, the first detectable assembledproducts are provirions whose shells are composed of 180 ${alpha}$ -precursorprotein subunits encapsidating the two positive-stranded genomicRNAs (25). Provirions are labile, and proteolytic maturationis required for infectivity (61), resulting in an increasedvirion stability (25). Provirion matures through an autocatalyticcleavage of the ${alpha}$ -protein, generating the mature coat proteinß and the ${gamma}$ -peptide and leading to the rearrangementof the capsid subunits (25). Additionally, as a result of proteolyticmaturation, dramatic conformational changes have been describedwithin a procapsid form of the Nudaurelia capensis ${omega}$ virus (N ${omega}$ V),a single-stranded RNA virus which infects insects of the lepidopteranorder. Electron cryomicroscopy and image analysis were usedto elaborate a model in which the procapsids are larger, morerounded, and porous and with more domains than the virion ofN ${omega}$ V (7). The large rearrangements were interpreted as subunitmovements within the capsid.

For IPNV, we have found that particles A are built of both matureand precursor viral proteins. Among precursor proteins, we wereable to identify the polyprotein and pVP2 as associated withimmature provirions. It is actually not known why precursorproteins must be cleaved within the immature particle, but oneconsequence is the higher compaction of the virion, possiblythrough conformational changes between subunits, resulting inan icosahedral and regular geometry. For another birnavirus,IBDV, virus-like particles (VLPs) have been formed in heterologoussystems (40). Composition analyses of the different kinds ofVLP showed variable pVP2/VP2 ratios in which the presence ofpVP2 correlated with spherical capsids and VP2 was predominantlyfound in regular icosahedral VLPs, indicating an incompletematuration in the spherical capsids (9, 40, 48). In a recentreport, Chevalier et al. have proposed that the efficient maturationof pVP2 requires a precise and favored assembly of the IBDVparticle (9). Furthermore, the C-terminal region of pVP2, whichis presumably cleaved during capsid maturation, was implicatedin the control of subunit interactions and flexibility duringthe assembly of IBDV VLPs (8, 9). Similarly, from the resultsshown for IPNV in this study, the presence of pVP2 correlatedwith immature provirions, whereas mature VP2 correlated withinfectious and icosahedral particles. However, we cannot ignorethe existence of subpopulations with different or intermediatedegrees of maturation within the particle A fraction (Fig. 5).Thus, the postassembly maturation of pVP2 seems to be a commonstep during the morphogenesis of different members of the birnavirusfamily.

The maturation cleavage of viral precursor proteins within thecapsid may also be a requirement for viral infectivity. In thepresent study, we have found that, at least, both pVP2 and theviral polyprotein are associated with uninfectious IPNV provirions,whereas infectious virions (particles B) lack these proteinprecursors. In addition, we have found different VP4 and VP4tpatterns in both particles (Fig. 3D). By mutagenesis of recombinantproteins, the cleavage sites of birnavirus pVP2 (38, 56) wereidentified and some of them were later shown to be essentialfor viral viability, indicating that maturation of IBDV pVP2is also critical for infectivity (12). On the other hand, theviral polyprotein is processed and cleaved at the pVP2-VP4 andVP4-VP3 junctions by the viral protease VP4 (1, 45, 46, 56),which is a Ser-Lys protease (56) and shares homology to bacterialLon proteases (3). VP4 has been described as a structural proteinfor IBDV (4, 40) and IPNV (42). Additionally, truncated formsof VP4 (VP4t) were previously identified in purified IPNV (42)as well as in our preparations of both particles A and B, althougha higher proportion of VP4t is associated with infectious virions.Whether VP4t correspond to a postassembly maturation productor to a degradation fragment generated to inhibit VP4 activityafter maturation has been completed remains to be elucidated.

Structural information about birnavirus particles has been obtainedon both homologous and heterologous systems, but details ofthe virion morphogenesis remain obscure. The external surfaceof the particle is formed of trimeric subunits of VP2 (23, 39),and the innermost layer is formed by trimeric subunits of VP3,the viral dsRNA, VP1, and VP4 (4, 18). VP3, as predicted earlier,interacts with both segments of genomic dsRNA through its carboxy-terminalregion (33, 64, 65), which also binds VP1 (40, 63, 64). Theassociation of VP3 with viral dsRNA was also observed afterextensive low-salt treatment of IPNV virions (29). Furthermore,EM analyses have shown that viral VPg-dsRNA complexes tend toself-associate where the interactions were found to take placeat the end of the segments (6, 51). In this scenario, as soonas it is synthesized, the newly replicated VPg-dsRNA may actas an initiation complex to trigger genome assembly by continuouslynucleating capsid proteins. VP3 may play a key role in stabilizingthe genomic dsRNA, where charged residues at its C terminusseem to be essential for this interaction, and to prompt properparticle assembly (9, 47, 64). In a proposed model for IBDVVLPs, VP3 needs to be activated by either genomic RNA or VP1to induce capsid assembly and pVP2 maturation (9, 48). As elucidatedin the present study, the assembly of immature provirions seemsto be a rapid and favored process. However, postassembly maturationappeared to be slow and inefficient, and it may reflect thatonly a fraction of particles A are correctly assembled and capableof undergoing the whole pathway of morphogenesis.

In the present study, we were unable to isolate stable morphogenesisintermediates prior to the detection of the replicated dsRNAassembled into particles A (Fig. 5). One possibility is thatsmaller capsid precursors are labile and/or their structureis sensitive to the conditions applied (i.e., NP-40). For differentRNA viruses, RNA replication complexes form on membrane structuresderived from diverse intracellular organelles (21, 24, 36, 49,55, 60). On the other hand, for reoviruses, replication andassembly take place within cytoplasmic inclusions which arenot membrane bound but associate with cytoskeletal elements(2, 53, 54). For IPNV, components or subcellular structuresassociated with viral RNA replication are unknown. However,since viral assembly occurs simultaneously with RNA replication,there must be a temporal and spatial coordination between theseevents. The existence of capsid precursors would support thevision of a discrete morphogenesis, as revealed for picornaviruses(see above). Another possibility is the existence of a preformedprocapsid. However, we favored the model of a continuous morphogenesisfor IPNV, initiated by viral dsRNA synthesis, where it promotesparticle assembly, immediately nucleating capsid componentsuntil the generation of provirions. The morphogenesis of IPNVis later completed, and probably for the other birnavirusesas well, when most of the molecules of viral precursor proteinshave been cleaved within the immature capsid, leading to thecompaction of the particle and the acquisition of infectivityto yield icosahedral virions (Fig. 5).

ACKNOWLEDGMENTS

We thank Eugenio Spencer, John T. Patton, and Marcelo López-Lastrafor contributions to this study and M. Teresa Castillo for technicalassistance in tissue culture. We thank J. Dubuisson, C. Wychowski,and Y. Rouille for comments on the manuscript and Sophana Ungfor artwork.

This study was supported by the following grants: FONDECYT 1950257,DICYT 02-9643SG, and FONDAP, Oceanografía y BiologíaMarina, Subprograma Peces, Chile.

FOOTNOTES

* Corresponding author. Present address: Unité Hépatite C, CNRS-UPR2511, IBL/Institut Pasteur de Lille. 1, rue du Professeur Calmette, B.P. 447, 59021 Lille Cedex, France. Phone: 333 2087 1092. Fax: 333 2087 1201. E-mail: rodrigo.villanueva{at}ibl.fr.

REFERENCES

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