A New Member of the Tetraviridae That Infects Cultured Insect Cells

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Virology 306 (2003) 359Ð370 www.elsevier.com/locate/yviro

Providence virus: a new member of the Tetraviridae that infects cultured insect cells

Fiona M. Pringle,a Karyn N. Johnson,a Cynthia L. Goodman,b Arthur H. McIntosh,b and L. Andrew Balla,*

a Department of Microbiology, University of Alabama at Birmingham, 845 19th Street South, Birmingham, AL 35294, USA b USDA, Agricultural Research Service, Biological Control of Insects Laboratory, 1503 South Providence, Columbia, MO 65203, USA Received 18 July 2002; returned to author for revision 13 September 2002; accepted 2 October 2002

Abstract We identified a new member of the Tetraviridae, Providence virus (PrV), persistently infecting a midgut cell line derived from the corn earworm (Helicoverpa zea). Virus purified from these cells also productively infected a H. zea fat body cell line, and a cell line from whole embryos of the beet armyworm, Spodoptera exigua. PrV is thus the first tetravirus shown to replicate in cell culture. PrV virions are isometric particles composed of two structural proteins (60 and 7.4 kDa) that encapsidate both the genomic (6.4 kb) and the subgenomic (2.5 kb) RNAs. The monopartite organization of the PrV genome resembles that of Nudaurelia ␤ virus and Thosea asigna virus, members of the genus Betatetravirus. The predicted sequence of the PrV structural proteins demonstrates homology to tetraviruses in both genera. The infectivity of PrV for cultured cells uniquely permitted examination of tetravirus RNA and protein synthesis during synchronous infection. The discovery of PrV greatly facilitates studies of tetravirus molecular biology. © 2003 Elsevier Science (USA). All rights reserved.

Keywords: Tetravirus; Tetraviridae; Cell culture; Insect virus; Providence virus; RNA virus; T ϭ 4

Introduction organization differs between these genera (Hanzlik and Gordon, 1999). Betatetraviruses such as N␤V and Thosea Tetraviruses originally attracted interest about 40 years asigna virus (TaV) have monopartite genomes that contain ago because of their ability to cause epizootics in pest insect about 6.5 kb. The RNA-dependent RNA polymerase species (Grace and Mercer, 1965; Hendry et al., 1968). (RdRp) is expressed from the genomic RNA, and the struc- Viruses in this family exclusively infect lepidopteran insects tural protein is expressed from a 2.5-kb subgenomic tran- (butterflies and moths) and are extremely host-specific, usu- script which is encapsidated in virions (Gordon et al., 1999; ally confined to a few closely related species (Hanzlik and Pringle et al., 1999). In contrast, the omegatetraviruses Gordon, 1997). Tetraviruses have single-stranded positive- Nudaurelia ␻ virus (N␻V) and HaSV have bipartite ge- sense RNA genomes. The complete nucleotide sequences nomes composed of two unique RNA molecules: a 5.3-kb have been determined for two members of the family, in- RNA that encodes the RdRp and a separate 2.5-kb RNA that cluding species from the two genera: Nudaurelia ␤ virus encodes the structural proteins (Agrawal and Johnson, (N␤V; Betatetravirus) (Gordon et al., 1999) and Heli- 1992; Hanzlik et al., 1993). coverpa armigera stunt virus (HaSV; Omegatetravirus) Tetraviruses are also interesting from a structural per- (Gordon et al., 1995; Hanzlik et al., 1995). The genome spective. The name tetravirus derives from the T ϭ 4 capsid architecture of members of this family, currently the only nonenveloped viruses known to have this symmetry * Corresponding author. Department of Microbiology, University of ␻ Alabama at Birmingham, 845 19th Street South, BBRB 373/17, Birming- (Hanzlik and Gordon, 1999). The structure of N V has ham, AL 35294. Fax: ϩ1-205-934-1636. been solved by both X-ray crystallography and electron E-mail address: [email protected] (L.A. Ball). cryomicroscopy (cryo-EM) (Munshi et al., 1996, 1998), and

0042-6822/03/$ Ð see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0042-6822(02)00052-1 360 F.M. Pringle et al. / Virology 306 (2003) 359–370 that of N␤V has been examined by transmission electron microscopy (TEM) and cryo-EM (Finch et al., 1974; Olson et al., 1990). Each tetravirus particle is composed of 240 copies of two structural proteins that range in size from 56 to 63 kDa and from 6 to 8 kDa, respectively (Ball et al., 2000). The structural proteins are produced by proteolysis of a capsid precursor in an autocatalytic postassembly cleavage reaction (Agrawal and Johnson, 1992, 1995). In the case of TaV, processing of the capsid precursor is predicted to yield a 17-kDa nonstructural protein of unknown function in addition to the large and small capsid proteins (Pringle et al., 1999, 2001). Recent studies of N␻V virus-like particles made using a baculovirus expression system have shown that during maturation N␻V provirions undergo large conformational changes (Canady et al., 2000, 2001). Despite the advent of molecular techniques that have begun to allow the characterization of tetraviruses, their inability to replicate in cell culture severely hinders these studies. Previous attempts to identify cell lines that support the replication of these viruses were unsuccessful (Hanzlik and Gordon, 1997; Moore and Tinsley, 1982). An extensive study that tested 14 cell lines for susceptibility to HaSV by either infection or transfection of viral RNAs failed to identify a cell line that supported HaSV replication (Baw- den et al., 1999). Since there is no cell-culture system, tetraviruses must be either purified from infected larvae or Fig. 1. Products of RNA replication detected in cell lines derived from Helicoverpa zea. FB33 cells (Lanes 1 and 2) and MG8 cells (Lanes 3 and produced by another method. The insect hosts are difficult 4) were infected with PaV (Lanes 2 and 4) or left untreated (Lanes 1 and or impossible to rear in the laboratory (Hanzlik and Gordon, 3); 24 h p.i. (Lanes 1 and 2) and 72 h p.i. (Lanes 3 and 4) RNAs were 1997), and heterologous systems for production have thus far metabolically labeled by incorporation of [3H]uridine in the presence of been unable to produce significant quantities of virus (Gordon actinomycin D. Total cellular RNAs were extracted, resolved by electro- et al., 2001; Venter, 2001). Therefore, several aspects of tet- phoresis on denaturing 1% agarose-formaldehyde gels, and visualized by fluorography. The size of the two PaV genomic RNAs (3.0 and 1.3 kb) are ravirus replication remain poorly characterized. indicated on the right, as are the estimated sizes of the RNAs identified in This article describes the isolation of a new virus that we MG8 cells. discovered persistently infecting a midgut cell line (MG8) derived from the corn earworm, Helicoverpa zea. Virus was purified from these cells and used to infect two other cell lines, specifically radiolabeled by incorporation of [3H]uridine in one from H. zea fat body (Kariuki et al., 2000) and one from the presence of actinomycin D. In the absence of deliberate larvae of the beet armyworm, Spodoptera exigua (Gelernter infection with an RNA virus, most cell lines show only and Federici, 1986). We present evidence that this virus is a minimal background labeling (Fig. 1, Lane 1; uninfected previously undescribed tetravirus. Because of the uncertainty BCIRL-HZ-FB33 cells; referred to here as FB33). Infection about its natural host range, we have named this new tetravirus with an RNA virus, such as the nodavirus Pariacoto virus Providence virus (PrV) derived from the location of the labo- (PaV), reveals the production of virus-specific bands by ratory where the MG8 cell line was generated. Since it repli- RNA-dependent RNA synthesis (Fig. 1, Lane 2; PaV-in- cates in cell culture, PrV provides a unique opportunity to fected FB33 cells). However, in the BCIRL-HZ-MG8 cell examine aspects of tetravirus biology that have previously line (referred to here as MG8), two prominent RNAs were been out of reach. We have taken a first step toward elucidating detected even in uninfected cells (Fig. 1, Lane 3). These the life cycle of a tetravirus by studying PrV macromolecular cells were originally derived from the midgut of larvae from syntheses during synchronous infection of FB33 cells. a laboratory colony of the corn earworm, H. zea (as described under Materials and methods). MG8 cells were susceptible to infection by PaV (Johnson and Ball, 2001), Results and discussion which provided 3.0- and 1.3-kb RNAs to use as size mark- Isolation of a virus persistently infecting a H. zea midgut ers (Fig. 1, Lane 4). By comparison with these RNAs and cell line the 6.0-kb dimer of PaV RNA1 (Johnson et al., 2000), the unidentified RNAs present in untreated and PaV-infected RNA molecules that are synthesized in a DNA-indepen- MG8 cells were estimated to be 6.4 and 2.5 kb. The size of dent manner, such as the products of viral RdRps, can be the larger RNA was also estimated to be 6.4 kb by com- F.M. Pringle et al. / Virology 306 (2003) 359–370 361 parison to an RNA molecular weight marker on ethidium bromide stained gels (data not shown). In addition to the unexpected RNAs seen in MG8 cells, when concentrated spent medium from very early passage MG8 cells (passage 8) was analyzed by SDSÐPAGE and isoelectric focusing, a major protein of approximately 60 kDa with an acidic pI (4.7) was the major product detected (Goodman and McIn- tosh, 1995). Although no previously described tetravirus has been reported to replicate in cultured cells (Bawden et al., 1999; Moore, 1991), these RNA and protein sizes are char- acteristic of tetraviruses (Gordon and Hanzlik, 1998). Fur- thermore, all known tetraviruses exhibit tropism for the midgut of lepidopteran insects (Hanzlik and Gordon, 1997), the source of the MG8 cell line. These circumstances sug- gested that MG8 cells might carry a persistent tetravirus infection and prompted us to attempt virus isolation. To determine whether infectious virus could be recov- ered from MG8 cells, a virus purification procedure that had been successfully used for the isolation of tetraviruses from insects (Pringle et al., 1999) was adapted for virus purification from cultured cells. Briefly, MG8 cells were lysed with a nonionic detergent and the lysate was pelleted through a 30% sucrose cushion. Virus was purified by centrifugation on 10Ð40% sucrose gradients, and gradient fractions were analyzed for the presence of putative viral proteins by SDSÐPAGE. A protein of approximately 60 kDa was consistently detected by Coomassie blue staining (Fig. 2A). Fractions containing the 60-kDa protein were combined and after further purification, the resulting preparation was characterized. Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry analysis of purified virus identified a small protein with a mass of 7439 Da (Fig. 2B), which was also detected when virions were examined by tricine SDSÐPAGE (data not shown). The sizes of the two structural proteins closely resemble the capsid proteins of other tetraviruses, which range in size from 56 to 63 and 6 to 8 kDa for the large and small capsid proteins, respectively (Gordon and Hanzlik, 1998). When sucrose gradient-purified material was negatively Fig. 2. Characterization of virus purified from MG8 cells. Virus was purified from MG8 cells on 10-40% sucrose velocity gradients and frac- stained and examined by TEM, nonenveloped isometric tions collected. (A) The proteins present in each fraction were resolved by particles with a diameter of about 40 nm were visualized SDSÐPAGE on 10% gels and visualized by staining with Coomassie blue. (Fig. 2C). Their structure was examined at higher resolution Fractions 1 to 5 (numbered from the bottom of the gradient) of a total of by cryo-EM and 3D image reconstruction (P. Natarajan and 10 are shown. The sizes of the molecular mass markers are shown on the J.E. Johnson, Scripps Research Institute, personal commu- left. The star in Lane 4 indicates the putative capsid precursor protein. The fractions containing the 60-kDa protein were combined and analyzed nication). The virions have an external capsid architecture further. (B) MALDI-TOF mass spectrometry analysis was performed di- that resembles that of N␤V and TaV (Olson et al., 1990; rectly on the purified sample. The spectrum is shown for the region in Pringle et al., 1999). Twelve capsomeres are arranged as which a protein of 7439 Da was identified. (C) The purified sample was four Y-shaped trimers on each triangular face of the virion, negatively stained and analyzed by TEM. The diameter of the virus par- yielding a T ϭ 4 icosahedral lattice composed of 240 sub- ticles was estimated to be 40 nm. The size bar represents 100 nm. units per virion. Each face contains three distinct pits, and adjacent faces are separated by deep surface grooves. A PVDF membrane, and excised separately. Their N-terminal detailed description of the structure will be presented else- sequences were determined to be PQMTR...and where. FAGTV...,respectively. Both amino acid sequences were The large and small capsid proteins from purified virions identified in a single open reading frame (ORF) of 2265 nt were resolved on tricine SDSÐPAGE gels, transferred to in a partial cDNA clone of PrV RNA. The sizes predicted 362 F.M. Pringle et al. / Virology 306 (2003) 359–370

During the course of this study, MG8 cells from both a low passage (passage 32) and a high passage (passage 100ϩ) of the cell line were examined for the presence of PrV. Virus was puriﬁed from both low- and high-passage cells, indicating that PrV persistently infected MG8 cells even after numerous passages. Preparations of virus from both low- and high-passage MG8 cells were found to be infectious (see below and data not shown). The presence of a major protein species of 60 kDa in concentrated spent medium from very early passage MG8 cells (passage 8) (Goodman and McIntosh, 1995) further suggests that PrV Fig. 3. Comparison of the PrV capsid protein precursor to those of other was present in these cells soon after the cell line was tetraviruses. The amino acid sequence of the PrV capsid protein precursor, from the proline at the N-terminus of the large capsid protein to the established and may have been present in the laboratory C-terminus of the small capsid protein, was predicted from the nucleotide colony of H. zea from which the cell line was prepared. The sequence of cDNA clones and compared to the amino acid sequences of the tetravirus HaSV was also discovered as an infection in a structural protein precursors of other tetraviruses by pairwise alignments laboratory colony of H. armigera (Hanzlik et al., 1993). using GAP. Similarity and identity scores are presented in a matrix.

␤ for the large and small capsid proteins from the nucleotide The genome organization of PrV resembles that of N V sequence of the cDNA were 60,649 and 7434 Da, respec- and TaV tively, which correlate well with the results of SDSÐPAGE ␤ and MALDI-TOF analysis of the virion proteins. Moreover, Viruses in the Betatetravirus genus (such as N V and the pI of the 60.6-kDa capsid protein predicted from its ORF TaV) have monopartite genomes and express their structural (4.78) agreed well with the experimentally determined proteins via a subgenomic RNA. Those in the Omegatetra- value of 4.7 (Goodman and McIntosh, 1995). virus genus (such as N␻V and HaSV) have bipartite ge- Within the ORF, the N-terminus of the large capsid nomes in which the larger genome segment (RNA 1) en- protein (PQMTR...)waspreceded by 123 codons that are codes the RdRp and the smaller segment (RNA 2) encodes predicted to encode a protein of 13.4 kDa. Other tetraviruses the capsid protein precursor (Gordon and Hanzlik, 1998). have two different strategies of capsid protein expression. In Although N␤V and TaV have monopartite genomes, both N␤V, N␻V, and HaSV, the large and small capsid proteins the genomic and the subgenomic RNAs are encapsidated are generated by cleavage of the capsid protein precursor at (Gordon et al., 1999; Pringle et al., 1999). a single site (Agrawal and Johnson, 1992; Gordon et al., To examine the genome organization of PrV, total RNA 1999; Hanzlik et al., 1993), whereas in TaV, the precursor from MG8 cells and RNA isolated from purified virions is cleaved twice to produce an unidentified nonstructural were analyzed by Northern blot hybridization. Two cDNA protein in addition to the large and small capsid proteins clones that contained small portions (544 and 705 nt) of the (Pringle et al., 1999). The presence of 123 codons preceding PrV genome were generated during this study. The 544-nt the start of the PrV large capsid protein suggests that it may cDNA contained the 3Ј end of the ORF that encodes the have a processing strategy similar to that of TaV. capsid proteins, whereas the 705-nt cDNA represented a The predicted amino acid sequence of the structural portion of the nonstructural region of the PrV genome (data portion of the PrV capsid protein precursor (from the pro- not shown). We anticipated that if PrV had a bipartite line residue at the N-terminus of the large capsid protein to genome similar to omegatetraviruses, probes made from the the C-terminus of the small capsid protein) was compared to 705- and 544-nt cDNAs would hybridize uniquely to the the structural protein sequences of other tetraviruses. The large and small RNA molecules, respectively. However, if results, presented as a similarity/identity matrix of pairwise alignments in Fig. 3, clearly established that PrV is related PrV had a monopartite genome that transcribed a sub- to the four other tetraviruses that have been characterized in genomic RNA, the 544-nt probe would hybridize to both this detail. The structural proteins of PrV were somewhat RNA species. Figure 4A shows that the probe from the more similar to those of the omegatetraviruses, HaSV and 544-nt cDNA hybridized to both the 6.4- and the 2.5-kb N␻V (47 and 46%, respectively), than to those of the RNAs. The probe from the 744-nt cDNA hybridized only to betatetraviruses, TaV and N␤V (35% to both). The sizes of the larger RNA molecule (Fig. 4B). Both RNAs were en- the viral RNAs and proteins, the T ϭ 4 icosahedral sym- capsidated in virus particles (Fig. 4, virion lanes). These metry of the nonenveloped virus particles, and the definitive results established that PrV has a monopartite genome or- homology of the structural protein sequences with those of ganization similar to N␤V and TaV. As is the case for N␤V other tetraviruses, all support the inclusion of this virus in and TaV, the 2.5-kb subgenomic RNA of PrV is encapsi- the family Tetraviridae. dated in virions. F.M. Pringle et al. / Virology 306 (2003) 359–370 363

demonstrates that both FB33 and Se-1 cells were susceptible to PrV infection (data not shown). PrV RNAs were detected in FB33 cells at m.o.i.s as low as 1 ϫ 103 particles per cell, but in Se-1 cells only at the highest m.o.i. We therefore chose FB33 cells as the host cell system for further analysis of PrV infection.

Synthesis of viral RNAs and proteins following synchronous infection

Cell culture provides ready access to virus replication cycles. Synchronous infections of FB33 cells were used as a first step toward analysis of RNA and protein production during a PrV infection. The time course of infection was analyzed by the following: (1) Northern blot hybridization to assess the accumulation of positive- and negative-sense genomic and subgenomic RNA molecules (Figs. 5A and 5B); (2) metabolic labeling of RNA to analyze RNA synthesis (Fig. 5C); and (3) Western blot to assay the accumulation of structural proteins (Fig. 6). Input positive-sense genomic and subgenomic RNA were detected by Northern blot hybridization at 6 and 12 h p.i. (Fig. 5A). There was a significant increase in the amount of RNA detected between 18 and 24 h p.i., and it reached a maximum at about 36 h p.i. (Fig. 5A). By metabolic label- Fig. 4. Analysis of the genome organization of PrV. RNA was extracted ing, RNA synthesis was first detected at 18 h p.i. (Fig. 5C). from MG8 cells (Lanes 1 and 3) or purified virus (Lanes 2 and 4), resolved The synthesis of the subgenomic RNA slightly lagged that on denaturing 1% agarose-formaldehyde gels, and transferred to nylon of the genomic RNA. Negative-sense RNAs were also not membranes. The membranes were hybridized with riboprobes transcribed detected until 18 h p.i., when both the genomic and the from cDNA clones containing small portions (544 nt, A; 705 nt, B) of the PrV genome. The probe generated from the 544-nt clone hybridized to both subgenomic RNAs were observed (Fig. 5B). At 36 h p.i. the genomic (6.4 kb) and the subgenomic (2.5 kb) RNAs, whereas the these negative-sense RNAs reached peak levels that were probe from the 705-nt clone hybridized only to the genomic RNA. The about 10-fold lower than the corresponding positive-sense sizes of the RNAs are indicated on the left. Schematic diagrams of the species (Fig. 5B). This is the first time that negative strands genome organization of viruses from the Betatetravirus and Omegatetra- have been detected for a tetravirus subgenomic RNA, and it virus genera are shown for comparison. raises the possibility that the PrV subgenomic RNA can be replicated. Evidence for replication of the subgenomic RNA Infectivity of PrV for other cell lines of the nodavirus Flock house virus has recently been presented (L. D. Eckerle and L. A. Ball, unpublished results), We tested the infectivity of PrV in a cell line derived but this issue needs further study. Positive- and negative- from the fat body of H. zea larvae (FB33) (Kariuki et al., sense RNA accumulation monitored over 5 days of infec- 2000) and in a cell line derived from S. exigua larvae (Se-1) tion mirrored the results seen in the shorter time course, and (Gelernter and Federici, 1986). Cells in six-well plates were RNA accumulation at later time points was maintained at a infected with serial 10-fold dilutions of purified PrV from 3 similar level to the 48 h samples in the shorter time course ␮g per well to 3 ϫ 10Ϫ4 ␮g per well, yielding multiplicities (data not shown). of infection (m.o.i.s) that ranged from approximately 1 ϫ Total protein from mock-infected and PrV-infected 105 particles per cell at the highest dose of virus to approx- FB33 cells was analyzed by immunoblotting for the pres- imately 10 particles per cell at the lowest dose. No cyto- ence of PrV-specific proteins using a polyclonal antibody pathic effect was evident by phase contrast microscopy at raised in rabbits against purified PrV (Fig. 6). The large any dose in either cell line. At 48 h p.i., cells were meta- capsid protein of the input virus was detected 6 h p.i. and bolically labeled with [3H]uridine in the presence of acti- this band increased in intensity from about 24 h p.i. Begin- nomycin D; then total cellular RNA was extracted, resolved ning at 36 h p.i., a larger capsid-specific protein accumu- by electrophoresis on denaturing agarose-formaldehyde lated, having a size appropriate for a precursor comprising gels, and visualized by fluorography. RNA molecules cor- the large and small structural proteins (approximately 68 responding in size to the 6.4-kb genomic and 2.5-kb sub- kDa). Synthesis of this 68-kDa precursor protein slightly genomic PrV RNAs were labeled in both cell types. Since lagged the appearance of the subgenomic RNA that is likely input RNA is not detected using this technique, this result to direct its translation. Although the 68-kDa precursor to 364 F.M. Pringle et al. / Virology 306 (2003) 359–370

large and small capsid proteins that comprise mature virions (Agrawal and Johnson, 1995; Johnson and Reddy, 1998). The absence of the 68-kDa precursor protein in purified PrV may be due to maturation of the virus during purification and/or storage. A small amount of the putative 68-kDa precursor to the large and small capsid proteins was detected in virus samples during purification (Fig. 2A, Lane 4). A species corresponding to the primary translation product of the 2265-nt ORF was not detected either in cells during the time course or in purified virions. However, processing of this precursor could be extremely rapid. Im- mediately upstream of the proline residue at the N-terminus of the PrV large capsid protein, there is a cis-acting processing signal that is active in a diverse range of viruses (e.g., TaV, Cricket paralysis virus, and Foot-and-mouth disease virus; see Donnelly et al., 2001).

Detection of virus particles in infected cells

To determine whether virions could be observed in infected cells, infected FB33 cells and persistently infected MG8 cells were sectioned and examined by TEM. FB33 cells were infected with puriﬁed PrV at an m.o.i. of approximately 1 ϫ 104 particles per cell and harvested at 3 days p.i. Persistently infected MG8 cells (passage 100ϩ) were harvested from 75-cm2 tissue culture ﬂasks, but were otherwise treated identically to FB33 cells. Sectioned cells were examined by TEM, and their appearance compared between the two cell lines and to mock-infected FB33 cells.

Fig. 5. Synthesis and accumulation of viral RNAs following infection of FB33 cells. (A and B) FB33 cells were infected at an m.o.i. of 104 particles per cell or mock-infected, and incubated at 28¡C until harvesting at 6, 12, 18, 24, 36, and 48 h p.i. Following extraction, total cellular RNAs were resolved on denaturing gels along with a virion RNA control (V) and transferred to nylon membranes. Genomic and subgenomic viral RNAs were detected by Northern blot hybridization using riboprobes synthesized from the 544-nt clone to detect positive- (A) or negative- (B) sense (10ϫ greater exposure) RNAs. The positive-sense RNA detected at 6 and 12 h p.i. was derived from the inoculum. Negative-sense species were detected from 18 h p.i. for both the genomic and the subgenomic RNAs. (C) Cells Fig. 6. Accumulation of PrV structural proteins during infection of FB33 infected as described for (A) were labeled by metabolic incorporation of cells. FB33 cells were infected at an m.o.i. of 104 particles per cell or [3H]uridine in the presence of actinomycin D fora3hperiod at 6, 12, 18, mock-infected and incubated at 28¡C until harvesting at 6, 12, 18, 24, 36, 24, 36, and 48 h p.i. and then harvested. Total cellular RNAs were resolved and 48 h p.i. Total cellular proteins were harvested at the times shown, on denaturing 1% agarose-formaldehyde gels along with labeled MG8 resolved by SDSÐPAGE on a 10% polyacrylamide gel, transferred to cellular extracts (C) and visualized by fluorography. PVDF membranes, and detected using a hyperimmune rabbit antiserum raised against purified PrV. The capsid protein detected at early time points was derived from the inoculum. A capsid-specific protein of appropriate the large and small capsid proteins accumulated in infected size to be an uncleaved precursor of the large and small capsid proteins was detected from 36 h p.i. Two markers were used on gels, 150 ng of purified cells, this protein was not detected in purified virions (Fig. PrV (V) and 150 ng of virus mixed with 5 ␮l of the 48 h p.i. mock-infected 6, lanes V and VϩC). In other tetraviruses, immature pro- protein sample (VϩC). No virus-specific protein bands were detected in virions undergo cleavage of the precursor protein into the mock-infected FB33 cells 48 h p.i. (lane M). F.M. Pringle et al. / Virology 306 (2003) 359–370 365

Fig. 7. Detection of PrV particles in MG8 and FB33 cells by TEM of cell sections. (AÐC) Sections of persistently infected MG8 cells. Virus particles were detected in association with a structure in the cytoplasm (A, B) or associated with the cell membrane (C). (D and E) Infected FB33 cells exhibited virus particles associated with a structure (D), free in the cytoplasm (D, E), or on the cell membrane (E). Mock-infected FB33 cells (F) did not exhibit particles that resembled virions. The size bar in each panel represents 250 nm. Arrows in (A) and (B) indicate virus particles within a cytoplasmic structure, and those in (E) indicate virus particles on the cell membrane.

Virus particles approximately 40 nm in diameter were iden- addition, single virus particles could be identified on the tified in both cell lines (Fig. 7). In each case, virus particles outside of the cell membrane (arrows, Fig. 7E). Mock- were detected only in the cytoplasm. The particles were infected FB33 cells did not contain any particles resembling associated with an unidentified cytoplasmic structure, which virions in the cytoplasm nor did they exhibit cytoplasmic in some sections appeared to be enclosed by a membrane. structures that contained particles (Fig. 7F). TEM of tissues isolated from tetravirus-infected whole lar- Infected Se-1 cells were also sectioned and examined by vae have also demonstrated the presence of virus particles TEM, but these cells contained very few virus particles, within the cytoplasm, often within cytoplasmic vesicles correlating well with the reduced amount of viral RNA (Moore, 1991; Moore and Greenwood, 1984). detected in these cells compared to the MG8 and FB33 cells In the sections of MG8 cells examined, almost every cell (data not shown). However, similar to the virus seen in had at least one virus-containing structure (Figs. 7A and 7B, MG8 and FB33 cells, virus in Se-1 cells was associated with arrows), and most cells had a number of the structures. a cytoplasmic structure (data not shown). Occasionally, a clump of virus particles was observed that was associated with the cell membrane (Fig. 7C), but it was TaV, N␤V, and HaSV did not infect FB33 cells unclear whether this represented viral endocytosis or re- lease. To determine whether FB33 cells could support infection The abundance of virus particles observed in infected by other tetraviruses, samples of TaV, N␤V, and HaSV FB33 cells varied more than in MG8 cells. Some FB33 cell were assessed for infectivity by metabolic labeling of RNA. sections did not appear to have any virus particles, whereas The HaSV preparation used in this study was verified as others had virus-containing cytoplasmic structures similar being pathogenic for its insect host (T. N. Hanzlik, CSIRO, to those seen in MG8 cells. One to two percent of the FB33 personal communication), although the infectivity of the cells examined contained numerous virus particles (Figs. N␤V and TaV preparations could not be verified for tech- 7D and 7E). In these cells, virus particles were visible in nical reasons. Using the method that detected the synthesis large arrays within cytoplasmic structures and also as indi- of PrV RNAs, no TaV-, N␤V-, or HaSV-specific bands vidual virus particles spread throughout the cytoplasm. In were identified in total RNA extracted from cells labeled 366 F.M. Pringle et al. / Virology 306 (2003) 359–370

48 h p.i. (data not shown). The lack of a detectable signal is was then placed in a petri dish in HBSS containing 100 units most likely due to the inability of these viruses to infect the penicillin/ml and 100 ␮g/ml streptomycin and then rinsed cells tested. Previous attempts to find a cell line that would twice in HBSS without antibiotics. The midgut tissue was support HaSV replication were similarly unsuccessful cut into small pieces with dissecting scissors and 1-1.5 ml (Bawden et al., 1999). trypsinÐEDTA (Sigma) solution added and allowed to in- cubate at room temperature for 3-5 min. Five milliliters of Conclusion TC-199MK medium (McIntosh et al., 1973) containing 10% heat-inactivated fetal bovine serum (FBS; 56¡C/30 This article describes Providence virus, the first tetravi- min) was added to the minced tissue. The tissue was re- rus found to replicate in cultured insect cells. PrV has a moved from the dish and centrifuged at 1000 rpm for 10 genome organization similar to the monopartite genomes of min in a Beckman Model TJ-6 table-top centrifuge. The viruses in the Betatetravirus genus of the Tetraviridae and supernatant was discarded and the pellet resuspended in 5 a predicted capsid processing strategy similar to that of ml ExCell-400 (JRH Biosciences) supplemented with 10% TaV. However, the structural proteins of PrV have closer inactivated FBS. The resuspended pellet was transferred to ␻ homology to the omegatetraviruses (HaSV and N V) than a 25-cm2 tissue culture flask. Cultures were routinely re-fed ␤ to the betatetraviruses (N V and TaV). PrV was found as a at 10 day intervals until the cells began to proliferate. persistent long-term infection of a cell line from the midgut In this study, MG8 cells from a low passage (passage 32) of H. zea, and the virus could also infect two other insect and a high passage (passage 100ϩ) were routinely used. cell lines. All other tetraviruses that have been tested failed Both low- and high-passage MG8 cells were grown in to replicate in cultured cells, indicating that PrV is unusual serum-free medium as described above, since it was ob- and presently unique in its ability to replicate in cell culture. served that the presence of 10% FBS caused the cells to Future research will focus on the development of a reverse become stressed, round up, and detach from the flask sur- genetic system to facilitate studies of PrV replication, as- face. Unless otherwise noted, virus or cells used in experi- sembly, and structure. ments were derived from passage 32.

Materials and methods Analysis of concentrated spent media from the MG8 cell line Insect cell cultures Spent media concentrates from passage 8 MG8 cells Insect cell lines derived from H. zea (corn earworm, were generated by dialyzing spent medium against 0.05 M Lepidoptera, Noctuidae) fat body (BCIRL-HZ-FB33, re- sodium phosphate buffer (pH 7.0) and then concentrating ferred to here as FB33) (Kariuki et al., 2000) and S. exigua using Aquacide II. Protein (10Ð22 ␮g) from these samples larvae (Se-1; beet armyworm, Lepidoptera, Noctuidae) was analyzed by denaturing electrophoresis on 8Ð25% (Gelernter and Federici, 1986) were maintained at 28¡Cin acrylamide gels using the PhastSystem (Amersham Bio- ExCell-401 medium (JRH Biologicals) supplemented with sciences) as described by Goodman et al. (1997). Isoelectric 10% heat-inactivated fetal bovine serum (FBS) and antibi- focusing using the Multiphor System (Amersham Bio- otics. A H. zea midgut cell line (BCIRL-HZ-MG8, referred sciences) was performed as described previously (McIntosh to here as MG8), which was developed as described below, and Ignoffo, 1983). was maintained at 28¡C in serum-free ExCell-401 supplemented with antibiotics. RNA labeling, extraction, and analysis Establishment of the MG8 cell line from H. zea The products of RNA replication were labeled by meta- 3 Fourth or ﬁfth instar larvae of H. zea were starved for bolic incorporation of [ H]uridine for3hinthepresence of approximately 4 h and surface sterilized by immersing the 20 ␮g/ml actinomycin D as previously described (Ball, larvae in 70% alcohol for 10-15 min followed by rinsing in 1992). Total RNA was extracted using an RNAgents kit Hanks’ balanced salt solution (HBSS). The larvae were (Promega) according to the manufacturer’s directions. pinned on sterile wax in a petri plate and dissected, and the RNAs were resolved by electrophoresis on 1% agarose- fat body, silk gland, tracheae, and Malphigian tubules formaldehyde gels and visualized by ﬂuorography (Laskey cleared away. A few milliliters of HBSS was added to the and Mills, 1975). In passage 100ϩ cells infected with the plate and the larvae were immersed in the solution. The nodavirus Pariacoto virus, the genomic RNAs of 3011 and outer part of the midgut was raised gently with forceps and 1311 nt, were used as size standards. The 0.24- to 9.5-kb a small opening cut in it to allow the extrusion of food. The RNA ladder (Invitrogen) was included as a size standard on midgut was cut at both ends and the whole peritrophic gels when RNAs were visualized by staining with ethidium membrane and any remaining food discarded. The midgut bromide. F.M. Pringle et al. / Virology 306 (2003) 359Ð370 367

Virus purification from MG8 cells Determining the similarity of the capsid precursor protein of PrV to those of other tetraviruses MG8 cells were grown as monolayers in tissue culture flasks at 28¡C for 5 to 7 days. Cells were lysed by rocking The sequence of a 2593-nt cDNA clone containing a for 15 min at room temperature in the presence of 0.5% portion of the PrV genome was determined using fluores- NP-40. Cellular debris was removed by centrifugation at cent dideoxynucleotides in an automated DNA sequencer 10,000 g for 20 min. Virus was pelleted from the superna- and analyzed using GCG software for the presence of ORFs. tant through a 30% sucrose cushion (in 50 mM sodium A 2265-nt ORF was identified that encoded the capsid phosphate buffer, pH 7.2) by centrifugation at 100,000 g for precursor protein (GenBank Accession No. AF548354). 2.5 h. Virus pellets were resuspended overnight at 4¡Cin50 The structural protein sequences predicted from this ORF mM phosphate buffer and then subjected to centrifugation were compared to the capsid protein sequences of TaV through a 10Ð40% sucrose velocity gradient, and fractions (GenBank Accession No. AF062037), HaSV (NC_001982), were collected and analyzed as described previously (Prin- N␤V (NC_001990), and N␻V (S43937). Pairwise align- gle et al., 1999). Virus was repelleted from the pooled ments were performed in GCG using GAP, with a gap gradient fractions and resuspended as described above. Pu- creation penalty of 8 and a gap extension penalty of 2. The rified virus was stored at Ϫ20¡C. similarity and identity scores from these alignments were used to prepare the similarity matrix shown in Fig. 3. Analysis of gradient fractions by SDSÐPAGE Calculation of number of PrV particles/␮g and the m.o.i. Gradient fractions were analyzed for the presence of viral proteins by SDSÐPAGE. Samples were boiled in 2ϫ The number of virus particles/␮g was estimated using sample buffer for 5 min before electrophoresis at 170 V the RNA (6.4 and 2.5 kb) and protein (60 and 7.44 kDa) through a 10% glycine SDSÐpolyacrylamide gel (Laemmli, sizes determined for PrV in this study. The amount of 1970). Proteins were stained with Coomassie blue. A broad protein in each particle was calculated based upon the pres- range molecular mass marker (New England Biolabs) was ence of 240 copies each of the large and small capsid included as a size standard. proteins. The molecular weight of RNA in each particle was estimated as the sum of the genomic and subgenomic

Matrix-assisted laser desorption ionization-time-of-flight RNAs. The estimated Mr values of protein and RNA in a mass spectrometry PrV particle were calculated to be 1.62 ϫ 107 and 0.29 ϫ 107 Da, respectively. The estimated total particle weight Samples were analyzed in the linear mode on a Voyager (RNA plus protein) was 1.91 ϫ 107 Da, with the RNA Elite mass spectrometer with delayed extraction technology comprising approximately 15% of the PrV virus particle. (PerSeptive Biosystems, Framingham, MA). The accelera- The calculated mass of the virus particle was used to esti- tion voltage was set at 25 kV, and 100 to 150 laser shots mate the number of particles/␮g of virus at 3.2 ϫ 1010.In were summed. Sinapinic acid (Aldrich) dissolved in aceto- a typical purification, approximately 1 ϫ 108 MG8 cells nitrile-0.1% trifluoroacetic acid (1:1) was used as the ma- yielded about 350 ␮g of purified PrV (approximately 1 ϫ trix. Virus samples were diluted 1:10 with the matrix, and 1 1013 particles). ␮l was pipetted onto a smooth plate. Two internal standards, insulin (5734 Da) and ubiquitin (8565 Da), were included to Infection of FB33 and Se-1 cells with PrV calibrate detection in the size range of the small capsid protein. Cells were seeded in 35-mm diameter tissue culture plates at a density of approximately 1 ϫ 106 cells/well in 2 N-terminal sequencing of PrV structural proteins ml complete ExCell-401 medium (containing 10% FBS and antibiotics) and allowed to attach at 28¡C. Cells were in- Purified virions were denatured and electrophoresed fected with 200 ␮l serum-free ExCell-401 medium contain- through a 10% tricine SDSÐPAGE gel (Schagger and von ing 3 ␮g purified PrV or serial 10-fold dilutions. Mock- Jagow, 1987). Proteins were transferred to Immobilon P infected wells received 200 ␮l of serum-free medium only. membrane (Millipore) by semidry blotting with the Milli- Infections were incubated at room temperature with rocking pore Graphite Electroblotter System. Proteins were visual- for 2 h; then the inoculum was removed from each well, 2 ized on the membrane by staining with 0.025% Coomassie ml of complete ExCell 401 medium was added, and plates blue in 40% methanol for 2 min. The membrane was were incubated at 28¡C. destained with 50% methanol and then washed in deionized To analyze the production of RNA and protein over the water. Bands corresponding to the large and small capsid course of infection, FB33 cells were seeded as described proteins were excised. Five cycles of sequencing were per- above and then duplicate wells were infected at an m.o.i. of formed on each protein using a Beckman Model PI 2090E approximately 1 ϫ 104 particles/cell. Duplicate mock-in- sequencer. fected wells received 200 ␮l of medium only. Infected and 368 F.M. Pringle et al. / Virology 306 (2003) 359Ð370 mock-infected wells were harvested at time points of 6, 12, rified virus alone, or mixed with 5 ␮l of 48 h p.i. mock- 18, 24, 36, and 48 h p.i. At each time point, duplicate wells infected cells, was analyzed in parallel. Proteins were trans- were harvested for either RNA analysis by Northern blot ferred to Immobilon-P membranes (Millipore) in Towbin hybridization or protein analysis by immunoblotting (see buffer (10 mM Tris base, 96 mM glycine in 10% methanol) below). In addition to time courses where RNA was de- using a semidry graphite electroblotter (Millipore) for2hat tected by Northern blot hybridization, a 48-h time course a constant current of 39 mA. Following transfer, the mem- where RNA was detected by metabolic labeling (as de- branes were stained in Ponceau S (Sigma); the position of scribed above) was also performed. In this time course, the viral large capsid protein was marked, and the mem- mock and infected wells were labeled for3hwith branes destained. PrV antiserum raised in rabbits (Lampire [3H]uridine in the presence of actinomycin D at 6, 12, 18, Biologicals) against purified virus was diluted 105-fold for 24, 36, and 48 h p.i. RNAs were resolved by electro- use as the primary antibody in the Immu-star chemilumi- phoresis on 1% agarose-formaldehyde gels and visual- nescent protein detection system (Bio-Rad). ized by fluorography. TEM of purified virus and sectioned cells Source of other tetraviruses and testing infectivity in FB33 cells Five microliters of purified virus was pipetted onto the surface of a carbon-coated copper EM grid (Electron Mi- Samples of other tetraviruses were kindly supplied by croscopy Sciences) and allowed to absorb for 2 min. The Vernon Ward, University of Otago, Dunedin, New Zealand sample was blotted off the grid and the grid was rinsed three (TaV), Don Hendry, Rhodes University, Grahamstown, times with 50 mM phosphate buffer, pH 7.2, and then twice South Africa (N␤V), and Terry Hanzlik and Karl Gordon, with 1% (w/v) uranyl acetate, blotting between each rinse. CSIRO Division of Entomology, Canberra, Australia A final drop of 1% uranyl acetate was left on the grid for 2 (HaSV). The infectivity of the TaV and N␤V virus stocks min and then removed and the grid allowed to dry. Grids for their respective insect hosts could not be determined due were examined by TEM on a Hitachi 7000. to unavailability of the insects. The infectivity of the viral MG8 cells (passage 100ϩ) and infected (m.o.i. ϭ 1 ϫ stock of HaSV was confirmed in H. armigera, the natural 104) and uninfected FB33 and Se-1 cells were harvested for host (T. Hanzlik, personal communication). FB33 cells cell sections and analysis by TEM. FB33 and Se-1 cells were infected or mock-infected and the RNA was metabol- were harvested at 3 days p.i. by scraping the surface of the ically labeled at 48 h p.i. as described above. Total RNA plate, transferring the cells and medium to microcentrifuge was analyzed for the presence of virus-specific bands. tubes, and pelleting by centrifugation in a microcentrifuge at 7000 rpm for 3 min. MG8 cells were scraped from a Northern blot hybridization tissue culture flask and pelleted by centrifugation. Cells were rinsed in phosphate-buffered saline, repelleted, and Total cellular RNAs were extracted and resolved on 1% resuspended in 2.5% glutaraldehyde in 0.1 M sodium caco- agaroseÐformaldehyde gels (3 ␮g/lane) as described above. dylate buffer for 3 min and then pelleted at 5000 rpm for 3 RNAs were transferred to Nytran nylon membranes (Schlei- min and left to fix for 60 min. Fixed cells were rinsed three cher & Schuell) by capillary blotting; then the membranes times in 0.1 M cacodylate buffer, incubated in 1% osmium were air-dried and fixed by exposure to UV light for 2 min. tetroxide in 0.1 M cacodylate buffer for 60 min, and then The two partial clones used in this study contained 705- and rinsed three times in cacodylate buffer followed by 10 min 544-nt inserts that were specific for parts of the PrV genome in 0.5% tannic acid in 0.1 M cacodylate buffer and a further that encode nonstructural and structural proteins, respec- three rinses with 0.1 M cacodylate buffer. The fixed cells tively. Strand-specific 32P-labeled RNA probes were pre- were dehydrated by washing with a series of solutions of pared from the T7 and SP6 promoters present in the vector increasing concentrations of ethanol up to 100%. Pellets using the method described previously (Albarino et al., were infiltrated with Polybed 812 resin and 100% ethanol in 2001). Membranes were probed overnight at 60¡C, washed, a 1:1 proportion overnight and then in 100% Polybed resin exposed to a phosphor screen overnight, and visualized for three incubations of 2 h each. Pellets were then poly- using a Molecular Dynamics Storm digital radioactivity merized in fresh Polybed resin at 60¡C overnight. Sections imaging system. were collected on grids and stained in 2% uranyl acetate and then in lead citrate before examination by TEM on a Hitachi Western blot analysis 7000.

Total cellular proteins were harvested by adding 200 ␮l of 2ϫ glycine SDSÐPAGE buffer to each 35-mm-diameter Acknowledgments plate of cells. Five microliters samples were resolved by electrophoresis on 10% glycine SDSÐPAGE gels using the The BCIRL-HZ-MG8 cell line was established by Q. method of Laemmli (1970). Approximately 150 ng of pu- Chen, Z. Yu, and H. Hong while at the Biological Control F.M. Pringle et al. / Virology 306 (2003) 359Ð370 369 of Insects Research Laboratory as visiting scientists in 1991. Gelernter, W. D., Federici, B. A., 1986. Continuous cell line from Spo- Thanks to Leigh Millican at the UAB High-Resolution doptera exigua (Lepidoptera: Noctuidae) that supports replication of Imaging Facility for cell sectioning and TEM, Lori Coward nuclear polyhedrosis viruses from Spodoptera exigua and Autographa californica. J. Invertebr. Pathol. 48, 199Ð207. for MALDI-TOF analysis, Kelly Morrison for N-terminal Goodman, C.L., Greenstone, M.H., Stuart, M.K., 1997. Monoclonal anti- sequencing, and the UAB Center for AIDS Research se- bodies to vitellins of bollworm and tobacco budworm (Lepidoptera: quencing core facility for the determination of DNA se- Noctuidae): biochemical and ecological implications. Ann. Entomol. quences. We also thank the members of the Ball and Dr. Soc. Am. 90, 83Ð90. Gail Wertz (UAB) labs for useful discussions. The mass Goodman, C.L., McIntosh, A.H., 1995. Characteristics of midgut-derived spectrometer was purchased with funds from an NIH Shared insect cell lines. In Vitro Cell. Dev. Biol. 31, 85A. Instrumentation Grant (S10 RR11329) and from a Howard Gordon, K.H.J., Hanzlik, T. N.Tetraviruses, 1998. In: Miller, L.K., Ball, L.A. (Eds.), The Insect Viruses. Plenum Press, New York, pp. 269Ð Hughes Medical Institute infrastructure support grant to 299. UAB. Its operation was supported in part by an NCI Core Gordon, K.H.J., Johnson, K.N., Hanzlik, T.N., 1995. The larger genomic Research Support Grant to the Comprehensive Cancer Cen- RNA of Helicoverpa armigera stunt tetravirus encodes the viral RNA ter (P30 CA13148-27). N-terminal sequencing was carried polymerase and has a novel 3Ј-terminal tRNA-like structure. Virology out by the Protein Analysis Shared Facility of the Cancer 208, 84Ð98. Center of UAB with support from the NIH (CA13148). This Gordon, K.H.J., Williams, M.R., Baker, J.S., Gibson, J.M., Bawden, A.L., Millgate, A.G., Larkin, P.J., Hanzlik, T.N., 2001. Replication-indepen- work was supported in part by NIH Grant RO1 AI18270 to dent assembly of an insect virus (Tetraviridae) in plant cells. Virology L.A.B. Mention of trade names or commercial products in 288, 36Ð50. this publication is solely for the purpose of providing spe- Gordon, K.H.J., Williams, M.R., Hendry, D.A., Hanzlik, T.N., 1999. Se- cific information and does not imply recommendation or quence of the genomic RNA of Nudaurelia ␤ virus (Tetraviridae) endorsement by the U.S. Department of Agriculture. The defines a novel virus genome organization. Virology 258, 42Ð53. Accession No. for the PrV sequence is AF548354. Grace, T.D.C., Mercer, E.H., 1965. A new virus of the saturniid Antherea eucalypti (Scott). J. Invertebr. Pathol. 7, 241Ð244. Hanzlik, T. N., Dorrian, S. J., Gordon, K. H. J., Christian, P. D., 1993. A novel small RNA virus isolated from the cotton bollworm, Helicoverpa References armigera. J. Gen. Virol. 74, 1805Ð1810. Hanzlik, T.N., Dorrian, S.J., Johnson, K.N., Brooks, E.M., Gordon, K.H.J., 1995. Sequence of RNA2 of the Helicoverpa armigera stunt virus Agrawal, D.K., Johnson, J.E., 1992. Sequence and analysis of the capsid (Tetraviridae) and bacterial expression of its genes. J. Gen. Virol. 76, protein of Nudaurelia capensis ␻ virus, an insect virus with T ϭ 4 icosahedral symmetry. Virology 190, 806Ð814. 799Ð811. Agrawal, D.K., Johnson, J.E., 1995. Assembly of the T ϭ 4 Nudaurelia Hanzlik, T.N., Gordon, K.H.J., 1997. The Tetraviridae. Adv. Virus Res. capensis ␻ virus capsid protein, post-translational cleavage, and spe- 48, 101Ð168. cific encapsidation of its mRNA in a baculovirus expression system. Hanzlik, T.N., Gordon, K.H.J., 1999. Tetraviruses (Tetraviridae). In: Web- Virology 207, 89Ð97. ster, R.G., Granoff, A. (Eds.), Encyclopedia of Virology, 2nd ed. Albarino, C.G., Price, B.D., Eckerle, L.D., Ball, L.A., 2001. Characteriza- Harcourt Brace & Company, London, pp. 1764Ð1773. tion and template properties of RNA dimers generated during Flock Hendry, D.A., Becker, M.F., van Regenmortel, M.H.V., 1968. A non- House virus RNA replication. Virology 289, 269Ð282. inclusion virus of the pine emperor moth Nudaurelia cytherea capensis Ball, L.A., 1992. Cellular expression of a functional nodavirus RNA Stoll. S. Afr. Med. J. 42, 117. replicon from vaccinia virus vectors. J. Virol. 66, 2335Ð2345. Johnson, J.E., Reddy, V., 1998. Structural studies of nodaviruses and Ball, L.A., Hendry, D.A., Johnson, J.E., Rueckert, R.R., Scotti, P.D., tetraviruses. In: Miller, L.K., Ball, L.A. (Eds.), The Insect Viruses. (2000). Tetraviridae, in: van Regenmortel, M.H.V., Fauquet, C.M., Plenum Press, New York, pp. 171Ð223. Bishop, D.H.L., Carstens, E.B., Estes, M.K., Lemon, S.M., Maniloff, Johnson, K.N., Ball, L.A., 2001. Recovery of infectious Pariacoto virus J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), from cDNA clones and identification of susceptible cell lines. J. Virol. Virus Taxonomy: Seventh Report of the International Committee on 75, 12220Ð12227. Taxonomy of Viruses, Academic Press, San Diego, pp. 757Ð764. Johnson, K.N., Zeddam, J.L., Ball, L.A., 2000. Characterization and con- Bawden, A.L., Gordon, K.H.J., Hanzlik, T.N., 1999. The specificity of struction of functional cDNA clones of Pariacoto virus, the first Al- Helicoverpa armigera stunt virus infectivity. J. Invertebr. Pathol. 74, phanodavirus isolated outside Australasia. J. Virol. 74, 5123Ð5132. 156Ð163. Kariuki, C.W., McIntosh, A.H., Goodman, C.L., 2000. In vitro host range Canady, M.A., Tihova, M., Hanzlik, T.N., Johnson, J.E., Yeager, M., 2000. studies with a new baculovirus isolate from the diamondback moth Large conformational changes in the maturation of a simple RNA virus, Plutella xylostella (L.) (Plutellidae: Lepidoptera). In Vitro Cell. Dev. Nudaurelia capensis ␻ virus (N␻V). J. Mol. Biol. 299, 573Ð584. Biol. Anim. 36, 271Ð276. Canady, M.A., Tsuruta, H., Johnson, J.E., 2001. Analysis of rapid, large- scale protein quaternary structural changes: time-resolved X-ray solu- Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly tion scattering of Nudaurelia capensis ␻ virus (N␻V) maturation. J. of the head of bacteriophage T4. Nature 227, 680Ð685. 3 14 Mol. Biol. 311, 803Ð814. Laskey, R.A., Mills, A.D., 1975. Quantitative film detection of H and C Donnelly, M. L. L., Hughes, L. E., Luke, G., Mendoza, H., ten Dam, E., in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335Ð341. Gani, D., Ryan, M. D., 2001. The “cleavage” activities of foot-and- McIntosh, A.H., Ignoffo, C.M., 1983. Characterization of five cell lines mouth disease virus 2A site-directed mutants and naturally occurring established from species of Heliothis. Appl. Entomol. Zool. 18, 262Ð “2A-like” sequences. J. Gen. Virol. 82, 1027Ð1041. 269. Finch, J.T., Crowther, R.A., Hendry, D.A., Struthers, J.K., 1974. The McIntosh, A.H., Maramorosch, K., Rechtoris, C., 1973. Adaptation of an structure of Nudaurelia capensis ␤ virus: the first example of a capsid insect cell line (Agallia constricta) in a mammalian cell culture me- with icosahedral surface symmetry T ϭ 4. J. Gen. Virol. 24, 191Ð200. dium. In Vitro 8, 375Ð378. 370 F.M. Pringle et al. / Virology 306 (2003) 359Ð370

Moore, N.F., 1991. The Nudaurelia ␤ family of insect viruses. In: Kurstak, Olson, N.H., Baker, T.S., Johnson, J.E., Hendry, D.A., 1990. The three- E. (Ed.), Viruses of Invertebrates. Dekker, New York, pp. 277Ð285. dimensional structure of frozen-hydrated Nudaurelia capensis beta Moore, N.F., Greenwood, L.K., 1984. Pathogenicity of a small RNA-virus virus, a T ϭ 4 insect virus. J. Struct. Biol. 105, 111Ð122. of insects determined by a virulent virus rather than a susceptible host. Pringle, F.M., Gordon, K.H.J., Hanzlik, T.N., Kalmakoff, J., Scotti, P.D., Microbios 40, 181Ð186. Ward, V.K., 1999. A novel capsid expression strategy for Thosea Moore, N.F., Tinsley, T.W., 1982. The small RNA viruses of insects. Arch. asigna virus (Tetraviridae). J. Gen. Virol. 80, 1855Ð1863. Virol. 72, 229Ð241. Pringle, F.M., Kalmakoff, J., Ward, V.K., 2001. Analysis of the capsid Munshi, S., Liljas, L., Cavarelli, J., Bomu, W., McKinney, B., Reddy, V., processing strategy of Thosea asigna virus using baculovirus expres- Johnson, J.E., 1996. The 2.8 A structure of a T ϭ 4 animal virus and sion of virus-like particles. J. Gen. Virol. 82, 259Ð266. its implications for membrane translocation of RNA. J. Mol. Biol. 261, Schagger, H., von Jagow, G., 1987. Tricine-sodium dodecyl sulfate-poly- 1Ð10. acrylamide gel electrophoresis for the separation of proteins in the Munshi, S., Liljas, L., Johnson, J.E., 1998. Structure determination of range from 1 to 100 kDa. Anal. Biochem. 166, 368Ð379. Nudaurelia capensis omega virus. Acta Crystallogr. D Biol. Crystal- Venter, P. A. (2001). Ph.D. Thesis, Rhodes University, Grahamstown, logr. 54, 1295Ð1305. South Africa.