P64, a Novel Major Virion DNA-Binding Protein Potentially Involved in Condensing the Spodoptera frugiperda Ascovirus 1a Genome

Abstract

We recently identified 21 structural proteins in the virion of Spodoptera frugiperda ascovirus 1a (SfAV1a), a virus with a large, double-stranded DNA genome of 157 kbp, which attacks species of the lepidopteran family Noctuidae. The two most abundant virion proteins were the major capsid protein and a novel protein (P64) of 64 kDa that contained two distinct domains not known previously to occur together. The amino-terminal half of P64 (residues 1 to 263) contained four repeats (a recently recognized motif with an unknown function) of a virus-specific two-cysteine adaptor. Adjoined to this, the carboxy-terminal half of P64 (residues 279 to 455) contained 14 copies of a highly basic, tandemly repeated motif rich in arginine and serine, having an 11- to 13-amino-acid consensus sequence, SPSQRRSTS(V/K)(A/S)RR, yielding a predicted isoelectric point of 12.2 for this protein. In the present study, we demonstrate by Southwestern analysis that SfAV1a P64 was the only virion structural protein that bound DNA. Additional electrophoretic mobility shift assays showed that P64 bound SfAV1a as well as non-SfAV1a DNA. Furthermore, we show through immunogold labeling of ultrathin sections that P64 is a component of virogenic stroma and appears to be progressively incorporated into the SfAV1a DNA core during virion assembly. As no other virion structural protein bound DNA and no basic DNA-binding proteins of lower mass are encoded by the SfAV1a genome or were identified by proteomic analysis, our results suggest that P64's function is to condense the large genome of this virus and assist in packaging this genome into its virion.

Following nucleic acid synthesis, viral genomes are condensed and packaged into virions by a variety of mechanisms (17, 23, 24, 30). Essential to this process is neutralization of the negative electrostatic charge on phosphate groups of the nucleotide backbone by positively charged ions that would otherwise resist condensation of viral genomes during encapsidation. Viruses typically accomplish this by sequestering positive charges in the form of divalent cations, such as Mg²⁺, or through polyvalent polyamine cations, such as spermine and spermidine or other small, basic cationic proteins that bind and condense nucleic acids with high affinity (7, 14, 19, 22). An example of the latter group is histones, which are abundant in the nucleus and high in arginine and lysine content, their primary role being to condense host chromosomal DNA and package it into nucleosomes (3). During virion assembly, histones are involved in condensation of viral genomes by several DNA viruses, including polyomaviruses, papovaviruses (8, 9, 20, 27), and herpes viruses (25), and by RNA retroviruses (31).

Despite their ubiquity in the nucleus, histones do not appear to be involved in genome condensation and packaging of most viruses. For example, viruses as diverse as hepatitis B virus (HBV), baculoviruses, and other DNA viruses that replicate in the nucleus encode cationic proteins rich in arginine and lysine, a characteristic they share with histones and protamines. The latter are small, arginine-rich proteins that replace histones on chromosomal DNA during the late stages of spermatogenesis (1). Similar proteins have been found in viruses. The carboxy terminus of the HBV core antigen (16), for example, and the small P6.9 (6.9-kDa) proteins of baculoviruses (38, 39) are protaminelike virion components known to bind and condense viral DNA for encapsidation.

Viruses of the family Ascoviridae are highly pathogenic to larvae of the lepidopteran family Noctuidae. The virions typically are large (130 to 150 nm by 200 to 400 nm) and have complex symmetry and organization consisting of an inner particle containing a protein/DNA core that after assembly is enveloped to form the virion (11, 12). Although the ultrastructure of ascoviruses is markedly different from those of baculoviruses, iridoviruses, and entomopoxviruses (33), all of these viruses have large, double-stranded DNA (dsDNA) genomes, typically >100 kbp, that must be condensed for packaging. However, whereas the protaminelike P6.9 protein of baculoviruses is known to be a key protein involved in condensing genomic DNA and packaging it into the virion, the proteins responsible for this function in the other DNA viruses that attack insects remain unknown. Sequence analysis of the Spodoptera frugiperda ascovirus 1a (SfAV1a) genome (4), the type species, revealed no genes coding for small, protaminelike peptides that could facilitate condensation and packaging of its large genome (157 kbp). Moreover, of 21 proteins revealed by our recent proteomic analysis of the SfAV1a virion (35a), no small, cellular histones or histonelike protamines were detected. The two most abundant proteins in the SfAV1a virion were the major capsid protein (MCP) and a protein with a mass of 64 kDa (ORF048 or P64) (4, 35a). As MCPs and core DNA-condensing and -packaging proteins generally occur in amounts proportionally greater than those of other structural proteins, the relative abundance of P64, and its high pI of 12.2, suggested that it might be a key protein involved in condensing and packaging the SfAV1a genome. In the present study, we characterized P64 and show that it is a novel protein containing two distinct domains with two distinct motifs. The amino-terminal portion contains four repeats of a well-conserved virus-specific two-cysteine adaptor motif (pfam08793.1) (18), whereas the carboxy-terminal portion contains 14 copies of a previously uncharacterized motif rich in arginine and serine residues (SPSQRRSTS[V/K][A/S]RR) and, to a lesser extent, several lysine residues. Through a combination of DNA-binding and gel shift assays, along with immunogold labeling electron microscopy, we demonstrate that P64 is a major virion protein in the SfAV1a DNA/protein core and may be involved in packaging this virus' genome into the virion.

MATERIALS AND METHODS

Virion purification.

Early fourth-instar Spodoptera exigua larvae were inoculated with Spodoptera frugiperda ascovirus 1a (SfAV1a) as described previously (13). The hemolymphs of morbid, diseased larvae were collected after 6 to 7 days and suspended in ice-cold phosphate-buffered saline (pH 7.4) containing 1% glutathione. The suspension was centrifuged at 2,000 × g for 10 min, and the supernatant was collected and centrifuged at 4°C for 1 h in a Beckman SW28 rotor at 105,000 × g. The pellet was resuspended in 1 ml ice-cold phosphate-buffered saline. SfAV1a virions were purified by isopycnic centrifugation in a CsCl gradient (30%, wt/wt) as described previously (35a).

Analysis of SfAV1a ORF48 (P64).

The predicted amino acid sequence of P64 was analyzed using various online programs, including BLAST at the NCBI website (http://www.ncbi.nlm.nih.gov/) and the Scratch protein predictor (http://www.ics.uci.edu/∼baldig/scratch/), to identify gene homologues, orthologues, conserved domains, motifs, and secondary structure.

Recombinant six-His-tagged P64 and anti-six-His-tagged P64 antibody.

The Bac-To-Bac Autographa californica multicapsid nucleopolyhedrovirus baculovirus expression (Bacmid) system (Invitrogen) was used for overexpression of p64 under the polyhedrin promoter. The sequence containing the open reading frame of the p64 gene (ORF48) (4) was amplified by PCR using the primer pair comprising P64Foward (5′-CGCGGATCCATGGCGTCAAAACGTAAA-3′) and P64Reverse (5′-CCGCTCGAGATCCTTCGACGATCAGG-3′), with BamHI and XhoI sites added to the primers (underlined). The amplicon was digested with BamHI and XhoI and ligated to the same sites in pFastBac HTb (Invitrogen) for production of recombinant six-histidine-tagged P64 (hereafter referred to as rP64). BTI-TN-5B1-4 (Tn5) cells (Invitrogen) were transfected with recombinant Bacmid, using TransIT-LT1 transfection reagent (Mirus Corp.), and rP64 was purified by affinity chromatography using nitrilotriacetic acid resin under denaturing and native conditions (Qiagen), according to the manufacturers' protocols. Antiserum against purified rP64 was raised in rats (Josman, LLC, Napa, CA).

Virion protein fractionation and Western blotting.

Protein concentrations were determined using a Micro BCA kit (Pierce). For various assays, 20 to 70 μg of SfAV1a virion proteins and 1 to 3 μg of purified rP64 were solubilized in 2× Laemmli buffer (21), fractionated in a 12% gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie brilliant blue R-250 or transferred to a polyvinylidene difluoride-plus membrane (Osmonics, Inc.) by using a Semiphor Transphor unit (GE Healthcare) set at 8 V for 1 h. Western blot analysis with primary rat anti-rP64 antibody and secondary anti-rat immunoglobulin G (IgG)-alkaline phosphatase conjugate (Sigma) was performed as described by Park et al. (29).

Southwestern blotting.

A modified Southwestern blotting technique (6, 32) was used to investigate SfAV1a virion protein-DNA interactions. Proteins in the purified SfAV1a virion were fractionated in a 12% gel by SDS-PAGE and electroblotted onto a polyvinylidene difluoride-plus membrane as described above. After transfer, virion proteins were fixed by drying the membrane at room temperature. Transfer of proteins onto the membrane was subsequently confirmed by staining the membrane with Ponceau S (28) and the polyacrylamide gel with Coomassie blue. To renature virion proteins, the membrane was soaked in absolute methanol for 5 s, rinsed once in Western transfer buffer (25 mM Tris, 192 mM glycine, 15% methanol [pH 8.4]) and renaturation buffer (50 mM Tris, 1% Triton X-100, 100 mM KCl, 10% glycerol, 1 mM ZnCl, 0.2% bovine serum albumin [BSA] [pH 7.5]), and then incubated in fresh renaturing buffer overnight at 4°C with gentle shaking (∼30 rpm). The membrane was soaked in 50 ml DNA-binding buffer (DBB) (50 mM Tris [pH 7.5], 0.1% Triton X-100, 100 mM KCl, 10% glycerol, 0.1 mM ZnCl, and 2% BSA) for 2 h at room temperature. After incubation, the buffer was removed and replaced with 25 ml of fresh DBB containing 7.5 × 10⁶ cpm of ³²P-labeled SfAV1a genomic DNA prepared by randomly primed labeling with hexameric nucleotides (Roche) and [α-³²P]dATP (Perkin-Elmer), according to the manufacturers' protocols. The DNA-binding reaction mixture was incubated at room temperature for 30 min, and the membrane was washed four times in 200 ml of fresh DBB at room temperature before autoradiography using Blue Lite Autorad Film (ISC BioExpress) was performed.

EMSAs.

Nonviral covalently closed circular dsDNA (pGEM-T Easy, 3 kb; Promega) or a 0.2-kb SfAV1a-specific linear dsDNA originating from the ORF048 (p64) gene obtained by PCR with the primer pair comprising TS64CentF and TS64CentR (5′-CCATCGACATGTTTAAGCACGAGTCGTTCCAA-3′ and 5′-GATGATCTCGAGCATGCTTGGACGCATG-3′, respectively) was used in electrophoretic mobility shift assays (EMSAs). Fifty nanograms (ng) of pGEM-T Easy plasmid DNA or 100 ng of the linear amplicon was used in each assay. Substrate dsDNAs and various quantities of rP64 (1.5 μg to 9 μg) in a total of 15 μl of 0.5× DBB (see above) were incubated at 25°C for 20 min, followed by incubation at 4°C for an additional 20 min. In addition, to release DNA from stable rP64-DNA complexes in reaction mixtures containing 9 μg of rP64 or 50 ng of pGem-T Easy, 5 μg or 10 μg of proteinase K (Invitrogen), respectively, was added after complex formation. Proteolytic digestion was performed at 25°C for 30 min. The controls contained all of the components of the standard reactions except that rP64 was replaced with 9 μg of BSA. After incubation, 2 μl of 50% glycerol was added, reaction tubes were stored on ice, and products were fractionated in a 0.7% agarose gel containing ethidium bromide (0.5 μg/ml) by electrophoresis in 1× Tris-borate-EDTA buffer (Fisher Scientific).

Immunogold labeling electron microscopy.

Purified virions and virion-containing vesicles were embedded in 2% agar and fixed with 0.1% glutaraldehyde and 2% formaldehyde in 0.05 M NaPO₄ (pH 7.4) for 1 h. The specimen was dehydrated through an ethanol series and embedded in LR-White (Polysciences). Ultrathin sections were cut with a Sorvall MT 5000 ultramicrotome, stained with lead citrate and uranyl acetate, and examined and photographed with an FEI Tecnai 12 electron microscope and a Gatan US 1000 camera. Ultrathin sections of inner particles and sectioned virions were observed and quantified as described by Wills et al. (37). Immunogold labeling was performed as described previously (34, 37), using gold-labeled rabbit anti-rat IgG (Ted Pella, Inc., CA) against the primary rat anti-rP64 antibody.

Phosphorylation of P64.

Different amounts of SfAV1a virion proteins (10 μg, 15 μg, and 20 μg) and purified rP64 (0.7 μg, 1.5 μg, and 3 μg) were fractionated by SDS-PAGE in a 12% gel and stained with Pro-Q Diamond phosphoprotein gel stain (Invitrogen). Destaining with Pro-Q Diamond phosphoprotein destaining solution (Invitrogen), detection with UV transillumination (300 nm), and calibration with ovalbumin and β-casein phosphoprotein markers in PeppermintStick phosphoprotein standards (Invitrogen) were performed according to the manufacturer's protocols, using an Alpha imager (Alpha Innotech Corp.). After detection by UV spectroscopy, the gel was stained with Coomassie blue to confirm the identities and locations of corresponding phosphoproteins.

RESULTS

SfAV1a P64 (ORF048) is rich in arginine and serine and contains two distinct motifs.

P64 is composed of 565 amino acid residues (Fig. 1). Analysis of its primary sequence showed that it is an unusual highly basic protein, with a predicted isoelectric point (pI) of 12.2. The high pI is primarily due to abundances of arginine and lysine (112 and 26 residues, respectively), which accounted for 26.2% of this protein's amino acids. P64 is also rich in serine (107 residues) and threonine (31 residues), which accounted for an additional 24.4% of its amino acids.

FIG. 1.

Domains and sequence motifs in the P64 structural protein of the Spodoptera frugiperda ascovirus 1a virion. Two domains were identified with the Scratch protein predictor program, the amino-terminal domain spanning residues 1 to 263 and the carboxy-terminal domain spanning residues 264 to 565. The four virus-specific two-cysteine adaptor motifs (highlighted in yellow) with conserved cysteines (bright red) are present in the amino-terminal domain. The 14 copies of the basic tandemly repeated motif (SPSQRRSTS[V/K][A/S]RR) composed of 11 to 13 amino acids and rich in serine and arginine (residues 279 to 455; alternating blue and dark red letters highlighted in pink) are present in the carboxy-terminal portion of P64. Amino-terminal sequences rich in arginine and/or lysine that do not conform to the basic repeat motif in the carboxy terminus are boxed. Predicted helices (H) and extended beta strands (E) are also shown.

Two domains, comprising residues spanning from positions 1 to 263 (the amino-terminal portion) and 264 to 565 (the carboxy-terminal portion), were predicted based on the Scratch protein predictor program. The P64 amino-terminal portion contained four well-conserved repeats of the virus-specific two-cysteine adaptor motif (pfam08793.1) (18), at positions 13 to 50, 58 to 94, 141 to 177, and 182 to 218. This adaptor motif is known to be fused to ovarian tumor/A20-like peptidases and serine-threonine protein kinases. Although the biochemical function of this motif is unknown, its occurrence in P64 suggests that it could function as an adaptor domain that facilitates intermolecular interactions among P64 molecules or other virion structural components (18).

The carboxy-terminal portion of P64 (Fig. 1) shares similarities with 16 viral proteins, 1 from the ascovirus HvAV3e (YP_001110913; E [expect] value of 2^e−38) and 15 from two vertebrate iridoviruses, a group of viruses phylogenetically related to ascoviruses (35). These were the Regina ranavirus virus (AAK54495; E value of 1^e−9) and the Ambystoma tigrinum virus (ACB11439, ACB11427, ACB11428, ACB11429, ACB11432, ACB11433, ACB11434, ACB11438, ACB11439, ACB11440, ACB11442, ACB11446, ACB11447, and YP_003846, with E values ranging from 4^e−12 to 9^e−8). Several mammalian and invertebrate proteins with various degrees of homology to sequences in the sperm nuclear basic protein PL-1 isoform (AAT45385) of the mollusk Spisula solidassina showed lower levels of identity (34 to 37%) and similarity (46 to 56%) (BAE00421, AAT453384, AAT45385, and AAH34980, with E values ranging from 3^e−4 to 0.005). An unusual feature in the carboxy terminus, not known to occur in other protein families, is the presence of 14 copies of a basic tandemly repeated motif (residues 279 to 455) rich in arginine and serine and having an 11- to 13-amino-acid consensus sequence, SPSQRRSTS(V/K) (A/S)RR. Predicted extended β strands were found in both domains, but notably, it appeared that this secondary structure overlapped and connected each of the 14 basic tandem repeats (Fig. 1).

rP64 binds covalently closed circular plasmid and linear DNAs.

The marked abundance of P64 (35a) in the SfAV1a virion (Fig. 2 and 3), together with its high arginine and lysine content (26.2%) and basic pI of 12.2, suggested that P64 is the major protein component that interacted directly with viral DNA to neutralize the large negative electrostatic charge for genome packaging. To determine whether P64 could potentially play a role in condensing SfAV1a DNA for packaging of the genome in virions, an anti-rP64 antibody was raised in rats against the rP64 antigen produced with a Bacmid expression vector (Fig. 2A) and used for Western and immunogold labeling studies.

FIG. 2.

Western blot analysis showing the position of P64 among proteins of the Spodoptera frugiperda ascovirus 1a (SfAV1a) virion. (A) 12% SDS-PAGE showing recombinant six-His-tagged P64 (rP64) protein produced and purified from insect (Tn5) cells and used for raising rat anti-rP64 antibody and protein profile of purified SfAV1a virions. (B) Western blot showing the specificity of the rat anti-rP64 antibody against purified rP64 and P64 (white arrowhead) in the virion protein profile. MW, molecular mass standards; kDa, kilodaltons.

FIG. 3.

Southwestern blot showing that the Spodoptera frugiperda ascovirus 1a P64 virion protein binds DNA. Purified recombinant six-His-tagged P64 (rP64) and SfAV1a virion proteins were fractioned by SDS-PAGE in a 12% gel, electroblotted, and incubated with ³²P-labeled SfAV1a DNA (A) or pBR328 DNA (B). The autoradiographic film was exposed for 45 min (m) and 3 h (h) and overexposed for 17 h to rule out DNA-binding activities of SfAV1a virion proteins other than P64. SfAV1a open reading frames (ORFs) encoding virion proteins (4, 35a), including the positions of P64 and MCP, are shown. MW, molecular mass standards; kDa, kilodaltons.

Western blot analysis showed that the anti-rP64 antibody bound specifically to purified rP64 and the P64 band observed in the profile of the purified SfAV1a virion protein (Fig. 2B). Nonspecific binding of the antibody to other virion proteins was not observed, thus confirming the identity of P64 based on previous proteomic and gene sequence analyses and its presence as a structural component in the SfAV1a virion (4, 35a).

Southwestern blot analysis using radiolabeled SfAV1a sequence-specific viral DNA showed that of all the virion proteins, only P64 bound the DNA substrate (Fig. 3A). To determine whether other SfAV1a virion structural proteins, particularly those with low molecular masses in the range typical for histones, protamines, and baculovirus P6.9 protein, bound DNA, the autoradiographic film was overexposed for 17 h. Even after prolonged exposure, no protein other than P64 was observed to bind viral DNA. In addition, in a similar Southwestern analysis, rP64 and native P64 were shown to bind digoxigenin-labeled pBR328 DNA (Roche) (Fig. 3A and B), suggesting that this protein has a non-sequence-specific mode of DNA-binding activity.

To provide additional evidence that P64 has intrinsic DNA-binding activity, EMSAs were performed to demonstrate that rP64 bound covalently closed circular plasmid dsDNA (pGEM-T Easy, 3 kb) and linear SfAV1a-specific dsDNA (0.2 kb) (Fig. 4). Increased shifts in pGEM-T Easy DNA mobility were observed with increasing amounts of rP64 (Fig. 4A, lanes 4 to 9). In these assays, stable DNA-protein complexes were also observed based on their presence in the gel close to the wells. DNA in these complexes was liberated following proteolytic cleavage with proteinase K (Fig. 4A, lanes 10 and 11). Shifts in DNA mobility were not observed in controls with BSA (Fig. 4A, lanes 2 and 3). A similar shift in the mobility of the linear dsDNA substrate was also observed (Fig. 4B).

FIG. 4.

EMSAs demonstrating that Spodoptera frugiperda ascovirus 1a P64 is a DNA-binding protein. (A, B) EMSAs demonstrating that six-His-tagged P64 (rP64) binds covalently closed plasmid dsDNA (pGEM-T Easy, 3 kbp, 50 ng per reaction) and linear dsDNA (0.2 kbp, 100 ng per reaction); DNA was incubated with rP64 for 40 min, and products were fractionated by electrophoresis in a 0.7% agarose gel and stained with ethidium bromide. (A) pGEM-T Easy control DNA at 25 ng (lane 1) and 50 ng (lane 12) without protein; with 9 μg BSA (lanes 2 and 3); or with 1.5 μg, 6 μg, and 9 μg of P64 (replicates; lanes 4 and 5, 6 and 7, and 8 and 9, respectively). Following incubation, reaction mixtures containing 6 μg (lane 10) or 9 μg (lane 11) of rP64 were also treated with proteinase K at 5 μg and 10 μg, respectively, resulting in the release of rP64-bound dsDNA complexes. (B) EMSA using the 0.2-kb linear dsDNA, without (lane 1) and with (lane 2) rP64.

Localization of P64 by immunogold labeling of SfAV1a virions.

Immunogold labeling electron microscopy of SfAV1a virion-containing vesicles and purified virions showed that the secondary gold-tagged antibody against the rat anti-rP64 antibody localized in virogenic stroma (Fig. 5A and B) and was also incorporated into maturing virions (Fig. 5C and D). In the eight sections examined, 109 of 126 (86%) gold particles (dots) were observed within the dense virogenic stroma network (Fig. 5A and B), whereas only 17 (14%) appeared to be loosely attached to or outside its periphery. The apparent progressive incorporation of the label from the virogenic stroma into developing virions suggested that P64 is packaged inside the SfAV1a virion.

FIG. 5.

Representative electron micrographs of the Spodoptera frugiperda ascovirus 1a virion-containing vesicles showing the location of P64 in virogenic stroma, developing virions, and SfAV1a mature virions. (A to D) Sections of virion-containing vesicle were reacted with primary rat anti-rP64 antibody, followed by detection with gold-labeled rabbit anti-rat IgG. Arrowheads indicate the presence of gold beads within virogenic stroma (A, B) and maturing SfAV1a virions (C, D). Bar, 100 nm. (E to G) Representative electron micrographs showing internal location of P64 in purified SfAV1a virion. Sections were reacted with primary rat anti-rP64 antibody, followed by detection with gold-labeled rabbit anti-rat IgG. Arrowheads indicate the presence of gold beads inside mature sectioned SfAV1a virions (E, F, G). Specific association of gold beads with the peripheries of intact SfAV1a virions was not observed (H). Bar, 200 nm.

In this regard, counts of immunogold dots on eight micrographs of purified virions chosen randomly showed that for 508 virions, 70 (73%; ∼1 dot per 7.3 virions) of 95 dots observed were located inside SfAV1a virions, whereas only 25 (27%; 1 dot per 20.3 virions) were located in close proximity to virions (Fig. 5E, F, and G). Labeled particles clearly attached to the external surfaces of SfAV1a virions were not apparent in these micrographs, suggesting that P64 was not associated with peripheral structures in the SfAV1a virion. This is supported by observations that the gold-labeled antibody did not localize with high affinity to the surfaces of intact virions in assays performed under identical conditions (Fig. 5H). In five negatively stained samples containing 1,236 intact SfAV1a virus particles, a total of 29 dots were observed (1 dot per 42.6 virions), and of these, only 5 (1 dot per 247.2 virions) appeared to be loosely attached to the virion surface. Thus, it is likely that these were random associations, as suggested by comparison to the results showing high affinities of the labeled antibody for virogenic stroma and internal structures of the SfAV1a virion (Fig. 5A to G). The general methods and results described for the localization of P64 are similar to those that demonstrated the internal localization of the ICP35 scaffolding virion protein of herpes simplex virus 1 (37).

Phosphorylation of P64.

Phosphoprotein analysis of the SfAV1a virion showed that P64 was not modified by phosphorylation or, if phosphorylated, was undetectable by the method used (Fig. 6). Of the other virion proteins, only the capsid protein was phosphorylated. Interestingly, rP64 produced in insect cells in vitro was modified by phosphorylation, suggesting that P64 could be differentially phosphorylated during SfAV1a virogenesis. Treatment of rP64 with calf intestinal phosphatase (Biolabs) to dephosphorylate the recombinant protein was unsuccessful (data not shown).

FIG. 6.

P64 is unphosphorylated in Spodoptera frugiperda ascovirus 1a virions. Different amounts of protein standards, purified SfAV1a virions, and recombinant six-His-tagged P64 (rP64) produced in insect cells were fractionated by SDS-PAGE in a 13% gel (A) and stained with Pro-Q Diamond phosphoprotein gel stain (Invitrogen) to detect the presence of phosphorylated proteins, which were visualized by UV transillumination at a wavelength of ∼300 nm (B). The protein standards included unphosphorylated β-galactosidase and BSA at 116.3 kDa (a) and 66.3 kDa (b), respectively, and the phosphoproteins ovalbumin and β-casein (arrowheads) at 45 kDa (c) and 23.6 kDa (d), respectively. After UV transillumination, the gel was stained with Coomassie blue to confirm the locations and identities of phosphoproteins. MW, molecular mass standards; kDa, kilodaltons.

DISCUSSION

Ascoviruses are insect viruses that have unique structural and biological characteristics that distinguish them from other viruses (11, 12). The genome sequences of three ascoviruses, including SfAV1a, have been reported over the past 2 years (2, 4, 36), but despite genomic sequence data, only a few proteins coded for by these viruses were identified as virion structural proteins (10, 35a). An interesting aspect of ascovirus structure is how the large genomes of these viruses are condensed and packaged into virions. Here, we have provided evidence suggesting that one of P64's functions is to condense SfAV1a genomic DNA for packaging during virion assembly. In addition to the sequence data showing that P64 contains a highly basic protaminelike, arginine-rich domain in the carboxy-terminal domain (Fig. 1), we show that P64 (i) is a major virion protein (Fig. 2), (ii) binds SfAV1a genomic DNA in vitro (Fig. 3 and 4), and (iii) is initially localized within virogenic stroma, followed by progressive packaging of what appears to be SfAV1a genomic DNA into the virion core, as observed by immunogold labeling (Fig. 5). This combination of data lends strong support to our conclusion that P64 is a novel DNA-binding protein involved in packaging of viral DNA into the virion during assembly. Of course, this does not exclude other proteins from participating in binding and packaging of the SfAV1a genome in the virion, and this occurrence is even likely. However, whether nuclear cations, histones, and/or protamine-like peptides participate in this process during early stages of SfAV1a virion assembly is unknown, but as the last two were not identified in proteomic analyses (35a) and are not encoded by the SfAV1a genome (4), it is unlikely that these play a role in virion assembly, as has been observed with other viruses (7, 14, 19, 22, 23). However, it must be noted that although DNA-binding proteins other than P64 were not observed, we cannot rule out the possibility that the SfAV1a virion contains other genome-sequestering-virus-encoded or nuclear proteins, as their activity potentially could have been abolished or suppressed by the experimental conditions used in the Southwestern assays described here.

Taken together with its abundance and location in the virion core, the modular organization of P64 into the two distinct domains, i.e., the virus-specific two-cysteine adaptor domain (pfam08793.1) (18) at the amino-terminus and the protaminelike domain at the carboxy terminus, suggests that P64 is a multifunctional protein that participates in the assembly and structure of the SfAV1a virion. In particular, its association with SfAV1a DNA in the stroma (Fig. 5) also suggests that P64 functions in early stages of virogenesis, not only in organizing and configuring DNA for virion assembly but also in recruiting or being recruited to other virion structural components during this process. In this regard, the amino-terminal virus-specific two-cysteine adaptor motif could play a role in virion assembly via protein-protein interactions. The specific role of this domain in virion structure and biology is not known at present. However, through comparative sequence analyses, Iyer et al. (18) have shown that the virus-specific two-cysteine adaptor motif is known to be fused to ovarian tumor/A20-like peptidases and serine-threonine protein kinases, suggesting both structural and regulatory roles for this motif through protein-protein interactions.

Nevertheless, the modular arrangement of P64 is not unlike that of the hepatitis B core antigen (HBcAg) protein (15). The virion of HBV is composed of an outer envelope containing lipids and virally encoded surface proteins that form a lipoprotein shell that encloses the nucleocapsid, or HBcAg, which contains the RNA genome of this virus. Interestingly, the capsid consists of dimers of a 183-residue protein that contains two domains with distinct functions, an amino-terminal assembly domain (residues 1 to 149) and a protaminelike carboxy-terminal domain (residues 150 to 183), responsible for polymerization into virion particles and RNA packaging, respectively (16). The protaminelike domain is dispensable for HBV capsid assembly (16), and though a nucleic acid binding motif appears to be present in the amino-terminal domain, the basic motif is required for RNA packaging (5, 40). Therefore, the HBcAg core protein has dual functions in HBV virion maturation. By analogy, similar roles are anticipated for P64 in genome packaging, assembly, and maturation of the SfAV1a virion.

Finally, the proportionally high level of serine and threonine residues, collectively 25% (Fig. 1), in P64 suggests that these amino acids potentially play crucial roles in its putative function, similar to those of histones and protamines. In the nuclei of host cells, modification of histones by acylation, phosphorylation, and methylation of serine and threonine residues is known to influence processes such as chromatin structure remodeling and organization, gene transcription and silencing, and DNA replication and repair, thereby elaborating epigenetic effects on virus nucleic acid structure and metabolism (1, 3, 23, 26, 27). In this regard, the potential for P64 to be modified by phosphorylation/dephosphorylation (Fig. 6) suggests that it could play a role in diverse epigenetic processes related to SfAV1a virogenesis, including gene transcription and genome packaging and release. Whether nascent P64 is phosphorylated is unknown, but it is not surprising that it is unphosphorylated in the mature SfAV1a virion, where it presumably assists in neutralizing negative electrostatic charges conferred by phosphate groups in DNA to facilitate efficient packaging of viral genomic DNA, which otherwise would resist condensation during its encapsidation. Indeed, if P64 functions as proposed here, the mechanism(s) related to its phosphorylation during disassembly of the SfAV1a virion and release of genomic DNA during infection is of interest and could involve both host and virus-encoded kinases. Further studies are required to determine whether nascent P64 is phosphorylated after cellular invasion and to determine the mechanism(s) that results in its dephosphorylation during SfAV1a virion assembly.