ABSTRACT
The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the Baculoviridae family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9 binds to DNA in a non-sequence-specific manner. By using a p6.9-null mutant of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), we demonstrate that P6.9 is not required for viral DNA replication but is essential for the production of infectious virus. Virion production was rescued by P6.9 homologs from a number of Alphabaculovirus species and one Gammabaculovirus species but not from the genus Betabaculovirus, comprising the granuloviruses, or by the P6.9 homolog VP15 from the unrelated white spot syndrome virus of shrimp. Mutational analyses demonstrated that AcMNPV P6.9 with a conserved 11-residue deletion of the C terminus was not capable of rescuing p6.9-null AcMNPV, while a chimeric Betabaculovirus P6.9 containing the P6.9 C-terminal region of an Alphabaculovirus strain was able to do so. This implies that the C terminus of baculovirus P6.9 contains sequence elements essential for virion formation. Such elements may possibly interact with species- or genus-specific domains of other nucleocapsid proteins during virus assembly.
Condensation and packaging of viral DNA are integral and necessary features of double-stranded (ds) viral assembly within infected eukaryotic host cells. To mediate DNA condensation during viral assembly, dsDNA viruses may functionally coopt host histone proteins, as do the Polyomaviridae (13, 40) and Papillomaviridae (36), or express their own protamine-like protein with putative DNA condensation functions, as do the Adenoviridae and Asfarviridae (6, 29). In contrast, members of the Baculoviridae and Nimaviridae, which are exclusively pathogenic for arthropods, predominantly within the orders Lepidoptera and Decapoda (1, 25), neither utilize host histones nor express their own histone-like proteins but instead express a single, functionally equivalent protamine-like protein, P6.9 (41, 56). The p6.9 gene, which is present in all 50 baculovirus genomes sequenced to date (46), encodes a small arginine- and serine-rich protein that, depending on the species, contains 49 to 109 residues (see the supplemental material). Evidence indicates that P6.9 is prevented from binding to DNA as a result of posttranslational phosphorylation of arginine and serine residues (23, 53, 54). Upon viral assembly in the host nucleus, P6.9 is dephosphorylated, promoting DNA binding and enabling condensation of the viral genome and packaging into the viral nucleocapsid (14, 22, 28). In a newly infected cell, and following rephosphorylation by a capsid-associated kinase, P6.9 dissociates from the viral DNA, thereby releasing the viral genome into the nucleus (14, 42, 54). At this stage, cellular histones bind to the viral DNA, forming nucleosomes and an active transcriptional complex (55).
The binding of P6.9 to DNA is apparently species independent, as evidenced by the facts that Heliothis zea single nucleopolyhedrovirus (HezeNPV) P6.9 can bind λ-DNA (22) and the P6.9 homolog VP15 of the unrelated white spot syndrome virus (WSSV) is able to bind Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA (56). Although DNA binding by P6.9 exhibits a degree of species and sequence independence, we were interested in learning whether P6.9 possesses any species specificity for overall virion formation. We report here the results of experiments designed to examine whether AcMNPV P6.9 can be functionally replaced either by P6.9-like proteins from phylogenetically distinct baculovirus species or by WSSV VP15. Furthermore, by constructing AcMNPV genomes encoding either P6.9 proteins with C-terminal deletions or point mutations, or chimeric P6.9 fusion proteins, we demonstrate that the C-terminal domain of P6.9 appears to be an essential genus-specific feature required for infectious virion formation.
MATERIALS AND METHODS
General molecular methods.Classical restriction enzyme (RE)-based genetic engineering techniques, preparation of media, and related procedures were performed as described elsewhere (38a) unless otherwise stated. All PCRs were performed in volumes of 50 μl containing 15 pmol of each oligonucleotide primer and 200 μM each deoxynucleoside triphosphate (dNTP) and were catalyzed with a high-fidelity DNA polymerase (Expand long-template PCR system [Roche] or Phusion [Finnzymes]). Recombineering, utilizing lambda phage recombinases supplied via plasmid pBAD-αβγ (31), was performed essentially as described previously (36). All plasmids and bacmid constructs generated during the study were sequenced and/or analyzed by PCR to confirm construction fidelity.
Cell culture and virus harvesting.The Spodoptera frugiperda cell line IPLB-Sf21 (47) was cultured at 27°C in plastic culture flasks (Nunc) in Grace's insect medium (pH 5.9 to 6.1; Invitrogen) supplemented with 10% fetal bovine serum (FBS). Viruses were harvested from culture supernatants following clarification (2,200 × g, 10 min) and filter sterilization (pore size, 0.45 μm).
Deletion of AcMNPV p6.9.The p6.9 sequence in the AcMNPV bacmid bMON14272 (27) was partially replaced, from 10 bp downstream of the translational start codon ATG to 68 bp upstream of the TAA stop codon, with a chloramphenicol resistance gene (cat) via recombineering (Fig. 1A). Therefore, recombineering primers were designed with 50- to 52-nucleotide (nt) 5′ extensions corresponding to sequences immediately flanking the p6.9 sequence to be deleted. The forward primer (5′-GTAACTTCGGCGACCTGTCGATGAACGGCTCCTGGATCTTCTGTATGTGCCCTCAGGTTTAAGGGCACCAATAACTGCCTTAAAAAAATT-3′) contains viral flanking sequences (5′ untranslated region [5′ UTR]) from nt 86730 to 86779 according to the AcMNPV-C6 complete genome sequence (3), and the reverse primer (5′-GCAAAGCGTAAAAAATATTAATAAGGTAAAAATTACAGCTACATAAATTACACCTGAGGTTCCTGTGCGACGGTTACGCCGCTCCATGAG-3′) contains viral flanking sequences (3′ UTR) from nt 86941 to 86890. The 3′ ends of the primers anneal to cat of pBeloBac11 (39, 48), and a Bsu36I site (underlined) was designed between the viral and cat sequences.
Schematic presentation of the p6.9-null AcMNPV bacmids. (A) The p6.9 locus in the AcMNPV bacmid bMON14272 is shown on top. A PCR-amplified cassette containing the chloramphenicol resistance gene (cat) was inserted by ET recombination into E. coli from nt 86779 to 86890 according to the AcMNPV genome numbering (3). Subsequently, gene cassettes containing the gfp marker controlled by the AcMNPV p10 promoter and baculovirus p6.9 genes controlled by the AcMNPV p6.9 promoter were transposed into the attTn7 sites of the polyhedrin locus of the bacmid. (B) Schematic presentation of the ChocGV (Co), CypoGV (Cp), and AcMNPV (Ac) P6.9 proteins with C-terminal modifications introduced into p6.9-null AcMNPV. Specific ChocGV, CypoGV, and AcMNPV sequences are highlighted in gray or black or are boxed, respectively. Alanine substitutions are underlined. (C) Sequence alignment of P6.9 homologs of AcMNPV (GenBank accession number NP_054130), HearNPV (NP_075157), SeMNPV (NP_037825), NeleNPV (YP_025228), CypoGV (NP_148870), ChocGV (YP_654488), CrleGV (NP_891924), and WSSV (AAK77778) introduced into p6.9-null AcMNPV. Amino acids highlighted in black, dark gray, and light gray are at least 75, 50, and 25% similar, respectively. The C-terminal sequences deleted from AcMNPV are indicated by domains I and II (black lines), and the conserved C-terminal sequence YxxRxY in domain I is shown. The grey lines represent four possible degenerated nuclear localization signals.
The resulting 1,050-bp PCR fragment, containing the cat gene flanked by the homology arms, was gel purified, digested with DpnI to eliminate residual pBeloBac11 template DNA, and gel purified once more. The purified PCR product (500 ng) was introduced, via electroporation, into Escherichiacoli DH10B cells containing bMON14272 and pBAD-αβγ. Following transient arabinose-mediated induction of phage recombinase activities, the deletion of the p6.9 sequence was confirmed by PCR using primers flanking the p6.9 locus. A single recombinant bacmid was designated AcBacΔp6.9.
Construction of p6.9 donor plasmids and generation of corresponding bacmids.A series of donor plasmids, containing either native p6.9 gene sequences from different viral species or mutant sequences modified at the C termini and generated by conventional PCR, were cloned into a common recipient plasmid backbone that was constructed as follows. First the polyhedrin promoter (polPROM) was deleted from pFastBac-1 (Invitrogen) by Bst1107I/StuI digestion, and the vector backbone was religated. The AcMNPV p6.9 promoter sequence, PCR amplified from pAcMP1 (18) with primers 5′-GGTCGACGTACCAAATTCCGTTTTGCGACG-3′ and 5′-GGTCGACGGATCCGTTTAAATTGTGTAATTTATG-3′ (underlined and italicized sequences are SalI and BamHI sites, respectively), was cloned, as a SalI fragment, into this polPROM minus vector. Subsequently, the p6.9 promoter sequence was removed as a SnaBI/BamHI fragment and was introduced, via the Bst1107I and BamHI sites, into pFastBacDUAL (Invitrogen), thereby deleting the vector's polPROM sequence. Finally, a green fluorescent protein (GFP) (smRS-GFP) (11) sequence was cloned, at a unique XmaI site, downstream of the p10 promoter in this pFastBacDUAL derivative to yield the final base donor plasmid pFB-GFP-p6.9.
Native and mutant p6.9 sequences were generated by PCR amplification using either total or cloned fragments of the appropriate genomic DNA as templates and forward and reverse primers (see Table S1 in the supplemental material) containing EcoRI and NotI sites at their respective 5′ ends. p6.9 sequences containing modified C termini were generated by including the respective sequence change within the 5′ end of the reverse primer (see Table S1 in the supplemental material). Initially, native p6.9 sequences of AcMNPV, Spodoptera exigua MNPV (SeMNPV), Helicoverpa armigera NPV (HearNPV), Neodiprion lecontei NPV (NeleNPV), Choristoneura occidentalis granulovirus (ChocGV), Cydia pomonella GV (CypoGV), and Cryptophlebia leucotreta GV (CrleGV), as well as the vp15 sequence of WSSV, were generated. Subsequently, PCR amplification using appropriate primer pairs (see Table S1 in the supplemental material) and either AcMNPV, ChocGV, or CypoGV DNA as a template generated the following series of modified p6.9 sequences: for AcMNPV, an isoleucine insertion after tyrosine 55 (Y55_X56insI), deletion of residues 50 to 55 (Y50_Y55del), deletion of residues 45 to 55 (T45_Y55del), and alanine substitution for residue 50 (Y50A), 53 (R53A), or 55 (Y55A); for ChocGV, deletion of valine 57 (V57del); for CypoGV, replacement of residues 41 to 47 by either 6 (H41_Y49delinsYRTRYY) or 11 (H41_Y49delinsTGRRSYRTRYY) C-terminal residues of AcMNPV P6.9 (Fig. 1B). All amplicons were cloned into pFB-GFP-p6.9, and the resulting donor plasmids were introduced into AcBacΔp6.9 via Tn7-mediated transposition (Bac-to-Bac; Invitrogen) (Fig. 1A). The fidelity of the bacmids generated was confirmed by PCR as described previously (52).
Transfection-infection assay.DNA (approximately 1 μg) of each recombinant bacmid was transfected (10 μl Cellfectin; Invitrogen) into 1.5 × 106Sf21 cells. Five days posttransfection (p.t.), cells were examined for GFP expression by fluorescence microscopy. An aliquot (500 μl) of the harvest supernatant was then used to infect a new batch of Sf21 cells (1.5 × 106 in 2 ml medium). At 72 h postinfection (p.i.), cells were again inspected for GFP expression.
One-step growth curves.For each recombinant virus, the production of budded virus (BV) was monitored by constructing a viral growth curve as follows. Sf21 cells (1.5 × 105 per well; 24-well plates) were infected at a multiplicity of infection (MOI) of 10 50% tissue culture infective dose (TCID50) units/cell and were then incubated (1 h, 27°C). After infection, the inoculum was removed, and the cells were washed three times (0.5 ml medium). At 0, 6, 12, 18, 24, 48, and 72 h p.i the infected cell supernatants were collected. For each time point p.i. and each virus sample, triplicate samples were generated. The concentration of infectious BVs in each sample was determined by an endpoint dilution assay on Sf21 cells (34).
Electron microscopy. Sf21 cells, collected at 72 h p.t. by centrifugation (2,000 × g, 10 min), were fixed (with 2.5% [wt/vol] glutaraldehyde in 0.1 M sodium phosphate, pH 7.2 [NaPi], for 16 h at 4°C), washed (twice in NaPi for 15 min each time), further fixed (1% OsO4 in NaPi, 2 h, room temperature [RT]), washed (as described above), and dehydrated (8 immersions [15 min each] in increasing concentrations of ethanol [30 to 100%]). Specimens were embedded in capsules and were polymerized (60°C, 48 h), and sections (thickness, 60 to 80 nm) were stained (2% [wt/vol] uranyl acetate, 15 min; lead citrate, 15 min) and observed by transmission electron microscopy (FEI Tecnai G2 microscope at 200 kV).
qPCR analysis of BV release and viral DNA replication.To isolate BV DNA virions, supernatants from harvested (0 and 48 h p.t.) cultures of Sf21 cells transfected with different bacmid DNAs (1.0 μg, 1.0 × 106 cells) were treated with equal volumes of polyethylene glycol 8000 (PEG 8000) (20% [wt/vol] in 1 M NaCl; 30 min, RT). Then 500-μl aliquots were collected by centrifugation (12,000 × g, 15 min), resuspended (20 μl H2O), and lysed by incubation (50°C, 1 h) in virus disruption buffer (80 μl; 10 mM Tris-HCl [pH 7.6], 10 mM EDTA, 0.25% sodium dodecyl sulfate [SDS]) and proteinase K (5 μl; 20 mg/ml). Lysed BVs were phenol extracted, and viral DNA was ethanol precipitated and resuspended (40 μl H2O). Total cellular DNA from pelleted and washed (three times, with 1 ml Grace's medium) Sf21 cells was isolated with a commercial system (Genomic DNA rapid isolation kit; BioDev, China) according to the manufacturer's instructions and was then incubated (4 h) with DpnI (10 U; New England Biolabs) to digest residual bacmid DNA. BV DNA (5 μl) and DpnI-treated cellular DNA (5 μl) were used as templates in quantitative PCR (qPCR) analyses, performed as described previously (49), to determine viral copy numbers.
Computer-assisted sequence analysis.Homologous sequences in the GenBank/EMBL databases were identified with the FASTA and BLAST programs (2, 35). Sequence alignments were performed with the ClustalW program (EMBL-European Bioinformatics Institute [http://www.ebi.ac.uk ]) and were edited with GeneDoc software (32). Putative nuclear localization signals were found with the PSORT II program (Human Genome Center, Institute for Medical Science, University of Tokyo, Tokyo, Japan) (http://psort.ims.u-tokyo.ac.jp ).
RESULTS
Disruption of p6.9 in an AcMNPV bacmid.Baculovirus P6.9 plays an important role in condensing the viral genome in the nucleocapsid (14, 22, 28). The binding of these small, positively charged proteins to DNA seems not to be sequence specific (22). However, P6.9 might still have a species-specific role in the viral assembly process. To investigate this possibility, the AcMNPV bacmid bMON14272 (27) was modified by deleting p6.9, generating AcBacΔp6.9, followed by insertion of either intact homologous sequences from other species or p6.9 sequences modified at their C termini.
Because p6.9 overlaps with late essential factor 5 (lef-5) in the AcMNPV genome (3), the p6.9 open reading frame (ORF) of the AcMNPV bacmid bMON14272 was only partially deleted to avoid potentially disrupting the function of lef-5. To confirm that this partial deletion had inactivated p6.9 and that its replacement with cat had not functionally compromised the adjacent lef-5 and p40 genes, AcBacΔp6.9 was fitted with cassettes from either the GFP-expressing but empty donor plasmid pFB-GFP-p6.9 or a derivative containing the complete AcMNPV p6.9 sequence under the control of the native p6.9 promoter. Although Sf21 cells transfected with either bacmid expressed GFP (Fig. 2 A and B, upper panels), infectious virions were produced only with the p6.9 rescue bacmid (Fig. 2A and B, lower panels). Electron microscopic analysis of p6.9-null AcMNPV-transfected cells showed that no nucleocapsids were formed (Fig. 3A). Instead, electron-lucent tubule-like structures were present in the nucleus. These structures were not present in cells transfected with the rescue bacmid, where normal nucleocapsid formation took place (Fig. 3B). Furthermore, the kinetics of virion production of the rescue bacmid, as determined by one-step growth curve analysis, was similar (P, >0.05 by a two-tailed Student t test) to that of the parental AcMNPV bacmid provided with the GFP cassette alone (Fig. 4A). Taken together, these data indicate that p6.9 was functionally inactivated by its partial deletion and that lef-5 and p40 were not affected by the presence of cat.
Transfection-infection assay for viral propagation. Sf21 cells were transfected with p6.9-null AcMNPV bacmids containing either no p6.9 gene (−) or native or modified p6.9 sequences of AcMNPV (Ac), SeMNPV (Se), HearNPV (Ha), NeleNPV (Nl), CrleGV (Cl), CypoGV (Cp), or ChocGV (Co), or the vp15 sequence of WSSV. Five days posttransfection, GFP-expressing cells were visualized by UV microscopy (upper panels). Supernatants from the transfected cells were used to infect Sf21 cells. The occurrence of GFP fluorescence, visualized 72 h p.i. (lower panels), indicates that infectious viruses were generated.
Electron microscopic observation of Sf21 cells transfected with p6.9-null AcMNPV (A) or p6.9-null AcMNPV rescued with AcMNPV p6.9 (B). Bars, 500 nm.
One-step growth curves. Sf21 cells were infected with the AcMNPV bacmid (parental) or with p6.9-null AcMNPV containing AcMNPV (Ac), SeMNPV (Se), HearNPV (Ha), or NeleNPV (Nl) p6.9 (A) or with p6.9-null AcMNPV containing either AcMNPV (Ac) p6.9 or CypoGV p6.9 with the H41_Y49delinsTGRRSYRTRYY modification (B) at an MOI of 10 TCID50/cell. Supernatants were harvested at the indicated times p.i., and viral titers were determined by endpoint dilution assays. Each data point represents the average for three independent infections. Error bars represent the standard errors of the means.
P6.9 proteins from alpha- and gammabaculoviruses rescue p6.9-null AcMNPV.Recently, a new baculovirus classification has been proposed, consisting of four genera: Alphabaculovirus (lepidopteran NPVs), Betabaculovirus (lepidopteran granuloviruses [GVs]), Gammabaculovirus (hymenopteran NPVs), and Deltabaculovirus (dipteran NPVs) (21). Based on phylogenetic criteria and the type of envelope fusion protein (GP64 or F), the genus Alphabaculovirus can be phylogenetically subdivided further into groups I and II, respectively (8, 15-17). To investigate whether p6.9 genes from phylogenetically distinct baculoviruses could rescue p6.9-null AcMNPV (Alphabaculovirus, group I), the p6.9 proteins of the group II Alphabaculovirus species SeMNPV and HearNPV, the betabaculoviruses CrleGV, CypoGV, and ChocGV, and the gammabaculovirus NeleNPV were inserted into the p6.9-null AcMNPV bacmid. In all these constructs, the p6.9 genes were under the control of the AcMNPV p6.9 promoter. Transfection of Sf21 cells with the p6.9-null AcMNPV bacmids carrying SeMNPV, HearNPV, or NeleNPV p6.9 resulted in the production of infectious virions (Fig. 2C to E), while those bacmids carrying betabaculovirus p6.9 genes from CrleGV, CypoGV, or ChocGV did not rescue p6.9-null AcMNPV (Fig. 2F to H).
In addition to these baculovirus p6.9 genes, the homolog vp15 (56) from an unrelated Decapoda virus, WSSV, was inserted into the p6.9-null AcMNPV bacmid. Despite the fact that VP15 is able to bind to AcMNPV DNA (56), it did not functionally substitute for P6.9 in the generation of infectious BV (Fig. 2I).
For the rescued viruses, one-step growth curves were made to determine whether the heterologous p6.9 genes had any effect on the kinetics of BV production (Fig. 4A). Surprisingly, only the virus carrying HearNPV p6.9 was significantly impaired in BV production at 72 h p.i. (P, <0.05 by a two-tailed Student t test), whereas virus production with p6.9 from SeMNPV or the more phylogenetically distinct NeleNPV (Gammabaculovirus) was similar (P, >0.05 by a two-tailed Student t test) to that of AcMNPV p6.9.
qPCR analysis of BV release and viral DNA replication.The p6.9-null AcMNPV bacmids that either had no gene (Δp6.9) or carried Betabaculovirus p6.9 or WSSV vp15 did not produce infectious BVs. However, to test whether noninfectious BVs are still produced, their respective viral copy numbers in cell culture supernatants at 0 and 48 h p.t. were determined by qPCR and compared with those for the p6.9 rescue bacmid and a previously constructed (49) gp64-null AcMNPV bacmid (Δgp64) that is unable to produce BVs (Fig. 5A) (30). At 48 h p.t., a significant increase in the number of viral DNA copies over that at 0 h p.t. was found only for the p6.9 rescue bacmid (P, <0.05 by a one-tailed Student t test). This demonstrates that neither infectious nor noninfectious BVs were produced with the p6.9-null AcMNPV bacmids carrying Betabaculovirus p6.9 or WSSV vp15. As a control, the numbers of viral DNA copies in the transfected cells were also analyzed by qPCR (Fig. 5B). For all bacmids, transfected cells contained significantly increased copy numbers (P, <0.05 by a one-tailed Student t test), indicating that the presence of P6.9 per se is not essential for viral replication.
Quantification of viral genome copies in supernatants (A) and Sf21 cells (B) at 0 and 48 h after transfection with p6.9-null AcMNPV bacmids containing either no p6.9 gene (−), the p6.9 gene of AcMNPV (Ac), CrleGV (Cl), ChocGV (Co), or CypoGV (Cp), or the vp15 gene of WSSV, or with a gp64-null AcMNPV bacmid (ΔGP64). Data are expressed as relative frequencies, calculated by dividing each data point by the mean at 0 h. Error bars represent 1 standard deviation. Data were analyzed by a one-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Each data point represents the average for three experiments.
The AcMNPV P6.9 C-terminal domain is important for virion production.Baculovirus P6.9 proteins, like protamines, are a rather heterogeneous protein class. Homology searches and comparisons with FASTA and BLAST programs revealed significant amino acid identities only among P6.9 proteins from closely related baculoviruses. Consequently, our P6.9 alignments (see the supplemental material) are based more on aligning conserved arginine and serine residues than on global similarities. However, the C-terminal domains of P6.9 proteins have a greater degree of identity within the baculovirus genera. For example Alpha-, Gamma-, and Deltabaculovirus P6.9 proteins terminate at a tyrosine (Fig. 1C), while those of Betabaculovirus contain an extra valine or isoleucine (Fig. 1C; see also the supplemental material). To explore whether these additional C-terminal hydrophobic amino acids in Betabaculovirus P6.9 proteins interfere with BV production by AcMNPV, two AcMNPV mutants, one expressing native AcMNPV P6.9 but with an additional isoleucine at the extreme C terminus (Y55_X56insI) and the other expressing ChocGV P6.9 with a deletion of the C-terminal valine (V57del), were constructed (Fig. 1B) and transfected into Sf21 cells (Fig. 2J and K, upper panels). BVs were produced from Sf21 cells transfected with AcMNPV expressing P6.9 bearing the additional C-terminal isoleucine (Fig. 2J, lower panel) but not from those bearing the C-terminally modified ChocGV P6.9 (Fig. 2K, lower panel), demonstrating that, at least in these two cases, the presence or absence of a C-terminal hydrophobic amino acid had little effect on P6.9 function.
In the Alpha- and Gammabaculovirus P6.9 C termini, the YxxRxY sequence is highly conserved (Fig. 1C; see also the supplemental material). To investigate whether these conserved amino acids are important for BV production, AcMNPV p6.9 sequences with single alanine substitutions at each conserved residue (Y50A, R53A, and Y55A) were made (Fig. 1B) and inserted into the p6.9-null AcMNPV bacmid. None of the three substitutions disrupted the formation of infectious virions (Fig. 2L to N). To take this one step further, two AcMNPV p6.9 constructs were made, one with a deletion of the 6 C-terminal amino acids (Y50_Y55del) and one with a deletion of 11 C-terminal amino acids (Y45_Y55del) (Fig. 1B and C). Despite the fact that the six deleted C-terminal amino acids included the three conserved amino acids Y50, R53, and Y55, the modified P6.9 construct Y50_Y55del was able to rescue p6.9-null AcMNPV (Fig. 2O). In contrast, deletion of an extra five, more poorly conserved residues, compromising a TGRRS sequence, resulted in a complete block of BV production (Fig. 2P).
These results imply that this 11-residue stretch of amino acids at the extreme C terminus of AcMNPV P6.9 is important for the formation of infectious AcMNPV virions. To further investigate the role of these conserved C-terminal residues, p6.9-null AcMNPV bacmids fitted with CypoGV p6.9 genes encoding P6.9 variants in which residues 41 to 47 were replaced by either the 6 (H41_Y49delinsYRTRYY) or the 11 (H41_Y49delinsTGRRSYRTRYY) C-terminal residues of AcMNPV P6.9 were transfected into Sf21 cells (Fig. 2Q and R, upper panels). The H41_Y49delinsYRTRYY modification did not rescue the loss of AcMNPV p6.9 (Fig. 2Q, lower panel), but interestingly, infectious AcMNPV virions were formed with the H41_Y49delinsTGRRSYRTRYY C-terminal modification (Fig. 2R, lower panel). However, a one-step growth curve for this virus revealed that it produced 10 to 60 times fewer infectious viruses over time (24 to 72 h) (P, <0.05 by a two-tailed Student t test) (Fig. 4B). These results indicate that the C-terminal region of AcMPNV P6.9 contains residues crucial for AcMNPV virion production that are present in Alpha- and Gammabaculovirus but absent in Betabaculovirus P6.9 proteins. Furthermore, the decreased virion production observed with the CypoGV P6.9 protein with the H41_Y49delinsTGRRSYRTRYY C-terminal modification suggests that Betabaculovirus P6.9 lacks additional important amino acid residues or domains associated with virion formation.
DISCUSSION
Protamines are typically short (50- to 110-amino-acid [aa]) proteins containing a high density of positively charged amino acids, most notably arginines (24). Although protamines lack significant secondary structure in solution, upon interaction with DNA, they readily wrap themselves into the major groove of the DNA helix (4, 5, 9, 19). Playing a functionally similar role, baculovirus P6.9 is a small, heterogeneous arginine- and serine-rich protein, ranging from 49 to 109 aa (Fig. 1C; see also the supplemental material), that, in its dephosphorylated form, is believed to bind to and condense viral DNA during nucleocapsid assembly and virion packaging (14, 22, 28, 54) in an apparently sequence independent manner (22). In this study, we investigated whether P6.9 might contain domains that interact with other components specific to the species or genus during viral assembly.
We demonstrate, by deletion of p6.9 from the Alphabaculovirus type species AcMNPV (21), that P6.9 is an essential gene for viral propagation (Fig. 2A and 4A). Electron microscopic observations of p6.9-null AcMNPV-transfected cells showed that viral nucleocapsids were not formed (Fig. 3A). Instead of nucleocapsids, electron-lucent tubular structures were present in the nuclei of p6.9-null AcMNPV-transfected cells. These tubular structures might be malformed nucleocapsids, in view of the fact that similar structures have been found previously with a non-BV-producing orf103-null and 38k-null AcMNPV (44, 57). Furthermore, the genome copy number still increases over time, indicating that P6.9 is not essential for viral replication (Fig. 5B). The replication efficiency seems to be higher in the presence of AcMNPV P6.9 (Fig. 5B). This is probably due to the ability to have secondary infections within 48 h, as evidenced by the fact that the control gp64-null AcMNPV bacmids, which are not able to form BVs, have replication efficiencies similar to that of the p6.9-null AcMNPV bacmid.
The defect in the production of infectious virions could be rescued by reinsertion of p6.9 from baculoviruses belonging to the genera Alpha- and Gammabaculovirus (Fig. 2B to D). Among P6.9 proteins from these two genera, only the insertion of the HearNPV p6.9 into p6.9-null AcMNPV gave a significant decrease in the titer at 72 h p.i. (Fig. 4A). In contrast, although betabaculoviruses are phylogenetically more closely related to alphabaculoviruses than are gammabaculoviruses, P6.9 proteins from betabaculoviruses were not capable of rescuing p6.9-null AcMNPV (Fig. 2E to G and 4A). This suggests that, in this particular case, Betabaculovirus P6.9 may have evolved to interact specifically with a Betabaculovirus-specific nucleocapsid protein during viral assembly.
The inability of Gammabaculovirus P6.9 to rescue p6.9-null AcMNPV is presumably not due to dysfunctional translocation of the protein to the nucleus, since these proteins contain multiple nuclear localization signals. Most likely, P6.9 needs to interact with one or more nucleocapsid proteins during viral assembly. The proteins VP39, VP91, VP1054, and Vlf-1 are good candidates, since they are present in the nucleocapsids of all baculoviruses (7, 46) and are important for viral assembly (26, 33, 38, 43). Baculovirus P6.9 proteins are a rather heterogeneous group of proteins, but their C-terminal domains are conserved within the genera and therefore may contain domains designed to interact specifically with other viral proteins. Neither mutations in the conserved C-terminal domain YxxRxY of AcMNPV P6.9 nor its complete deletion abolished the ability to rescue p6.9-null AcMNPV (Fig. 2J to O). However, deletion of 5 additional amino acids, which are less conserved, eliminated BV production (Fig. 2P), whereas a CypoGV P6.9 protein whose C-terminal domain was replaced with these 11 C-terminal amino acids of AcMNPV P6.9 rescued the p6.9-null AcMNPV phenotype (Fig. 2R). These results support the view that the C terminus of P6.9 is important for viral assembly and, to a certain degree, acts in a sequence-specific manner within the genera. The latter hypothesis could be tested by future protein-protein interaction studies using P6.9 as the bait. The amount of infectious virus produced with the hybrid P6.9 was 60 times lower than that produced with the native AcMNPV P6.9 (Fig. 4B); this could imply that Betabaculovirus P6.9 lacks additional important crucial amino acids or domains or interacts erratically with alpha- and gammabaculoviruses.
The majority of dsDNA viruses infecting Eukaryota use cellular histones or virally expressed protamine-like proteins to condense their DNA (6, 13, 37, 38, 40), while baculoviruses have evolved a different and more compact way to condense their genomes by expressing proteins homologous to protamines. P6.9 proteins, though, lack cysteine residues used to link protamines together by multiple disulfide bridges in spermatids of eutherian mammals (4). GenBank comparisons with FASTA and BLAST programs showed that P6.9 proteins are, however, more similar in amino acid composition to the protamines of squids and marsupials, which also lack cysteine residues (data not shown).
Baculoviruses are not the only dsDNA viruses using protamine-like proteins to condense their genomes. The white spot syndrome virus (WSSV) uses a similar protein, named VP15 (56), which is one of its major nucleocapsid proteins (45). VP15 is 31% identical to AcMNPV P6.9 and has been shown to bind both WSSV and AcMNPV DNA (56). Despite its homology, VP15 was not able to rescue the p6.9-null AcMNPV (Fig. 2I), probably because it is similar to the betabaculovirus P6.9 proteins lacking the C-terminal domain needed for AcMNPV virion assembly. Construction and testing of a VP15 hybrid with the C-terminal end of AcMNPV P6.9 would prove this point. Another striking difference that might explain the inability of VP15 to assemble AcMNPV BVs is the fact that VP15 is rather lysine rich (21%), while the majority of P6.9 proteins contain few or no lysines (maximum, 7%). VP15 is detected only in a dephosphorylated form in WSSV-infected shrimp tissue, as well as when overexpressed in insect cells (45, 56). This could suggest that VP15 uses a different mode of action to bind to and be released from DNA.
The genomes of Heliothis zea virus 1 and Gryllus bimaculatus nudivirus also encode a protamine-like protein (10, 50). These viruses were previously called “nonoccluded baculoviruses.” At the moment they are unclassified (12), but since they share a few common genes with baculoviruses, including the P6.9 gene, it has been proposed that they be placed in the genus Nudivirus within the Baculoviridae (51). Chilo iridescent virus (CIV), also known as invertebrate iridescent virus 6, contains two genes encoding protamine homologs (20). No experimental data as to whether CIV uses these proteins to condense its genome are available, but so far it looks as though the use of protamine-like proteins in viral DNA packaging is evolutionarily conserved and also includes those dsDNA viruses infecting invertebrates of the phylum Arthropoda.
ACKNOWLEDGMENTS
We thank D. Zuidema (Wageningen University, Netherlands) for advice during the research, B. Arif (Great Lakes Forestry Centre, Sault Ste. Marie, Canada) for kindly providing the ChocGV HindIII-E and NeleNPV HindIII-D constructs, J. A. Jehle (State Education and Research Center for Agriculture, Viticulture and Horticulture [SLFA], Neustadt an der Weinstraße, Germany) for kindly providing genomic CypoGV and CreleGV DNA, G. P. Pijlman for constructing pFB1-p6.9, M. F. Boyong for assistance as an undergraduate student, and C. T. Dolphin (King's College, London, United Kingdom) for editing the manuscript.
This research was supported by grants from the Royal Netherlands Academy of Arts and Sciences (KNAW) and the Chinese Academy of Science (CAS) (Program Strategic Scientific Alliances projects 04-PSA-BD-02 and 2004CB720404) and by Intervet International BV, Boxmeer, Netherlands (WSSV).
FOOTNOTES
- Received 13 January 2010.
- Accepted 21 May 2010.
- Copyright © 2010 American Society for Microbiology
