Construction of the DlEPV EcoRI library

Details of the EcoRI DlEPV DNA library construction and sequencing of cloned fragments have been described (Lawrence 2002). Briefly, DlEPV DNA was extracted from virions that were harvested from female wasp venom glands and purified by sucrose density gradient centrifugation (Lawrence 2002). Upon digestion with EcoRI (Roche Molecular Biochemicals,  www.roche.com), the resulting DlEPV DNA fragments were cloned into the pBluescript® II KS (+/-) cloning vector (pBS; Stratagene,  www.stratagene.com ) using T4 DNA ligase (Roche) and the manufacturer's and standard (Sambrook et al. 1989) protocols. The clones were used to transfect supercompetent DH5-α Escherichia coli cells (Gibco-BRL,  www.lifetech.com/ www.invitrogen.com), amplified, and selected on ampicillin - Xgal (Gibco- BRL) agar plates at 37 °C for 18 h as previously described (Lawrence 2002). Recombinant plasmids were isolated from bacterial cells by alkaline lysis (Sambrook et al. 1989) and the presence of the DlEPV DNA inserts verified by EcoRI digestion and subsequent electrophoresis (Lawrence 2002). The clones (RI) were arbitrarily numbered and the RI-1 clone was selected for further analysis.

DNA labeling, hybridization, and detection

To verify the fidelity of the RI-1 DNA insert to the DlEPV genome, a 3 µg sample of the isolated insert was labeled with digoxigenin (DIG) by random priming using the DIG-High Prime® labeling protocols (Roche). DlEPV genomic DNA was digested with EcoRI, HindIII, and BamHI (Roche) and the resulting fragments electrophoresed into a 0.8% agarose gel at 30 V for 18 h and transferred to nitrocellulose membrane by the capillary method. The DNA was then fixed to the membrane by UV cross-linking at 50 mJoules. The blot was probed with 100 ng of the DIG-RI-1 insert diluted in 5 µl hybridization buffer [5X SSC (750 mM NaCl, 75 mM sodium citrate solution, pH 7.0), 0.1% (w/v) N-lauroylsarcosine, 0.2% (w/v) SDS, 1% blocking reagent (Roche)] at 65°C for 16 h. Hybridization was followed by two 5 min washes at RT with 2x washing buffer (2x SSC, 0.1% SDS) and two 15 min washes with 0.5x washing buffer. The hybridization signal was visualized using the DIG chemiluminescent detection protocol and exposure to LumiFilm (Roche).

Sequencing of the open reading frame within the DlEPV RI-1 clone

Forward and reverse sequencing of the open reading frame within the RI-1 clone were accomplished by primer walking, with fluorescence-labeled dideoxynucleotides and Taq DyeDeoxy terminator cycle sequencing protocols (Applied Biosystems, Perkin-Elmer Corp.,  home.appliedbiosystems.com) and the extension products analyzed with a model 377A DNA sequencer (Applied Biosystems), as previously described (Lawrence 2002). Sequences were assembled and further analyzed with the Sequencher 3.0 software (Gene Codes Corp.,  www.genecodes.com).

Sequence analysis of the RI-1 open reading frame

The amino acids deduced from the partial sequence of RI-1 by the Sequencher program were compared with homologs in the GenBank, PIR, and SWISS-PROT databases using the Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990). A multiple sequence alignment of the RI-1 open reading frame protein and its homologs was performed using the CLUSTALW 1.81 program (Thompson et al. 1994), with gap initiation and extension penalties of 10 and 0.2, respectively. Aligned sequences were imported into the Phylogenetic Analysis Using Parsimony (PAUP*®) program (Swofford 1998) to generate a phylogenetic tree using the neighbour joining method and 1,000 bootstrap trials to assess tree reliability. Pairwise comparisons of the DlEPV RI-1 open reading frame nucleotides and deduced amino acids with those of homologs identified by BLAST, were expressed as percent nucleotide identities, amino acid identities, or amino acid similarities [identities + homologous (conservative, sensuMount 2001) substitutions].

Figure 1.

Electrophoretic analysis of the EcoRI digested DlEPV RI-1 clone. A 75 µl aliquot of the digested clone was applied to the gel. DNA fragment sizes were verified using a BioRad® λ high molecular weight DNA size standard (λ). The upper band corresponds to the RI-1 insert of approximate 4.5 kb. The lower band is the pBluescript® cloning vector of 2.96 kb.

Rifampicin-like proteins occur in other large DNA non-poxvirus families including the insect-infecting Iridoviridae and Ascoviridae (Iyer et al. 2001; Stasiak et al. 2001 Stasiak et al. 2003). Thus pairwise amino acid comparisons, separate from those made with the poxviruses, were performed between the RIF sequence of DlEPV, orthologs/homologs from the insect iridovirus IIV-6, the Diadromus pulchellus ascovirus 4a (DpAV4a) from a parasitic wasp of the same name, and other non-pox DNA viruses.

Purification, sequencing and analysis of the RI-1 insert

The size of the RI-1 insert was verified to be ∼ 4.5 kb (Figure 1). Hybridization of the DIG-probe to the insert and the restricted DlEPV genomic DNA in the Southern blot, verified their fidelity to the DlEPV genome (Figure 2). The single hybridized fragment, with the same size as the positive control (∼4.0), obtained with the EcoR1 digested genomic DNA confirmed the absence of an EcoR1 restriction site within the fragment (Figure 2). The four bands detected in blots of the HindIII digest (Figure 2) were also consistent with the presence of three HindIII sites within the sequence (Figure 3). Although no BamHI sites (therefore one band) were predicted, two bands were observed (Figure 2), suggesting the presence of a second site in the unsequenced portion of the clone. Sequencher also predicted XbaI, DraII, SpeI, and Bsp106 restriction sites within the RI-1 fragment (Figure 3) but these enzymes were not evaluated.

Figure 2.

Autoradiograph of Southern hybridization of digested DlEPV genomic DNA with a 4.5 kb specific probe generated from the DlEPV R1-1 insert. Lanes 1–2: empty; Lane 3: 1 µl of the DlEPV R1-1 undigested 4.5 kb insert (positive control); Lane 4: 2 µl salmon sperm DNA (negative control); Lane 5: 5 µl EcoRI digested DlEPV genomic DNA; Lane 6: 5 µl HindIII digested DlEPV genomic DNA; Lane 7: 5 µl BamHI digested DlEPV genomic DNA.

The sequenced portion of the RI-1 fragment was determined by Sequencher to contain one complete open reading frame of 1,640 bases, encoding a putative protein of 546 amino acids and an apparent partial open reading frame. The rif open reading frame had 529 bases (5′) and 174 bases (3′) immediately flanking its translational start and stop codons, respectively (Figure 3). Thus, the sequenced portion of R1-1 comprised 2.34 kb (GeneBank accession # EF541029) of the ∼4.5 kb R1-1 insert. The analyses below will focus only on the complete open reading frame and sequences immediately flanking it (Figure 3).

The translation initiation codon (ATG) of the open reading frame starts at 530 nucleotides from the 5′ end of the fragment and the translational stop codon (TAA) starts at 2,168 nucleotides (Figure 3). Immediately preceding the translational initiation codon is a highly A/T rich (87%) 30 nucleotide sequence. Three of these bases immediately preceding the ATG and in combination with it, form the consensus poxvirus late transcriptional start signal (TAAATG) (Rosel et al. 1986; Moss 1996, 2001) (Figure 3). Potential poxvirus early transcription termination signals (TTTTTnT) occur at 236 and 315 nucleotides upstream of the late translational start codon and 168 nucleotides downstream of the translational stop codon of the open reading frame (Figure 3).

Alignment of all deduced poxvirus sequences revealed almost no conserved amino acids within the first 253 amino acids of the DlEPV sequence, except for a short region [LPE(I)/(V)KG] between amino acids 53–58 in which valine was substituted in the chordopoxviruses for isoleucine in the entomopoxviruses (Figure 4a). Two additional motifs, HTN(L)/(I)/(V)L(M)/(V)/(S)F(GT)/(SR)/(TR)R and GD(N)/(L)RS, occur within DlEPV amino acids 326–370 (region I) and 383–441 (region II) respectively (Figure 4a). These regions of 43 and 58 amino acids have ∼28 and 26% conserved residues respectively, and correspond to the same two regions in the H. armigera entomopoxvirus RIF that had 56 and 53% conserved amino acids respectively, when that virus was aligned with vaccinia and swinepox (Osborne et al. 1996). When only entomopoxviruses were aligned, the conserved amino acids in regions I and II of the DlEPV RIF increased to ∼44 and 38% respectively (Figure 4b). Interestingly, when each entomopoxvirus sequence was individually aligned with DlEPV, the percent conserved residues increased even further to as high as 79 and 41% in regions I and II respectively (alignment not shown). In addition at least 10% of 40 residues at the N-terminus and 20% of 50 residues toward the C-terminus were conserved between DlEPV and each of the other (beta) entomopoxviruses (data not shown).

Table 1.

Pairwise comparison of amino acids and nucleotides of the rifampicin resistance homologs of DlEPV and other poxviruses. The lower left triangle represents the percent similarities (= amino acid identities plus homologous substitutions). Numbers in parentheses represent percent amino acid identities. The upper right triangle represents percent nucleotide identities.

Figure 3a.

Locations of restriction enzyme recognition sites within a ∼2.54 kb sequenced portion of the RI-1 DNA fragment predicted by the Sequencher 3.0 program.

Regions I and II had motifs common to both chordopoxviruses and entomopoxviruses but contained substitutions that distinguished the two virus subfamilies (Figure 4a). A closer analysis of the entomopoxviruses revealed that within the motif in region I, DlEPV had a single substitution that distinguished it from the betaentomopoxviruses (Figure 4b). However, all residues in the motif in region II were conserved among all entomopoxviruses (Figure 4b).

Figure 3b.

DNA sequence of the RI-1 open reading frame and an immediately preceding region (539 nt) containing putative poxvirus early transcriptional stop (TTTTTnT) and late promoter (TAAATG) sequences (highlighted in black). Restriction enzyme recognition sites, shown in (a), are underlined. The putative translational stop codon (TAA) is indicated by an asterisk (*). The sequence has been assigned GeneBank accession # EF541029.

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Figure 4a.

ClustalW 1.81 multiple sequence alignment of the deduced amino acid sequence of the putative rifampicin resistance protein homologs from Amsacta moorei entomopoxvirus (AmEPV), Heliothis armigera entomopoxvirus (HaEPV), Melanoplus sanguinipes entomopoxvirus (MsEPV), Molluscum contiguosum poxvirus (MOLCV), swinepox virus (SPV), Myxoma poxvirus (MYXV), vaccinia virus (VACV), variola virus (VARV), and Diachasmimorpha longicaudata entomopoxvirus (DlEPV). A colon (:) represents amino acid homologous (“conservative”, sensu Mount 2001) substitutions. A period (.) identifies amino acid non-homologous substitutions. Asterisks indicate identical amino acids conserved in all sequences. Underlined sequences represent regions I and II in HaEPV and DlEPV with the highest percent conserved amino acids previously identified for HaEPV by Osborne et al. (1996). For the three motifs identified within the RIF sequence, Blue = conserved in all poxviruses; Red = conserved only among chordopoxviruses; Green = conserved only among EPVs. Other colors = conserved in some members of a subfamily.

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Figure 4b.

ClustalW 1.81 multiple sequence alignment of the deduced amino acid sequence of a selected region of the putative rifampicin resistance protein homologues from entomopoxviruses, showing regions I and II (underlined in HaEPV and DlEPV) of highest percent conserved sequences (Osborne et al. 1996) and their component motifs. Virus names, symbols, and color codes are as described in Fig. 4a.

Overall, pairwise comparison of amino acids of DlEPV RIF with each homolog revealed that DlEPV shared slightly more amino acid identities with the betaentomopoxviruses than with chordopoxviruses (Table 1). However, the betaentomopoxviruses shared 1.5–2 times more amino acids among themselves than they did with DlEPV and the lepidopteran entomopoxviruses shared more with each other than they did with the M. sanguinipes entomopoxvirus (Table 1). The percent similarities between DlEPV and all poxvirus RIF sequences and between the betaentomopoxviruses and chordopoxviruses were about the same (on average ∼44%) (Table 1). However, similarities among the betaentomopoxviruses were 1.5– 2 times higher than with DlEPV. The lepidopteran entomopoxviruses had greater similarity with each other than with the M. sanguinipes entomopoxvirus (Table 1).

The nucleotides conserved between DlEPV and the betaentomopoxviruses were 1.5 to > 5X fewer than those conserved among the betaentomopoxviruses themselves, with the lepidopteran entomopoxviruses sharing more with each other than with the M. sanguinipes entomopoxvirus (Table 1). Nevertheless, both DlEPV and the betaentomopoxviruses had few (0-≤ 20%) nucleotide identities with the chordopoxviruses, except in the case of the A. moorei entomopoxvirus and swinepox (Table 1). Thus, the DlEPV putative RIF protein is closer to (but distinct from) homologs of the lepidopteran and orthopteran entomopoxviruses than to those of chordopoxviruses (Table 1). This is further seen in the phylogenetic tree that assigns DlEPV to a different clade from the M. sanguinipes entomopoxvirus and from the H. armigera and A. moorei entomopoxviruses (Figure 5). DlEPV had ∼20% and 26.4% similarity respectively, with IIV-6 and DpAV4a, two non-pox double stranded DNA viruses of insects ≤22.96 with non-pox double stranded DNA viruses of other organisms (Table 2).