Nucleotide sequence analysis of the ClbiNPV genome
The genome of ClbiNPV has a size of 135,454 bp [GenBank: DQ504428
], slightly smaller than that of Spodoptera exigua
NPV (SeMNPV, 135,611 bp) [15
]. ClbiNPV has a highly AT rich genome. Its overall G+C content is 37%, similar to that recorded for Agrotis segetum
GV (AgseGV) and Ecotropis obliqua
NPV (EcobNPV) [16
], and higher only than those of Adoxophyes honmai
NPV (AdhoNPV, 35%) [17
] and Adoxophyes orana
NPV (AdorNPV, 34%) among the Alphabaculovirus (see Additional file 1
According to convention [18
], the adenine residue at the translational ATG start codon of the polyhedrin gene (polh
) was considered to be nucleotide number 1 of the genome, and successive nucleotides were numbered in the direction of the polh
gene (see Additional file 2
). Analysis of the ClbiNPV genome sequence led to the identification of 139 putative ORFs with 50 or more amino acids and minimal overlapping of adjacent ORFs. There are 60 ORFs with the same orientation as the polyhedrin gene, and 79 with the reverse orientation. Within 150 bp upstream of the ATG start codon, 34 ClbiNPV ORFs have baculovirus early promoter motifs (CAGT), 51 have late promoter motifs (TAAG), and 29 carry both these motifs.
Of the 139 ClbiNPV ORFs identified, 126 are homologous to at least one other baculovirus, and the 30 core genes that are probably shared by all baculoviruses are conserved in the ClbiNPV genome [19
]. Thirteen ORFs (Clbi5, Clbi6, Clbi18, Clbi31, Clbi35, Clbi42, Clbi47, Clbi49, Clbi56, Clbi57, Clbi70, Clbi75 and Clbi129) are unique to ClbiNPV; they account for 9% of the whole genome. Three baculovirus-repeated ORFs (bro
genes) were identified in ClbiNPV (ORF55, 115 and 131) and were designated bro-a
, respectively, based on their order in the genome. No typical homologous regions (hrs) were detected in ClbiNPV, which is similar to Chrysodeixis chalcites
NPV (ChchNPV). The relative locations of these ORFs are shown diagrammatically in linear format in Figure . Their orientations, sizes and other details are shown in Additional file 2
Figure 1 Linear map of the 139 predicted ORFs for the complete ClbiNPV genome. Arrows indicate ORFs and the direction of transcription. The names of putative genes are shown above or below the arrows. Numbers refer to the nucleotide position in kb (kilobases) (more ...)
Phylogenetic analyses and gene content
The sequences of individual baculovirus genes such as polyhedrin/granulin (polh
), DNA polymerase
have previously been used for phylogenetic analysis [1
]. Among those conserved baculovirus genes, pif
in AcMNPV), encoding a per os
infectivity factor, and lef
-8, encoding a subunit of the baculovirus RNA polymerase, proved to be particularly reliable baculovirus markers for phylogenetic analyses at the virus family level [2
]. A combined phylogenetic analysis of the pif
-2 and lef
-8 sequences further showed a clear, highly-supported classification among the four genera, and Alphabaculoviruses (lepidopteron-specific NPV) can be subdivided into Groups I and II. Phylogenetic analysis placed ClbiNPV in Group II (Figure ). ORF 139 in the ClbiNPV genome encodes a typical F protein, and ClbiNPV does not encode GP64, which is consistent with its classification as a Group II Alphabaculovirus.
Figure 2 Phylogenetic analysis using the predicted amino acid residues of 48 baculovirus PIF-2 and LEF-8 proteins. An NJ (neighbor-joining) tree is shown. Numbers above or below the nodes are bootstrap values showing the statistical reliability of bootstrapping (more ...)
The gene order of ClbiNPV was compared to AcMNPV [12
], OrleNPV and LdMNPV [20
] by gene parity plots [21
]. ClbiNPV shared 102, 108 and 101 ORFs with AcMNPV, OrleNPV and LdMNPV, respectively. In general, gene order is conserved between ClbiNPV and LdMNPV, and between ClbiNPV and OrleNPV, although three inverted areas, involving Clbi25-32, Clbi43-60 and Clbi132-139, are identified when compared with OrleNPV. The genomes appear less collinear than AcMNPV (Figure ).
Pairwise comparison of gene content and position of ClbiNPV with AcMNPV, OrleNPV and LdMNPV using gene parity plot analysis. Genes present in only one of the two viruses in the pair-wise comparison appear on the X or Y axes.
Overlapping ORF pairs in the sequenced Alphabaculovirusgenomes
Overlapping ORFs in ClbiNPV were searched and 26 pairs were found. The overlapping ORF pairs in all sequenced Alphabaculovirus genomes were further analysed (see Additional file 3
In Group I Alphabaculoviruses, the numbers of overlapping ORF pairs range from 21 (BmNPV) to 42 (AgMNPV). Except for ac68/ac69 and ac73/ac74 in OpMNPV, and ac43/ac44 in HycuNPV, nine (ac43/ac44, ac68/ac69, ac73/ac74, ac80/ac81, ac81/ac82, ac82/ac83, ac95/ac96, ac98/ac99 and ac102/ac103) appear in all Group I Alphabaculoviruses.
In Group II Alphabaculoviruses, the numbers of overlapping ORF pairs range from 18 (EcobNPV and OrleNPV) to 42 (AgipMNPV). Except for several NPVs, six overlapping ORF pairs (ac53a/ac54, ac57/ac59, ac67/ac68, ac80/ac81, ac82/ac83, ac89/ac90) are conserved, and four pairs (ac81/ac82, ac95/ac96, ac98/ac99 and ac102/ac103) exist in all Group II Alphabaculoviruses.
Altogether, overlapping ORF pairs ac81/ac82, ac95/ac96, ac98/ac99 and ac102/ac103 were conserved in all Group I and Group II Alphabaculoviruses. These overlapping ORF pairs are from the coding regions of ac81, tlp20, helicase, ac96, 38K, lef-5, p12 and p45, most of which have conserved functions in NPVs.
It was interesting that the numbers of overlapping ORFs in all Alphabaculoviruses were consistent with the phylogenetic tree constructed using the combined pif-2 and lef-8 sequences, meaning that closely-related NPVs have similar numbers of overlapping ORF pairs.
DNA photolyase-like gene sequence with a 1-bp deletion
DNA photolyase is a monomeric protein that directly repairs lethal and carcinogenic UV-induced DNA lesions. It has been found in a variety of pathogens and other organisms [22
]. However, among the baculoviruses, this enzyme exists only in ChchNPV [23
], Trichoplusia ni
NPV (TnSNPV) [25
] and Spodoptera litura
The complete ClbiNPV genome sequence analysis revealed the presence of a photolyase-like gene sequence 1,694 bp in length, which has the highest similarity to ChchNPV phr-2 using translated BLAST searches. Compared with photolyases among baculoviruses, it is interesting that there was a 1-bp deletion mutation (Figure ). In order to confirm this deletion, we analyzed the DNA fragment library of ClbiNPV. There were seven colonies including the region with the mutation, and they all contained the 1-bp deletion. To ensure that this mutation was genuinely present in the original sample, PCR and sequencing of the gene region around this mutation were performed using the original ClbiNPV DNA. The sequencing results confirmed that the mutation occurred in this region. This 1-bp deletion mutation disrupted the sequence into two small ORFs, labelled phr-1 (Clbi58) and phr-2 (Clbi59) on the basis of their position in the ClbiNPV genome relative to the polyhedrin gene (Figure ). The ORF of Clbiphr-1, corresponding to the 3' end of ChchNPV phr-2, is 306 bp long and encodes a polypeptide of 101 amino acids with a predicted molecular mass of 12.1 kDa. An early baculoviral transcription initiation motif (CAGT) was found 48 bp upstream of the putative translational start site (ATG), suggesting that Clbiphr-1 might be an early gene. In addition, a GATA motif (TGATAA) was found at position -129 bp relative to the translational start codon and might be involved in the transcriptional regulation of this gene. Clbiphr-2, corresponding to the 5' portion of ChchNPV phr-2, is 1,119 bp long and encodes a polypeptide of 372 amino acids with a predicted molecular mass of 42.0 kDa. No motifs characteristic of early (CAGT) or late (TAAG) baculovirus transcription initiation were found in the sequence upstream of Clbiphr-2. Two poly(A) motifs (AATAAA) are present at the end of the ORF of Clbiphr-2. Both the cyclobutane pyrimidine dimer (CPD)-DNA photolyase domain and the flavin adenine dinucleotide (FAD)-binding domain, characteristic of photolyases, are conserved in the ClbiNPV sequence. The CPD-DNA photolyase domain is located between amino acids 1 to 89 in Clbiphr-1, and an FAD-binding domain is located between amino acids 121 and 287 in Clbiphr-2.
Figure 4 ClbiNPV genome organization around the DNA photolyase locus. ClbiNPV encodes a photolyase-like gene sequence, which has a 1-bp deletion when compared with photolyases of other baculoviruses. This deletion generates a premature termination codon at 51,497 (more ...)
Baculoviruses are attractive candidates for biological control of insect pests [26
]. One major factor limiting their successful use in biological control is their sensitivity to inactivation by ultraviolet (UV) radiation [29
]. The most significant cellular target of UV is DNA. When DNA is exposed to UV, it is damaged by producing pyrimidine dimers [30
], which may block the activities of DNA or RNA polymerases and result in nucleotide misincorporation or inhibit polymerase progression during DNA replication or transcription [31
]. DNA photolyase is a photo-reactivating enzyme that can repair the toxic effects of UV-induced DNA damage. van Oers et al. [23
] suggested that the presence of a CPD-DNA photolyase gene in ChchNPV might be a remnant of the evolutionary history of baculoviruses, or a recent adaptation to a current ecological niche in Chrysodeixis chalcites
or an alternative host, which might have given ChchNPV a competitive advantage. However, the functional significance of this gene in ChchNPV infection has not been proved. We analyzed the photolyase genes in baculoviruses and found that they are almost all early genes expressed before virus DNA replication. Therefore, we speculate that baculovirus photolyases might play a critical role in repairing DNA damage caused by UV, enabling the replication of virus DNA to complete successfully. In ClbiNPV genome, the photolyase-like gene sequence was split into two ORFs by the 1-bp deletion. However, at present, we cannot give direct evidence that this mutant affects the function, since no insect cell line permitting ClbiNPV infection has been found. Our further research will focus on confirming the expression pattern of the ClbiNPV photolyase gene and detecting photolyase activity in baculoviruses.