Structure and evolution of a proviral locus of Glyptapanteles indiensis bracovirus

Abstract

Background: Bracoviruses (BVs), a group of double-stranded DNA viruses with segmented genomes, are mutualistic endosymbionts of parasitoid wasps. Virus particles are replication deficient and are produced only by female wasps from proviral sequences integrated into the wasp genome. Virus particles are injected along with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and therefore perpetuation of proviral DNA. Here we describe a 223 kbp region of Glyptapanteles indiensis genomic DNA which contains a part of the G. indiensis bracovirus (GiBV) proviral genome.

Results: Eighteen of ~24 GiBV viral segment sequences are encoded by 7 non-overlapping sets of BAC clones, revealing that some proviral segment sequences are separated by long stretches of intervening DNA. Two overlapping BACs, which contain a locus of 8 tandemly arrayed proviral segments flanked on either side by ~35 kbp of non-packaged DNA, were sequenced and annotated. Structural and compositional analyses of this cluster revealed it exhibits a G+C and nucleotide composition distinct from the flanking DNA. By analyzing sequence polymorphisms in the 8 GiBV viral segment sequences, we found evidence for widespread selection acting on both protein-coding and non-coding DNA. Comparative analysis of viral and proviral segment sequences revealed a sequence motif involved in the excision of proviral genome segments which is highly conserved in two other bracoviruses.

Conclusion: Contrary to current concepts of bracovirus proviral genome organization our results demonstrate that some but not all GiBV proviral segment sequences exist in a tandem array. Unexpectedly, non-coding DNA in the 8 proviral genome segments which typically occupies ~70% of BV viral genomes is under selection pressure suggesting it serves some function(s). We hypothesize that selection acting on GiBV proviral sequences maintains the genetic island-like nature of the cluster of proviral genome segments described herein. In contrast to large differences in the predicted gene composition of BV genomes, sequences that appear to mediate processes of viral segment formation, such as proviral segment excision and circularization, appear to be highly conserved, supporting the hypothesis of a single origin for BVs.

Figure 1

Structural organization of GiBV proviral locus 1. Proviral genome segments are labeled 1p-8p, with the square and pointed ends representing the 5' and 3' ends, respectively, relative to the putative excision motif. Inter-segmental regions are labeled isg1-isg7, and sequence regions outside the proviral genome segment sequences are labeled I-IV. The flanking tandem repeat regions (solid black squares) are labeled L1R1 and L1R2, and their structure is shown in the open boxes as black boxes in parentheses followed by the copy number of repeat as a subscript. The 2 BAC sequences were joined in isg4 (*) allowing the entirety of each proviral segment sequence to originate from a single BAC clone. Colored boxes represent genes; grey boxes are non-packaged genes, light green boxes are hypothetical proteins without gene family assignment, and the remaining colors represent different gene families.

Figure 2

Neighbor-joining clustering of the regions of proviral locus 1 based on relative dinucleotide frequencies. All proviral genome segments (1p-8p) group together, as do the regions outside the flanking repeats (I and IV). The scale represents the normalized Euclidean distance between regions. Regions < 500 bp (isg1–3, 5) and the flanking repeats (L1R1 and L1R2) were excluded from the analysis, as they have skewed dinucleotide frequencies.

Figure 3

Nucleotide conservation extended 30 bp in both directions around the GCT excision site. A) 5' motif of proviral genome segments in GiBV proviral locus 1, in which sequence to the left of the motif represents inter-segmental sequences and sequence to the right of the motif represents proviral genome segment sequences. B) 3' motif of proviral genome segments GiBV proviral locus 1, in which the positions of inter-segmental and proviral genome segment sequences are reversed with respect to A). C) Extended motif from the 8 viral genome segments in proviral locus 1. D) Extended motif from all 30 CcBV viral genome segments. E) Extended motif from 13 of 15 MdBV viral genome segments.

Figure 4

Histogram of dN/dS ratios of 39 genes in the viral genome segments.