Homologous sequences in the Campoletis sonorensis polydnavirus genome are implicated in replication and nesting of the W segment family

Abstract

Polydnaviruses (PDVs) are double-stranded DNA viruses with segmented genomes that replicate only in the oviducts of some species of parasitic wasps and are required for the successful parasitization of lepidopteran insects. PDV DNA segments are integrated in the genomes of their associated wasp hosts, and some are nested; i.e., smaller segments are produced from and largely colinear with larger segments. To determine the internal structure of nested viral segments, the first complete nucleotide sequence of a PDV genome segment and its integration locus was determined. By restriction mapping, Southern blot, and sequence analyses, we demonstrated that the Campoletis sonorensis PDV segment W is integrated into wasp genomic DNA. DNA sequence analysis revealed that proviral segment W terminates in two 1,185-bp direct long terminal repeats (LTRs) in the wasp chromosome, while only one LTR copy is present in the extrachromosomal (viral) W. The results suggest that terminal direct repeats are a general feature of PDV DNA segment integration but that the homology and size of the repeats can vary extensively. Segment W contains 12 imperfect direct repeats of six different types between 89 bp and 1.9 kbp with 65 to 90% homology. The orientation and structure of the repeats suggest that W itself may have arisen through sequence duplication and subsequent divergence. Mapping, hybridization, and sequence analyses of cloned R and M demonstrated that these segments are nested within segment W and that internal imperfect direct repeats of one type are implicated in the homologous intramolecular recombination events that generate segments R and M. Interestingly, segment nesting differentially increases the copy number of genes encoded by segment W, suggesting that the unusual genomic organization of PDVs may be directly linked to the unique functions of this virus in its obligate mutualistic association with parasitic wasps.