Endogenous non-retroviral RNA virus elements in mammalian genomes

Abstract

Retroviruses are the only group of viruses known to have left a fossil record, in the form of endogenous proviruses, and approximately 8% of the human genome is made up of these elements. Although many other viruses, including non-retroviral RNA viruses, are known to generate DNA forms of their own genomes during replication, none has been found as DNA in the germline of animals. Bornaviruses, a genus of non-segmented, negative-sense RNA virus, are unique among RNA viruses in that they establish persistent infection in the cell nucleus. Here we show that elements homologous to the nucleoprotein (N) gene of bornavirus exist in the genomes of several mammalian species, including humans, non-human primates, rodents and elephants. These sequences have been designated endogenous Borna-like N (EBLN) elements. Some of the primate EBLNs contain an intact open reading frame (ORF) and are expressed as mRNA. Phylogenetic analyses showed that EBLNs seem to have been generated by different insertional events in each specific animal family. Furthermore, the EBLN of a ground squirrel was formed by a recent integration event, whereas those in primates must have been formed more than 40 million years ago. We also show that the N mRNA of a current mammalian bornavirus, Borna disease virus (BDV), can form EBLN-like elements in the genomes of persistently infected cultured cells. Our results provide the first evidence for endogenization of non-retroviral virus-derived elements in mammalian genomes and give novel insights not only into generation of endogenous elements, but also into a role of bornavirus as a source of genetic novelty in its host.

Figure 1. Bornavirus N-like elements in mammalian genomes

a.. Alignment between predicted amino acid sequences of BDV N and two human Bornavirus N-like elements. Black arrowheads indicate predicted frameshift sites in LOC55096 b. Sequence alignments of transcription signal sites (S1/S2 and T1) at both the 5’ and 3’ ends of the Bornavirus N ORF. A schematic representation of Bornavirus genome structure is shown. c and d. Low-stringency Southern blot hybridizations of DNA from various mammalian species using human EBLN-1 and mouse EBLN chr.11 (c) and TLS EBLN (d) as probes. d shows the result using genomic DNA of several squirrel family members. Dots and arrowheads on the right side of the murine and shrew lanes in panel (c) indicate the positions of reproducible positive signals. Vero and COS7 cells are derived from African green monkey. Mr; molecular marker.

Figure 2. Phylogenetic tree of exogenous Bornaviruses and mammalian EBLNs

The bootstrap probability is indicated for each interior branch. The scale bar indicates the number of amino acid substitutions per site. Animals belonging to the same order are indicated by the same color. Strain and sequence accession numbers are given for each sequence.

Figure 3. Reverse transcription and integration of BDV RNA in mammalian cells

a. PCR amplification of BDV N-specific cDNA in BDV-infected cells. OL and 293T (human), Vero (monkey), C6 (rat), MDCK (dog). b. Schematic representation of the Bornavirus genome and mRNAs. Regions for the PCR amplification are indicated by red bars. c. Region-dependent amplification of BDV cDNA in infected OL cells. The numbers on the left side of the panels correspond to the amplification regions in panel b. Ctrl indicates the results of RT-PCR using RNA from BDV-infected OL cells. d. Integration of BDV DNA. Genomic DNA was isolated from BDV-infected OL cells at the indicated days after infection, and Alu-PCR was performed with (+) or without (−) the Alu primer. UI, genomic DNA from uninfected cells. dpi, days postinfection.

Figure 4. Structures of BDV N integration events in OL cells

a. Structure of BDV N cDNA. The numbering corresponds to nucleotide positions in the BDV genome. The BDV N transcript runs from nucleotide positions 44 to 1193. b and c. Structures of BDV N integrations detected by Alu-PCR (b) and inverse PCR (c). The integrated structures of BDV N are indicated. Gray rectangles in the N cDNA indicate truncated regions. Black lettering, host genome sequences; green lettering, inserted nucleotides; red lettering, predicted TSDs. The blue box indicates the position and length of the poly (A) sequence. The pre-integration form of chromosome 7 is shown in panel c.