Novel endogenous retrovirus in rabbits previously reported as human retrovirus 5

Abstract

Human retrovirus 5 (HRV-5) represented a fragment of a novel retrovirus sequence identified in human RNA and DNA preparations. In this study, the genome of HRV-5 was cloned and sequenced and integration sites were analyzed. Using PCR and Southern hybridization, we showed that HRV-5 is not integrated into human DNA. A survey of other species revealed that HRV-5 is present in the genomic DNA of the European rabbit (Oryctolagus cuniculus) and belongs to an endogenous retrovirus family found in rabbits. The presence of rabbit sequences flanking HRV-5 proviruses in human DNA extracts suggested that rabbit DNA was present in our human extracts, and this was confirmed by PCR analysis that revealed the presence of rabbit mitochondrial DNA sequences in four of five human DNA preparations tested. The origin of the rabbit DNA and HRV-5 in human DNA preparations remains unclear, but laboratory contamination cannot explain the preferential detection of HRV-5 in inflammatory diseases and lymphomas reported previously. This is the first description of a retrovirus genome in rabbits, and sequence analysis shows that it is related to but distinct from A-type retroelements of mice and other rodents. The species distribution of HRV-5 is restricted to rabbits; other species, including other members of the order Lagomorpha, do not contain this sequence. Analysis of HRV-5 expression by Northern hybridization and reverse transcriptase PCR indicates that the virus is transcribed at a low level in many rabbit tissues. In light of these findings we propose that the sequence previously designated HRV-5 should now be denoted RERV-H (for rabbit endogenous retrovirus H).

FIG. 1.

Cloning the HRV-5 genome and proviral flanking sequences. (A) HRV-5 was cloned as a series of overlapping PCR fragments (see supplementary material), which are shown in relation to the complete cloned genome. Information for fragment 1 was published previously (15). Fragments 2, 9, and 10 were cloned using degenerate primers for conserved regions of NC and IN. Fragments 3, 4, 8, and 11 were cloned using the Vectorette procedure (1). Fragments 5, 6, and 7 were cloned using a 5′ rapid amplification of cDNA ends modified for amplifying from genomic DNA. Fragment 12 was cloned using specific primers. Each new fragment was confirmed as contiguous with the previously identified sequence by PCR across the junction using specific primers. The dotted line represents DNA flanking the provirus that was cloned as part of fragment 11 (clone 3A). (B) Diagram of HRV-5 genome assembled from the various PCR fragments and cloned as plasmid pHRV5.6 (GenBank accession no. AF480924). The genome has ORFs for gag, pro, and pol and two LTRs but lacks env. The locations of putative protein-coding domains are marked, including MA, CA, NC, deoxyuridine triphosphatase (DU), PR, RT/RNase H (RT), and IN. Regulatory motifs including a polypurine tract (PPT), a primer binding site (PBS; specific for tRNA-His), and heptanucleotide frameshifting sequences (fs) between gag/pro and pro/pol are present. The locations of three PCR products containing integration sites are indicated (5A, 5B, and 3A).

FIG. 2.

HRV-5 is an ERV of rabbits. Genomic DNA from several mammalian and avian species was tested for the presence of HRV-5 sequences. (A) Southern blots probed with the MA domain of HRV-5 gag. Kbp, kilobase pairs. (B) PCR products obtained from the same species with primers derived from HRV-5 PR/RT, IN, and LTR and with degenerate primers for a gammaretrovirus pol sequence (γ). Following PCR, the HRV-5 products were transferred to a nylon membrane and were probed with an internal fragment specific for each product. Lanes: w, water; 1, fat-tailed dunnart; 2, quail; 3, Syrian hamster; 4, Chinese hamster; 5, rat; 6, mouse; 7, bat; 8, marmoset; 9, macaque; 10 African green monkey; 11, gibbon; 12, chimpanzee; 13, human (donor blood); 14, human (HeLa); 15, horse; 16, mink; 17, cat; 18, dog; 19, cow; 20, pig; 21, rabbit (SIRC); 22, rabbit (EREp), 23, rabbit spleen; 24, European hare; 25, black-tailed jackrabbit; and 26, Afghan pika. HRV-5 sequences were detected in rabbit DNA from three sources. (C) Southern blot of several species of Lagomorpha probed with the MA domain of HRV-5 gag.

FIG. 3.

HRV-5 sequences amplified from rabbit DNA. Sequences amplified from rabbit DNA by PCR (Fig. 2B) are shown aligned with the reference clone (HRV-5). (A) Deduced amino acid sequence of the pro ORF amplified by PR/RT primers (clones A to F). U46939, previously published clone (15). (B) Deduced amino acid sequence of the pol ORF amplified by PR/RT primers. (C) Deduced amino acid sequence of the region of pol amplified by IN primers (clones G to L). (D) Nucleotide sequence amplified by LTR primers (clones M to Q). Both the 5′ (HRV5five) and 3′ (HRV5three) LTRs of the reference clone are shown. The sequences of several representative plasmid clones are presented. A dash indicates identity with the reference clone (GenBank accession no. AF480924); a period indicates the presence of a gap in the alignment. Differences are marked and stop codons are indicated by an asterisk. Primer sequences are omitted.

FIG. 4.

Sequences flanking HRV-5 proviruses are present in rabbit genomic DNA. Southern blots of rabbit and human DNA were hybridized with probes derived from the three HRV-5 integration site sequences cloned from patient DNA. Each of the integration site clones (3A, 5A, and 5B) hybridizes only with rabbit DNA (from spleen or SIRC cells) and not with human DNA (from blood or HeLa cells), confirming that they are derived from rabbits only. Clones 5A and 5B appear to be single-copy elements, while clone 3A is a rabbit LINE element present at a high copy number.

FIG. 5.

HRV-5 RNA expression in rabbit tissues. The expression of HRV-5 RNA was analyzed in several rabbit tissues by RT-PCR with primer sets from gag (NC) and pol (IN). Lanes: 1, spleen; 2, kidney; 3, liver; 4, ovary; 5, stomach; 6, colon; 7, pancreas; 8, heart; 9, skeletal muscle; 10, lung; 11 skin; 12, brain; 13, placenta; 14, mammary gland; 15, lymph node; 16, SIRC (rabbit corneal epithelial cell line), 17, human L363 cell RNA; w, PCR water control; +, positive control (rabbit DNA); 18, water control from the DNase treatment step; and 19, water control from the primer-annealing step. Experiments were performed with or without RT in the cDNA synthesis reaction to control for contamination with cellular DNA. PCR primers for rabbit GAPDH were used as controls for RNA quality.