The nucleotide sequence of koala (Phascolarctos cinereus) retrovirus: a novel type C endogenous virus related to Gibbon ape leukemia virus

Abstract

A novel retrovirus, morphologically consistent with mammalian C-type retroviruses, was detected by electron microscopy in mitogen-stimulated peripheral blood mononuclear cell cultures from 163 koalas and in lymphoma tissue from 3 koalas. PCR amplified provirus from the blood and tissues of 17 wild and captive koalas, and reverse transcriptase-PCR demonstrated viral mRNA, viral genomic RNA, and reverse transcriptase activity in koala serum and cell culture supernatants. Comparison of viral sequences derived from genomic DNA and mRNA showed identity indicative of a single retroviral species-here designated koala retrovirus (KoRV). Southern blot analysis of koala tissue genomic DNA using labelled KoRV probes demonstrated banding consistent with an endogenous retrovirus. Complete and apparently truncated proviruses were detected in DNA of both clinically normal koalas and those with hematopoietic disease. KoRV-related viruses were not detected in other marsupials, and phylogenetic analysis showed that KoRV paradoxically clusters with gibbon ape leukemia virus (GALV). The strong similarity between GALV and KoRV suggests that these viruses are closely related and that recent cross-host transmission has occurred. The complete proviral DNA sequence of KoRV is reported.

FIG. 1

Graphic representation of KoRV provirus showing positions of primer pairs used to amplify proviral DNA. The primer pair U3F-U5R flanks the entire provirus, including both 5′ and 3′ LTRs, such that each primer binding site is duplicated in the provirus, whereas the primer pair KRVf-KRVr excludes amplification of repeated regions of the LTRs. The lines under each primer pair represent expected amplimers.

FIG. 2

Complete nucleotide sequence of a KoRV provirus. The boundaries of the major sections of the genome are shown by arrowheads: 5′ LTR (U3-R-U5, boundaries based on GALV [GenBank accession no. M26927]), gag, pro-pol, env, and 3′ LTR. Sequences important in transcription and reverse transcription are shown in boxes: CAAT box, TATA box, Cys-His box, polyadenylation signal (PAS), polypurine tract (PPT) and tRNA^pro primer binding site (PBS). Primer sequences described in the text are underlined. Note that nucleotides 1 to 8411 were sequenced from one clone. Nucleotides 8412 to 8431 were inferred from the 5′ LTR.

FIG. 3

(A) PCRs using primer pairs KRVf-KRVr and U3F-U5R resolved on a 1% agarose gel stained with ethidium bromide. Lanes: 1 and 9, 1-kb-ladder molecular weight markers; 2, no-DNA PCR control; 3, kangaroo DNA PCR control; 4, lymphoma DNA amplified with primer pair KRVf-KVRr; 5, blood DNA from the same koala amplified with the same primers; 7 and 8, the same samples amplified with primer pair U3F-U5R, showing amplimers representing full-length and truncated proviruses, as well as LTRs only (0.5-kb bands); 10 and 11, KRVf-KRVr amplimers from blood DNA from two different koalas. (B) Southern blot of a similar PCR gel probed with a KoRV pol probe. Lanes: 1 digoxigenin-labelled molecular weight marker; 2, kangaroo DNA PCR control; 3, dog lymphoma DNA PCR control; 4, koala DNA U3F-U5R PCR products; 5, koala DNA KRVf-KRVr PCR products.

FIG. 4

Maximum likelihood phylogeny for the pol gene of KoRV, GALV or GALVX, MLV-type viruses (MMLV, FMLV, and FeLV), and PERV. The transition/transversion ratio (3.1) and gamma shape parameter (0.44) were estimated from the data [D. L. Swofford, PAUP∗: Phylogenetic Analysis Using Parsimony (^∗ and other methods), version 4. Sinauer Assoc., Sunderland, Mass., 1999]. The results are the same for the gag and env genes and for all alternative alignments of the pol gene (see Materials and Methods).

FIG. 5

Spectral plot showing the relative support for branches in the pol phylogeny (Fig. 4). The frequency of patterns in the sequence data that support a partition (clade) is given above the x axis, and the frequencies of patterns that support a conflicting bipartition are given below. The KoRV-GALV clade has strong support and little conflicting signal. Spectral analysis was performed with the Spectrum package (9).

FIG. 6

Southern blot of koala genomic DNA digests. Lanes: 1, digoxigenin-labelled molecular weight marker; 2, dog genomic DNA digest (control); 3, kangaroo genomic DNA digest (control); 4 to 7, DNA digests from a koala with lymphoma; 4, 10 μg of bone marrow DNA digested with PstI; 5, 10 μg of bone marrow DNA digested with HindIII; 6, 10 μg of lymphoma DNA digested with HindIII; 7, 10 μg of lung DNA digested with HindIII; 8, 2 μg of bone marrow DNA digested with HindIII from a koala without lymphoma.