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. 2015 Feb 2:5:8164.
doi: 10.1038/srep08164.

Multiple invasions of an infectious retrovirus in cat genomes

Sayumi Shimode  1 , So Nakagawa  2 , Takayuki Miyazawa  1
Affiliations

Multiple invasions of an infectious retrovirus in cat genomes

Sayumi Shimode et al. Sci Rep. .

Abstract

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of host germ-line cells. While most ERVs are defective, some are active and express viral proteins. The RD-114 virus is a replication-competent feline ERV, and several feline cell lines produce infectious RD-114 viral particles. All domestic cats are considered to have an ERV locus encoding a replication-competent RD-114 virus in their genomes; however, the locus has not been identified. In this study, we investigated RD-114 virus-related proviral loci in genomes of domestic cats, and found that none were capable of producing infectious viruses. We also found that all domestic cats have an RD-114 virus-related sequence on chromosome C2, termed RDRS C2a, but populations of the other RDRSs are different depending on the regions where cats live or breed. Our results indicate that RDRS C2a, the oldest RD-114-related provirus, entered the host genome before an ancestor of domestic cats started diverging and the other new RDRSs might have integrated into migrating cats in Europe. We also show that infectious RD-114 virus can be resurrected by the recombination between two non-infectious RDRSs. From these data, we conclude that cats do not harbor infectious RD-114 viral loci in their genomes and RD-114-related viruses invaded cat genomes multiple times.

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Figures

Figure 1
Figure 1. Detection of RD-114 proviruses in the genomes of feline cell lines by Southern blotting analyses.
(A) Restriction endonuclease maps of an infectious molecular clone of RD-114 virus, termed pSc3c. Probes for gag, pol and env genes are shown as bars. (B–D) Southern blotting analyses of several feline cell lines. Genomic DNAs were digested with HindIII, SacII, EcoRI or SphI and then subjected to Southern blotting analyses using gag (B), pol (C) and env (D) probes. HindIII-digested fragments corresponding to pSc3c are shown by white arrowheads. Asterisks indicate RD-114 viral env locus detected in common in all cell lines examined.
Figure 2
Figure 2. Structures of RDRSs.
(A) Structures of the genomes of full length RDRSs. Open triangle indicates a deletion of nucleotides and filled triangle indicates an insertion of nucleotides. Green, orange and blue boxes indicate gag, pol and env ORFs, respectively. Numbers (%) above the diagrams indicate homologies with RD-114 infectious clones in each gene. (B) Schematic LTR structures of RDRSs and RD-114 virus clones, pSc3c and pCRT1. DR-A, direct repeat A; DR-B, direct repeat B; CAAT, CAAT box; TATA, TATA box; TSS, transcription start site; poly A, poly A signal. (C) Features of LTRs of RDRS proviruses and estimated integration time.
Figure 3
Figure 3. The maximum likelihood phylogenetic trees of RDRSs.
We used nucleotide sequences of gag, pol, env and full length excluding LTR. The general time-reversible (GTR) model of nucleotide substitution with the addition of invariant sites (I) and a gamma distribution of rates across sites (Γ) was used to infer the phylogenies. A2, RDRS A2; C2a, RDRS C2a; E3, RDRS E3; D4, RDRS D4; C2b, RDRS C2b; BaEV M7, Baboon endogenous virus strain M7.
Figure 4
Figure 4. Possession of RD-114 viral infectious-type env and RDRSs.
(A) Population of RDRS proviruses in feline PBMC. Regions that the cats originally lived in were indicated based on a previous study. (B) Position of reverse primer to detect infectious-type env indicated by blue characters and arrowheads. Red characters are the RDRS C2a-specific insertion. Numbers are defined as positions of pSc3c. (C) Cat's journey and process of RDRV integration. Virus particle with blue hexagonal shape and orange hexagonal indicate the oldest RDRV and the new RDRVs, respectively. The picture of cats and the map were drawn by S.S. using the Microsoft PowerPoint.
Figure 5
Figure 5. Comparison of sequences of RDRS C2a integration sites in domestic cat's (Felis catus) and other Felidae species' genomes.
(A) Alignment of sequences of 5′ and 3′flanking regions of RDRS C2a in domestic cat (Felis catus) and their corresponding sites' sequences in leopard cat (P. bengalensis), Serval cat (L. serval), Snow leopard (P. uncia) and Tiger (P. tigris). Asterisks indicate conserved nucleotides between four species. Target site duplications (TSDs) for SINEC_Fc2-like sequence are shaded in black. (B) Schematic view of RDRS C2a integration site and adjacent sequences of Felidae genomes. Lime green arrow indicates RDRS C2a. Pink and pink-red arrows indicate SINEC_Fc2 and C SINEC_Fc2-like sequence, respectively, and aqua arrow indicates LINE-like sequences. Short orange lines at both sides indicate primer positions used for PCR.
Figure 6
Figure 6. Infectivity of RDRS C1.
(A) Differences between RDRS C1 and an infectious molecular clone, pSc3c. Numbers indicate nucleotide locations at pSc3c. Shaded regions are functional domains of pol region predicted by Pfam (PR, aspartyl protease; RT, reverse transcriptase; RH, RNaseH; IN, integrase core domain [Pfam accession number: PF00077, PF00078, PF00075 and PF00665, respectively]). Differences between RDRS C1 and pSc3c are indicated by vertical lines (point mutations) and filled triangle (insertion). RDRS C1 mutants have mutations marked by crosses. (B, C) LacZ marker rescue assay performed using TE671 cells as target cells. Infection with LacZ pseudotype viruses (RD-114 virus, RDRS C1 wild-type and RDRS C1 mutants) were visualized by X-Gal staining (B) and the virus titers were expressed as f.f.u./ml (C). Assays were performed in triplicate and the data are shown as the mean viral titers ± standard errors.
Figure 7
Figure 7. Recombination between RDRS A2 and C1.
(A) Growth of RDRV AC in HEK293T cells. The RDRS plasmids were transfected into HEK293T cells and supernatants were inoculated into HEK293T(LacZ) cells, and then virus production was monitored by the LacZ marker rescue assay. Assays were performed in triplicate and the data are shown as the mean viral titers ± standard errors. (B) Comparison of nucleotide sequences of pSc3c, RDRS A2, C1 and RDRV AC virus. The nucleotide positions of pSc3c are shown. Dark grey arrowheads indicate positions of primers used for PCR. Small letters indicate nucleotides. Capital letters in parentheses indicate amino acids. Dots indicate differences between RDRV AC and the other clones. Colors of dots are dependent on where are mutations from (Red, derived from A2; Blue, from C1; Gray, from A2 or C1; Black, undetermined). (C) LTR structures of RD-114 virus clones (pSc3c and pCRT1), RDRS A2, C1 and RDRV AC.
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