Poxviruses as possible vectors for horizontal transfer of retroposons from reptiles to mammals

Abstract

Poxviruses (Poxviridae) are a family of double-stranded DNA viruses with no RNA stage. Members of the genus Orthopoxvirus (OPV) are highly invasive and virulent. It was recently shown that the taterapox virus (TATV) from a West African rodent is the sister of camelpox virus and therefore belongs to the clade closest to the variola virus (VARV), the etiological agent of smallpox. Although these OPVs are among the most dreaded pathogens on Earth, our current knowledge of their genomes, their origins, and their possible hosts is still very limited. Here, we report the horizontal transfer of a retroposon (known only from reptilian genomes) to the TATV genome. After isolating and analyzing different subfamilies of short interspersed elements (SINEs) from lizards and snakes, we identified a highly poisonous snake (Echis ocellatus) from West Africa as the closest species from which the SINE sequence discovered in the TATV genome (TATV-SINE) was transferred to the virus. We discovered direct repeats derived from the virus flanking the TATV-SINE, and the absence of any snake-derived DNA flanking the SINE. These data provide strong evidence that the TATV-SINE was actually transferred within the snake to the viral genome by retrotransposition and not by any horizontal transfer at the DNA level. We propose that the snake is another host for TATV, suggesting that VARV-related epidemiologically relevant viruses may have derived from our cold-blooded ancestors and that poxviruses are possible vectors for horizontal transfer of retroposons from reptiles to mammals.

Fig. 1.

TATV-SINE locus aligned with consensus sequences of six Sauria SINE subfamilies. (A) Alignment of genomic regions of six OPVs showing the TATV-SINE insertion and 50 bp of flanking regions, including the single insertion site (boxed in light gray), of closely related OPVs (28). (B) SINE sequences isolated from genomes of varanid lizards comprise four different subfamilies (VAR^α-type, VAR^β-type, VAR^γ-type, and VAR^δ-type sequences). VAR^α-type members share an 8-bp insertion (unshaded box, positions 81–88) with both subfamilies isolated from genomes of snakes (AFE and EOC). The EOC Sauria SINE subfamily and the TATV-SINE are closely related (diagnostic nucleotides are boxed). Each CpG site is marked with an asterisk. Nucleotide positions 28 and 304 are marked with a plus (see Fig. 2 for further explanation). The two deletions in the TATV-SINE sequence (positions 344 and 361) could have been created upon retrotransposition of the SINE. Functional regions are boxed in dark gray: the 5′ end RNA polymerase III-specific internal promoter sequences (A-Box and B-Box) and the 3′ end stem-loop structure for RT recognition (12).

Fig. 2.

Phylogenetic tree of Sauria SINEs and TATV-SINE obtained by the maximum-likelihood method. The relationships of investigated Sauria SINE subfamily consensus sequences of lizards and snakes are illustrated. Sauria SINEs of lacertid lizards (POM, Podarcis muralis, common wall lizard) and iguanian lizards (ACA, Anolis carolinensis, green anole) served as outgroups and are boxed in gray (12). We categorized EOC SINE members into five subsubfamilies to show the precise origin of the TATV-SINE (EOC-I to EOC-V). Subsubfamily EOC-I differs from the EOC consensus sequence by two diagnostic nucleotides that are shared with the TATV-SINE (positions 28 and 304 in Fig. 1B; see also positions 51 and 330 in SI Fig. 6). Numbers below the branches represent puzzle support values for Sauria SINE subfamilies. Numbers above the branches correspond to diagnostic nucleotides (dn) in subfamilies/subsubfamilies.

Fig. 3.

Distribution of Sauria SINE subfamily EOC and subsubfamily EOC-I. (A) Genomic DNA from snakes, lizards and rodents was amplified by PCR, using primers EOCconsF and EOCconsR (see Material and Methods). A discrete PCR product of ≈340 bp was generated in all three major lineages of advanced snakes (lanes 1–4 Viperidae; lane 5 Elapidae; lane 6, Colubridae) but not in other snakes (lane 7), lizards (lanes 8–10), or rodents (lane 11). (B) Phylogenetic relationships of snakes. (C) Genomic DNA from snakes, lizards, and rodents was amplified by PCR, using Sauria SINE-specific subsubfamily primers EOC-I-F and EOC-I-R (see Material and Methods). A discrete PCR product of ≈315 bp was generated only in E. ocellatus (lane 1). Lane 1 E. ocellatus; lane 2, E. coloratus; lane 3, Macrovipera lebetina; lane 4, Crotalus horridus; lane 5, Notechis scutatus; lane 6, Natrix tesselata; lane 7, Boa constrictor; lane 8, V. griseus; lane 9, V. timorensis; lane 10, P. muralis; lane 11, T. kempi. M, size marker.

Fig. 4.

Overlapping geographical distributions of the rodent T. kempi and the snake E. ocellatus in Africa. The distributions of T. kempi and E. ocellatus are shown in light gray and dark gray ellipses, respectively. TATV was isolated from Kemp's gerbil, caught in Benin (black) at the time of an epidemic of human smallpox (30). Kemp's gerbil and the carpet viper are distributed in the entire Benin region.

Fig. 5.

Horizontal retrotransposition of a Sauria SINE to a poxvirus in the cell of a highly poisonous snake. SINEs and LINEs are transcribed through internal promoter sequences by RNA polymerases. Each SINE family recruits the enzymatic machinery for retrotransposition (RT and EN) from the corresponding LINE family through an identical 3′ tail sequence (6). Reverse transcription and integration of the Sauria SINE into the viral genome occurred within the body of E. ocellatus. Direct repeats (DR) derived from the virus flanking the Sauria SINE show that the horizontal transfer was actually a horizontal retrotransposition.