Yedavalli VRK et al. (2021), A novel class III endogenous retrovirus with a ...

XB-ART-58274

Retrovirology 2021 Jul 14;181:20. doi: 10.1186/s12977-021-00564-2.

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A novel class III endogenous retrovirus with a class I envelope gene in African frogs with an intact genome and developmentally regulated transcripts in Xenopus tropicalis.

Yedavalli VRK , Patil A , Parrish J , Kozak CA .

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BACKGROUND: Retroviruses exist as exogenous infectious agents and as endogenous retroviruses (ERVs) integrated into host chromosomes. Such endogenous retroviruses (ERVs) are grouped into three classes roughly corresponding to the seven genera of infectious retroviruses: class I (gamma-, epsilonretroviruses), class II (alpha-, beta-, delta-, lentiretroviruses) and class III (spumaretroviruses). Some ERVs have counterparts among the known infectious retroviruses, while others represent paleovirological relics of extinct or undiscovered retroviruses. RESULTS: Here we identify an intact ERV in the Anuran amphibian, Xenopus tropicalis. XtERV-S has open reading frames (ORFs) for gag, pol (polymerase) and env (envelope) genes, with a small additional ORF in pol and a serine tRNA primer binding site. It has unusual features and domain relationships to known retroviruses. Analyses based on phylogeny and functional motifs establish that XtERV-S gag and pol genes are related to the ancient env-less class III ERV-L family but the surface subunit of env is unrelated to known retroviruses while its transmembrane subunit is class I-like. LTR constructs show transcriptional activity, and XtERV-S transcripts are detected in embryos after the maternal to zygotic mid-blastula transition and before the late tailbud stage. Tagged Gag protein shows typical subcellular localization. The presence of ORFs in all three protein-coding regions along with identical 5' and 3' LTRs (long terminal repeats) indicate this is a very recent germline acquisition. There are older, full-length, nonorthologous, defective copies in Xenopus laevis and the distantly related African bullfrog, Pyxicephalus adspersus. Additional older, internally deleted copies in X. tropicalis carry a 300 bp LTR substitution. CONCLUSIONS: XtERV-S represents a genera-spanning member of the largely env-less class III ERV that has ancient and modern copies in Anurans. This provirus has an env ORF with a surface subunit unrelated to known retroviruses and a transmembrane subunit related to class I gammaretroviruses in sequence and organization, and is expressed in early embryogenesis. Additional XtERV-S-related but defective copies are present in X. tropicalis and other African frog taxa. XtERV-S is an unusual class III ERV variant, and it may represent an important transitional retroviral form that has been spreading in African frogs for tens of millions of years.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: furin trna

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	Fig. 1. Cloning of XtERV-S. Positions are shown for PCR products PCR1-3 (grey boxes) and the locations of the primers (dashed lines and black arrowheads). Also shown are the PCR product sizes and the positions of restriction sites used for cloning and assembly of the provirus in the pBluescript SK(+) vector
	Fig. 2. The complete nucleotide and deduced amino acid sequence of the XtERV-S proviral genome. The sequence is shown from the beginning of the 5′ U3 region to the end of the 3′ U5. The LTR sequence is in black italics and its inverse repeats are double underlined. The gag, pol and env ORFs are in red, blue and purple, respectively, and termination codons are marked by an asterisk. The Orf-x2 sequence is in light green. The positions of the functional motifs are bolded and highlighted and include the following in order: PBS (primer binding site); basic regions of Gag; MHR (major homology region); GQR motif; PSAP late domain; PR (protease); RT/RNH (reverse transcriptase/RNase H); CWIC (isomerase domain); furin site; ISD (immunosuppressive domain); MSD (membrane spanning domain); PPT (polypurine tract); polyA (polyadenylation signal). Arrows indicate the splice donor and acceptor sites. The dUTPase region of pol is underlined and bolded
	Fig. 3. Hydrophobicity plot of the XtERV-S Env. The SU and TM subunits of envelope are separated by a furin site (RNWKR) at position 251–255. The SU CWIC domain (position 40–43) and its interacting TM CX6CC (position 344–352) domains are indicated in red. The TM subunit contains the following: FP (fusion peptide), two heptad repeats (HR1, HR2), ISD (immunosuppressive domain), MSD (membrane spanning domain), CT (cytoplasmic tail). N-linked glycosylation sites are marked with a Y.
	Fig. 4. Additional XtERV-S-related copies in X. tropicalis. A Schematic representation of XtERV-S-related deleted ERVs. Most of the 19 deleted copies are present in a single copy while Xt-S4—Xt-S14 are structurally similar. Identical line and bar colors represent sequence similarities. Dotted lines represent deletions. B Dot plot comparison of XtERV-S and the 5′ end of Xt-S5 (~ 1400 nt) shows similarities in the 3′ half of the LTR and the N-terminal region of gag
	Fig. 5. Geographic distribution of X. tropicalis, X. laevis and P. adspersus. The areas represented by red and blue highlighting and black stripes represent the natural habitats of X. tropicalis, P. adspersus and X. laevis, respectively [98]. The phylogenetic tree from Timetree [58] places the divergence of P. adspersus and Xenopus sp. at ~ 204 mya and the divergence of two X. tropicalis and X. laevis at 57 mya
	Fig. 6. Unrooted phylogenetic trees of representative retroviruses (Additional file 9: Table S2) based on a MUSCLE multiple alignment and neighbor-joining method. Asterisks indicate bootstrap values greater than 70. Horizontal branch lengths are proportional to the degree of amino acid substitutions per site. The three trees represent RTpol (A), the MHR region of CAgag (B) and a segment of TMenv (C). The RT tree identifies the clusters representative of the seven RV genera. The arrowheads in the TMenv tree identify sequences with the N-linked glycosylation site associated with a heptad “stutter” (Additional file 7: Fig. S7)
	Fig. 7. XtERV-S is transcriptionally active. A Functional analysis of the XtERV-S LTR cloned into a luciferase reporter vector and transfected into 293T cells. Luciferase expression is compared to the promoter-less vector pGL3 basic and to vectors using the Moloney mouse leukemia virus (MoMLV) LTR and CMV promoter. B Confocal examination of GFP-tagged XtERV-S Gag protein in 293T cells shows accumulation in the cytoplasm at the plasma membrane (red arrows) and localization in nucleus (yellow arrows). C ESTs mapping to XtERV-S are expressed embryonically in X. tropicalis. ESTs map to the gag, pol and env regions. D RNAseq reads map mostly to LTR and the gag-pol ORFs. The chart shows the total number of reads per kilobase per million (RPKM) mapped to the provirus and the reads mapping to LTR, gag, pol, and env. The developmental stages and events are indicated, including MBT (mid-blastula transition); the red arrow represents the transcriptional transition from maternal mRNA to zygote genome transcribed mRNAs; the green arrow represents the beginning of the tadpole stage

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Bamunusinghe, Recombinant Origins of Pathogenic and Nonpathogenic Mouse Gammaretroviruses with Polytropic Host Range. 2017, Pubmed