Fish retrovirus query gene component maps. The GPS accesses a database populated by retroid genome sequences. This query library contains all of the genes and noncoding component sequences, which the GPS uses to identify and reconstruct potential retroid agents found in genome databases. All fish retroviruses used as queries are presented in phylogenetic order, with those that are novel indicated in boldface. The complexity of intra- and intergenic, as well as interpolycistronic, variation in accumulation of stop codons (asterisks) and frameshifts (a shift to another line) is illustrated. Stop codons (within separate boxes), with or without a frameshift, are usually found between the gag/pol and pol/env polycistrons. Stop codons that are shown within a gene box are intragenic, while those within a separate box are accompanied by an intragenic frameshift. Note that genes with intragenic frameshifts are labeled multiple times; these do not indicate duplications. If a potential retroid agent encodes all of the genes in a specific query-component library, it is considered full-length. Those gene abbreviations not defined in the text as expected retroviral genes are as follows: CNPase, 2′,3′-cyclic nucleotide 3′-phosphodiesterase homolog; UNK, unknown region; UTR, untranslated region; ORF, open reading frame; CYCD/CYDh, cyclin D homologues; and Macro, the viral homologues of the cellular macro domain.

Estimates of LTR insertion times. Insertion time estimates are shown for all 46 homologous LTR data sets for D. rerio (A), O. latipes (B), G. aculeatus (C), and T. nigroviridis (D). The y axis shows the years in log scale to display the complete range of insertion times. The x axis indicates the datasets and potentially active copies per total sample size [e.g., DRERV1.1(1/5)]. Sets with single samples are displayed with single values in million of years, while sets with multiple samples have both the maximum and the minimum insertion dates of the set shown. The maximum and minimum 95% CI for multiple sample sets are indicated by thick and thin lines, respectively. The CIs overlap when the maximum and minimum insert dates are close to one another. For single sample sets, the 95% CIs are indicated on the single insertion values by a complete overlap of thick and thin lines. Identical LTRs have no change and are, therefore, the most recent integrations. The distance values, SEs, insertion times, and CIs are calculated as described in Materials and Methods.

Multiple alignment of the catalytic motifs of the CNPase homologues. Nine of the eleven novel fish retroviruses encode a CNPase homologue. The protein sequence alignment of the two histidine motifs at residues 244 and 374 are shown for the nine fish retrovirus containing a CNPase homolog and other CNPase genes. The alignment was rendered in Jalview and highlighted by conservation. The highest level of conservation is highlighted in black and the lowest in light gray; white indicates no conservation. Accession numbers not found in the text are as follows: Homo sapiens, PDB 1WOJ; Rattus norvegicus, PDB 2ILX; T. nigroviridis, CAG02336; C. auratus, PDB 2I3E; D. rerio, XM_001332834; Arabidopsis thaliana, PDB 1FSI; T4 bacteriophage, 15389; shrimp white spot syndrome virus, 17158251; fowlpox virus, 9634695; Chilo iridescent virus, 15078840; human coronavirus, 608643; Berne virus, 58777; human rotavirus, 6009563; and avian rotavirus, 2754604.

Relationship of gene sequences of full-length and representative indel mutant genomes. The y axis is the retroviral components (as defined in the text and Fig. 1); the x axis is the set of full-length and example indel mutants. Percent nucleotide identities are represented by bubble size and labeled on each bubble. Copy numbers are shown in parentheses next to the retrovirus name. The percent identities are the average pairwise sequence identity over each gene set. Note that not all queries have all components, as shown by blank regions. A deletion or insertion mutation (InDel) in the same region or gene is represented by a smaller bubble. Although most of the deletions overlap the same region, some are in a different position; therefore, no relationship values are given.

Fish retrovirus phylogeny. RT amino acid sequences from sequenced fish retroviruses were used to reconstruct the evolutionary history of the fish retroviruses. The consensus MrBayes3.1 tree with posterior probabilities at each node and branch lengths scaled to expected substitutions per site is shown. This tree was generated over 80,000 iterations using an eight-category gamma distribution rate with the mixed amino acid model. Six prior constraints were invoked on relationships that have a 100% confidence: (i) OLERV2, OLERV3, and ERV3_Tet; (ii) GAERV3, DRERV4, and GAERV2; (iii) OLERV1, SSSV, ZFERV, DRERV2, GAERV1, and ERV4_Tet; (iv) ERV2_Tet and DRERV1; (v) WDSV, WEHV1, and WEHV2; and (vi) HIV-1 and mouse mammary tumor virus (MMTV). Asterisks indicate queries that encode a CNPase region and tildes show those with macro domains (Fig. 1). All fish retrovirus acronyms are as defined in the text. Mouse mammary tumor virus and HIV-1 serve as outliers.