Schematics of the wt and mutants SnRV clones.

(A) Scaled schematics of SnRV wt clone, based on [3]. Black boxes, 5’ and 3’ LTRs. Dashed lines, introns. Gray arrows and bars, exons. Black bar, the first 14 residues of 3’ORF, overlapping the env ORF. For simplicity, only one of the two putative 3’ORF mRNAs (transcript c; [3]) is shown. Upper line, map scale (in nucleotides). (B-D) SnRV mutants. The mutations’ location is portrayed relative to the map in (A). The schemes depict the junctions between the SnRV nucleotides and cognate amino acids (uppercase and bold letters) and the nested non-viral sequences (nucleotides in non-bold lowercase letters). Red arrows mark four nucleotides in the Env^mut clone (B) and a single nucleotide in the Tins clone (C), inserted to make out-of-frame mutations. Asterisks, stop codons. Green boxes (C, D) and a grid (C) illustrate the GFP and the pac ORFs, respectively.

SnRV wt molecular clone is infectious.

(A, B) Kinetics of SnRV spread. BF-2 cells were infected with equal amounts - normalized by qRT-PCR with SnRV pol-derived primers (A) or RT activity (B) - of SnRV wt, SnRV Env^mut, or SnRV from E-11 cultures (SnRV E-11). On the indicated days postinfection, the culture supernatants were sampled (140 µl) to quantify viral RNA levels by qRT-PCR (A), or harvested (10 ml) for pelleting the virions by ultracentrifugation to determine the pellets’ RT activity by SG-PERT (B). The fold change (log₂) between each time point and (A) the initial time point (day 3 postinfection) or (B) the background sample (a pellet of a supernatant of naïve BF-2 culture) was calculated. Values represent the mean ± SD of three technical repeats. (C) Phenotypic changes in SnRV-infected BF-2 cells. Sub-confluent BF-2 cultures were infected (SnRV wt or SnRV Env^mut), or not. The cultures were passaged for 18 days postinfection, stained with crystal violet, and imaged by light microscopy. Bar; 100 µm.

Effects of 3’ORF replacements on the levels of SnRV genomic RNA and virions.

Genomic RNA levels (quantification by qRT-PCR with pol-derived primers) in cell extracts (normalized to actin RNA levels, A) or culture supernatant (140µl, B) of BF-2 cultures chronically infected with the wt virus (wt) or the 3’ORF14GFP clone (3’ORF14GFP-i), or stably transfected with the 3’ORF14GFP clone (3’ORF14GFP-e). (A, B) The graphs show the fold change in the SnRV genomic RNA levels in wt and 3’ORF14GFP-e relative to the 3’ORF14GFP-i (set to 1) cultures. Each bar represents the average of three technical repeats. ****p ≤ 0.0001, ns - not significant; one-way ANOVA. (C) SG-PERT assay for SnRV RT activity. The supernatants of the indicated cultures (10 ml each) were subjected to ultracentrifugation, and the RT activity of the pellets was determined as in Fig 2B. The dashed line marks the background level obtained from supernatant pellets of naïve BF-2 cultures (BF-2). Each bar represents the average of three technical repeats. (D, E) SnRV clones (wt, Tins, or 3’ORF14Puro) were electroporated into BF-2 cells, and three days posttransfection, the SnRV genomic RNA levels were quantified in cell extracts (D) and culture supernatants (E), by qRT-PCR and normalized to the transfection efficiencies. Transfection efficiencies were quantified by measuring the leaky transcription (in the fish cells) of the yeast LEU2 gene, located in the pGREG525 plasmid backbone shared by the different SnRV constructs. Graphs show the fold change in the normalized genomic RNA of wt and Tins clones, relative to 3’ORF14Puro clone (set to 1); n = 4, * p ≤ 0.05, ** p ≤ 0.01, ns - not significant; one-way ANOVA.

Spreading, expression, and induction of an elongated phenotype of wt and Tins clones in BF-2 cells.

(A) Spreading of wt and Tins clones in BF-2 cultures. The levels of the viral genomic RNA in the culture supernatants were quantified by qRT-PCR and are presented as the average, with standard error bars, of the 1/Ct values of two samples per each time point from two independent spreading assays. (B) Sequence chromatograms from the two independent spreading assays (Spread 1 and 2) of the Tins clone. Supernatants of Tins-infected cultures from (A) were collected 48 days after the initial infection. The region containing the overlap between the env (yellow) and 3’ORF (blue) genes and the T insertion was amplified by RT-PCR and sequenced. Bold and regular letters represent the expected and the actual sequences, respectively. Red rectangles mark the inserted T. Red squares with white asterisks represent stop codons. Amino acids appear in a 1-letter code. (C) MA-GFP expression of SnRV MA-GFPwt, MA-GFPTins, or MA-GFP3’ORF14Puro clones. Equal amounts of the indicated clones were electroporated into BF-2 cells together with a mCherry-expressing plasmid (transfection efficiency control). Three days posttransfection, the transfected cultures were analyzed using flow cytometry for GFP and mCherry signals. The graph depicts the GFP expression levels (normalized to mCherry expression) of the indicated clones relative to the MA-GFP3’ORF14Puro clone (which was set to 1); n = 3. (D) Naïve, or wt or Tins -infected BF-2 cells were stained and imaged, and their average cell area was calculated. 300-500 cells were monitored for each kind of cell. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001; one-way ANOVA.

SnRV promoter activity in fish and mammalian cells.

The indicated fish (A-C) or mammalian (D-F) cells were transfected with the GFP reporter gene under the control of the SnRV promoter (SnRV, circles), the tilapia β-actin promoter (Act, squares), or CMV promoter (CMV, rectangles). The Y-axes present the GFP mean fluorescence relative to the SnRV promoter sample (which was set to 1), quantified by FACS one day posttransfection, except for the RTgill-W1 cells (C) that were analyzed two days posttransfection. n = 3 or 4 for fish or mammalian cells, respectively. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, ns - not significant; one-way ANOVA.

Transient expression of SnRV promoter in zebrafish embryos.

The SnRV LTR-GFP plasmid was injected into zebrafish zygotes and monitored at one-hour intervals. Expression began at the blastula stage (A) and was continually evident throughout development and organogenesis (B). Bars = 500 µM.

Expression of SnRV promoter in sensory organs of transgenic zebrafish larvae.

Lateral (A, D) and dorsal (B, C) views of a 6 dpf Tg(SnRVLTR:EGFP) larva (A, B) or wt (not transgenic) siblings (C, D), showing GFP expression (A, B) and DASPEI signal (C) in the olfactory epithelium (arrows) and neuromasts hair cells (arrowheads). Non-transgenic larva was used for DASPEI staining due to the overlapping excitation and emission spectra of GFP and DASPEI. (D) Control, unstained larva imaged as in A and B. Asterisks mark regions with autofluorescence of the yolk sack. Bars = 200 µM.

Maternal inheritance of the GFP reporter.

Brightfield (A, C) and fluorescent images (B, D) of a fertilized zebrafish egg (A, B) and a four-cell stage embryo (C, D) derived from the transgenic female. Images depict the accumulation of GFP at the animal pole (B), and the blastomeres (D). Bars = 500 µM.

Expression of SnRV promoter in the adult olfactory epithelium and gonads.

Lateral views of dissected (A) and undissected (B) transgenic males, showing GFP signal in the testis (A; arrowheads) and the olfactory epithelium (A, B; arrows). (C) An isolated ovarian biopsy showing a GFP signal in oocytes of different developmental stages. (D) A lateral view of an undissected adult transgenic female showing the ovarian-generated GFP signal.