Five most common events in each genomic RNA in the final passage of each replicate.

<p>Five most common events in each genomic RNA in the final passage of each replicate.</p

Comparison of Illumina HiSeq to Oxford Nanopore MinION read data.

<p>Example of how reads generated from the Illumina HiSeq would map to a reference genome. The <i>Bowtie</i> alignment is able to map 150bp reads along the reference with a relatively even coverage distribution. Unmapped reads are then aligned with <i>ViReMa</i> to accurately identify recombination events. The low error rate of high-throughput sequencing allows us to precisely define the boundaries of the junctions. Below the reference genome is an example of how reads generated from Oxford Nanopore Technologies’s MinION sequencing would map to a reference genome. The MinION is able to generate full length reads at the expense of a high error rate, which is ~7%. Full length analysis allows us to determine what recombination events a genome contains. Due to the error rate the exact boundaries of the recombination event are imprecise.</p

TCID50, specific infectivity and cytopathic effect (CPE) of each passage for replicate 2.

<p><b>(A)</b> The 50% tissue culture infectious dose (TCID50) of each passage of replicate 2 is shown (inocula used in serial passaging in blue squares). Additionally, virus was purified and quantified by OD_260nm before infection allowing normalization particles per cell across all replicates for further TCID50 analysis (in red circles). <b>(B)</b> TCID50/ml values were used to calculate effective MOI values during serial passaging. *Passage 4 sample was not available. <b>(C)</b> 10⁵ cells were infected with quantified and serially diluted virus. The number of virus particles per cell plated is indicated on the x-axis. After 4 days of infection, the remaining number of cells was analyzed by flow-cytometry to count remaining viable cells. Cell death is indicated by the difference in count compared to non-infected wells (0 particles/cell). Each line indicates a different passage from replicate 2 as indicated in the color key.</p

Recombination profiling of conserved regions in the DI-RNAs.

<p>Conservation map represents the frequency with which specific nucleotides in the FHV genome are deleted after recombination. Dashed lines indicate boundaries of functional RNA motifs. Odd numbered passages from replicate 2 are represented here. See <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006365#ppat.1006365.s003" target="_blank">S3 Fig</a></b> for all replicates and all passages. <i>Cis-RE</i>: cis-Regulatory Element; <i>DSCE/PSCE</i>: Distal/Proximal Subgenomic Control Elements; <i>intRE</i>: internal Response Element; <i>3’ RE</i>: 3’ Response Element; <i>5’ SL</i>: 5’ Stem Loop</p

RNA recombination is characterized using RNAseq during serial passaging of FHV in cell-culture.

<p><b>(A)</b> Frequency of recombination events and <b>(B)</b> Shannon Diversity Index for all passages and replicates. Percent recombination is calculated from total number of FHV recombination events per total number of reads mapped to FHV. <b>(C)</b> The frequency of each nucleotide found both upstream and downstream of the 5’ and 3’ sites of RNA recombination events are plotted. Only recombination events with fewer than 10 reads were included in this analysis to avoid over-sampling of highly replicated DI-RNAs. The composition (and therefore expected frequency) of nucleotides in the FHV genome is given by the colored horizontal lines in each plot. <b>(D)</b> A recombination event can retain a putative open reading frame by deleting 3n nucleotides. The frequency of conservation of the reading frame is calculated from the ratio of the number of recombination events that delete 3n nucleotides to the total number of recombination events mapped to that RNA.</p

Parallel ClickSeq and Nanopore sequencing elucidates the rapid evolution of defective-interfering RNAs in Flock House virus

<div><p>Defective-Interfering RNAs (DI-RNAs) have long been known to play an important role in virus replication and transmission. DI-RNAs emerge during virus passaging in both cell-culture and their hosts as a result of non-homologous RNA recombination. However, the principles of DI-RNA emergence and their subsequent evolution have remained elusive. Using a combination of long- and short-read Next-Generation Sequencing, we have characterized the formation of DI-RNAs during serial passaging of Flock House virus (FHV) in cell-culture over a period of 30 days in order to elucidate the pathways and potential mechanisms of DI-RNA emergence and evolution. For short-read RNAseq, we employed ‘ClickSeq’ due to its ability to sensitively and confidently detect RNA recombination events with nucleotide resolution. In parallel, we used the Oxford Nanopore Technologies’s (ONT) MinION to resolve full-length defective and wild-type viral genomes. Together, these accurately resolve both rare and common RNA recombination events, determine the correlation between recombination events, and quantifies the relative abundance of different DI-RNAs throughout passaging. We observe the formation of a diverse pool of defective RNAs at each stage of viral passaging. However, many of these ‘intermediate’ species, while present in early stages of passaging, do not accumulate. After approximately 9 days of passaging we observe the rapid accumulation of DI-RNAs with a correlated reduction in specific infectivity and with the Nanopore data find that DI-RNAs are characterized by multiple RNA recombination events. This suggests that intermediate DI-RNA species are not competitive and that multiple recombination events interact epistatically to confer ‘mature’ DI-RNAs with their selective advantage allowing for their rapid accumulation. Alternatively, it is possible that mature DI-RNA species are generated in a single event involving multiple RNA rearrangements. These insights have important consequences for our understanding of the mechanisms, determinants and limitations in the emergence and evolution of DI-RNAs.</p></div

Serial passaging of Flock House virus in <i>Drosophila melanogaster</i>.

<p>S2 cells were transfected with pMT vectors containing either FHV RNA1 or RNA2 and induced to generate genetically homogeneous viral particles. Virus was allowed to propagate for three days after which cells and the supernatant were collected. A fraction (1:10) was used to further infect a new population of S2 cells in a series of passages. The remaining fraction was collected for analysis. In total, three replicates of nine passages were collected.</p

The frequency of deletions in the FHV genomic RNAs found by MinION nanopore sequencing.

<p><b>(A)</b> Gel electrophoresis analysis of cDNA copies for RNA1 and RNA2 for each passage in replicate 2 shows full-length viral genomic RNAs in early passages with increasing quantities of smaller defective RNAs bands in later passages. <b>(B)</b> Full length genomes were sequenced with the MinION and the number of deletions per genome were counted for each passage. Due to the higher error rate of the nanopore data only deletions ≥25nts were counted. <b>(C)</b> The Shannon Diversity Index of all RNA1 or RNA2 genes characterized with MinION nanopore sequencing is shown for each passage.</p

Number of ClickSeq reads gathered from each passage of replicate 2.

<p>Number of ClickSeq reads gathered from each passage of replicate 2.</p

HTLV-1 Tax interacts with Drosha <i>in vitro</i>.

<p><b>A</b>) HeLa whole cell lysates (1 mg) were incubated with purified GST-fusion proteins (∼5 µg) bound to Glutathione-Sepharose beads: GST-Alone, GST-Tax wild type, GST-Tax N-terminus, 1–244, GST-Tax C-terminus, 244–336, and GST-Tax truncated, 288–353. Beads were washed gently, proteins eluted, and western blotted for the presence of Drosha. CEM whole cell extract serves as a positive Drosha control. <b>B</b>) 293T cell lysates were prepared from cells alone or cells transfected with pACH.Tax (5 µg) or pACH.M22 (5 µg) in the presence of the proteasome inhibitor PSI (10 µM). Approximately 1 mg of lysate was incubated with α-IgG, α-Drosha, or α-Tax overnight at 4°C. The next day, 50 µl of a 30% Protein A + G slurry was used to IP Drosha or Tax and subsequently western blotted for the presence of Drosha and Ubiquitin. Multiple isoforms of Drosha as detected by the antibody are indicated as Drosha I (170 kDa), Drosha II (156 kDa), and Drosha III (115 kDa). <b>C</b>) Graphical depiction of the GST-Tax constructs and the regions which they span for the Tax protein. Relative Drosha binding is based on densitometry counts. <b>D</b>) C81 cells were treated with the proteasome inhibitors PSI and ALLN at increasing concentrations (0.1, 1.0, and 10 µM) for 48 hours. Cell lysates were western blotted for the presence of Drosha. β-Actin serves as a loading control. Drosha recovery as a percentage of CEM Drosha levels is indicated from densitometry counts normalized to β-Actin using ImageJ.</p