Figure 2: Chikungunya vDNA is required for small viral RNA production in cultured cells.

vDNA contributes to the production of vpiRNAs in mosquito cell lines. Infected U4.4 cells were (a) left untreated (clear bars) or (b) treated with 5 mM AZT (green bars) for 3 days. Graph represents the size distribution of the total number of CHIKV-specific small RNA reads (ranging from 18 to 33 nts) normalized to the total number of reads. (c) vsiRNAs in U4.4 cells show homogenous coverage over the entire genome. (d) vsiRNA coverage in AZT-treated cells. Uncovered regions are represented as grey lines. The coverage of vpiRNAs on the CHIKV genome in (e) untreated cells or (f) AZT-treated cells. Most of the CHIKV vpiRNA reads belong to the 3′-terminal region on the viral genome. The sense and anti-sense small RNAs are in red and green, respectively. (g-h) Relative nucleotide frequency per position of the 27–29 nt viral small RNAs that map to the sense and anti-sense strand of the viral genome, red and green respectively. The intensity varied in correlation with the frequency. A nucleotide bias (U1 and A10) is observed. (i,j) Accumulation of viral and cellular small RNAs in U4.4 cells 3 days post CHIKV infection in the presence (AZT pos) or absence (AZT neg) of AZT, assessed as (i) mapping of small RNAs corresponding to CHIKV vsiRNA (green), endo-siRNA (purple) belonging to the gene GAPW01000199 or to miRNA (blue). For miRNA each dot represents one miRNA. For siRNAs each dot represents the coverage of a region of 20 bases of the target RNA. The lines for miRNA and endo-siRNA are superposed. (j) Mapping of small RNA corresponding to CHIKV vpiRNA (orange), endo-piRNA (red) belonging to the gene GAPW01000199 or to miRNA (blue). For miRNAs each dot represents one miRNA. For piRNAs each dot represents the coverage of a region of 20 bases of the target RNA. For (i,j) lines represent the linear trendline of each set of values. The equation and R² value of each regression are also mentioned.