Fig 1.
RNA secondary structures in the CHIKV 3’UTR.
(A) Schematic representation of the genomic organization of CHIKV (left) and its 3’UTR (right) for the ECSA, IOL, and Asian epidemic lineages and the Caribbean strain. Colored boxes represent different DRs. The DR3 region corresponding to SLY is colored in pink. (B) Sequence alignment (middle) and nucleotide conservation plot (bottom, in gray) of the 80 nt-long DR3 region that folds into SLYa and SLYb, for ten representative ECSA, IOL, and Asian lineages isolates. Pink arcs and boxes indicate the nucleotide pairings that form the stems of each Y. Conserved nucleotides are in black, and variable nucleotides are in red, blue, green and orange. The black box indicates the nucleotide position 45/49 that covariates among lineages. (C) Consensus RNA secondary structures predicted for SLYa and SLYb. Left, minimum-free energy foldings with the corresponding consensus sequences. Middle, base-pair probabilities. For unpaired regions, the color represents the probability of being unpaired. Right, the SHAPE-constrained thermodynamic model for SLYa and SLYb. Nucleotide positions are numbered every 10 nucleotides. SLY, stem-loop Y; S1, stem 1; S2, stem 2; S3, stem 3; L1, loop 1; L2, loop 2; B1, bulge 1; B2, bulge 2; B3, bulge 3; B4, bulge 4.
Fig 2.
Replication and adaptation to mammalian cells of CHIKV-Caribbean and mutant viruses with SLY deletions.
(A) Left, schematic representations of the 3’UTRs of the WT virus and mutant viruses with deletions of one or two SLY copies (ΔSLYa, ΔSLYb and ΔSLYab). Right, immunofluorescence stainings for WT and mutant viruses in mammalian BHK cells on day 3 post-transfection. Images correspond to one representative experiment out of three biological replicates. (B) Viral yields in cell culture supernatant on day 3 post-transfection for WT and mutant viruses. The symbols and bars depict the means ± standard deviations of the means from three independent experiments. Data were compared with a two-tailed, unpaired t-test. Top, in vitro transcribed (IVT) RNAs used for transfection in the experiments shown in Figs 2 and 4. (C) Comparative growth kinetics for WT and ΔSLYab viruses in human fibroblasts and Huh-7 cells. Cells were infected with MOI = 0.1 and data were compared with a two-tailed, unpaired t-test. The symbols and bars depict the means ± standard deviations of the means from two independent experiments. (D) Analysis of the 3’UTRs of the WT and mutant viruses restricted to replication in mammalian cells. Top, schematic representation of viral passages and analysis of the 3’UTRs. Middle, bar graphs showing the frequencies of viral variants in WT and mutant viral populations P1 and P5 in mammalian cells. The ratio of 3’UTR deletion variants to total clones is indicated inside the bars and corresponds to the cumulative data from three independent experiments. Data were compared pairwise with Fisher’s exact test on cumulative data. Bottom, table with the proportion of 3’UTR deletion variants from three independent experiments (n1, n2 and n3) in the WT, ΔSLYa, ΔSLYb and ΔSLYab populations. (E) Representative agarose gels for PCR amplification of the 3’UTRs of individual clones recovered from WT and mutant viral populations passaged five times in BHK cells. The sizes of DNA bands in the ladder (base pairs) are indicated on the right. (F) Schematic representation of the alignment of the 3’UTRs of viral variants comprising the WT P5 population. Red lines indicate deletions within viral variants. The corresponding sequence for each variant is shown in S1 Fig.
Fig 3.
RNA recombination in the CHIKV 3’UTR through template switching between homologous templates.
(A) Schematic representation of copy-choice recombination mechanism between WT (top) or ΔSLYab (bottom) viruses differently marked with XbaI (WT-XbaI and ΔSLYab-XbaI) or AvrII (WT-AvrII and ΔSLYab-AvrII) at the 5’ and 3 ‘ends of their 3’UTRs, respectively. After cotransfections, recombinant viruses arise at the 3’UTR (XbaI/AvrII and −/−). (B) Immunofluorescence of WT, WT-XbaI, WT-AvrII, ΔSLYab, ΔSLYab-XbaI, and ΔSLYab-AvrII viruses on day 3 post-transfection of BHK cells (top) and assessment of the presence of XbaI or AvrII marker sites after four successive passages in BHK cells (bottom). Depending on the orientation of each inserted fragment into the blunt plasmid, digestion of XbaI positive clones generated 439 or 363 nt products, and digestion of AvrII positive clones generated 193 or 181 nt products. (C) For illustration, undigested and digested products from eight representative clones are shown for each cotransfection mixture. Recombinant 3’UTR clones are indicated with an inverted gray triangle under each agarose gel. (D) Only clones spanning the full-length fragments were numbered to calculate recombination frequencies. Left, bar graphs showing the relative abundance of recombinant and nonrecombinant full-length 3’UTRs from WT and ΔSLYab viral populations. Nonrecombinant XbaI and AvrII 3’UTR variants are indicated in orange and violet plain colors, respectively, and recombinant XbaI/AvrII and −/− 3’UTR variants are indicated in squared violet/orange and gray/white patterns, respectively. Data were compared with Fisher’s exact test on cumulative data from two biological replicates. Key: ns, not significant. Right, table showing the proportion of 3’UTR variants from two independent experiments (n1 and n2).
Fig 4.
Replication of WT CHIKV and viruses with SLY deletions in mosquito cells.
(A) Left, schematic representation of the CHIKV-Caribbean WT virus and mutant viruses with deletions of one or two SLY copies. Right, immunofluorescence staining for WT and mutant viruses in mosquito C6/36 cells on days 3, 6, and 9 post-transfection. Images correspond to one representative experiment out of three biological replicates. The data were analyzed and presented as described in Fig 2. (B) Viral yields in cell culture supernatants on day 6 post-transfection for WT and mutant viruses. The symbols and bars depict the means ± standard deviations of the means from three independent experiments. Data were compared with a two-tailed, unpaired t-test. (C) Comparative growth kinetics of WT and ΔSLYab viruses in C6/36 cells. Cells were infected with MOI = 0.1, and data were compared with a two-tailed, unpaired t-test. The symbols and bars depict the means ± standard deviations of the means from two independent experiments.
Fig 5.
Replication of recombinant viruses with mutations that maintain or disrupt the SLY structure in mosquito cells.
(A) Left, schematic representation of the mutations introduced into the SLY in order to generate recombinant viruses that maintain (MutL1 and RecS3) or disrupt (MutS1 and MutS3) its predicted Y-shape RNA structure. In each case, the sequences for WT and mutated SLYs are indicated inside gray and colored boxes, respectively. Right, a schematic representation of the 3’UTRs with SLYa mutations inserted into ΔSLYb (top) and vice versa (bottom). (B and C) Left, immunofluorescences for the mutant ΔSLYb, MutS1_ΔSLYb, MutS3_ΔSLYb, MutL1_ΔSLYb, and RecS3_ ΔSLYb viruses (B) and ΔSLYa, MutS1_ΔSLYa, MutS3_ΔSLYa, MutL1_ΔSLYa, and RecS3_ ΔSLYa viruses (C) in C6/36 and BHK cells at the indicated time points. Images correspond to one representative experiment, with data analyzed as described in Fig 2. Right, viral yields in cell culture supernatants on days 3, 6 and 9 post-transfection. The symbols and bars depict the means ± standard deviations of the means from two independent experiments. Data were compared with a two-tailed, unpaired t-test. Agarose gels with IVT RNAs used for transfection in a representative experiment are shown.
Fig 6.
RNA secondary structures in the 3’UTR of Sindbis group viruses.
(A) Left, schematic representation of the genomic organization of SINV and its 3’UTR with the RSEs indicated as light blue boxes and regions folding into SLYs in white boxes. Right, phylogenetic relationship between the Sindbis group members (SINV, Babanki ⦍BABV⦎, Whataroa ⦍WHAV⦎, Western equine encephalitis ⦍WEEV⦎, and Aura ⦍AURAV⦎ viruses) and their 3’UTRs. (B) Sequence alignment (middle) and nucleotide conservation plot (bottom, in gray) of the RSEs that fold into SLYa, SLYb, and SLYc for SINV (NC_001547.1), BABV (MF409178.1), WHAV (NC_016961.1), AURAV (NC_003900.1), and WEEV (NC_003908.1). Pink arcs and boxes indicate the nucleotides that pair to form the stems of each Y. Conserved nucleotides are in black, and variable nucleotides are in blue, red, green, and orange. Black boxes indicate the nucleotides that covary among viruses. Key: S, stem; L, loop. (C) Predicted RNA secondary structures for the RSEs in the 3’UTRs of SINV, BABV, WHAV, AURAV and WEEV. Base-pair probabilities are color-coded, and the minimum-free energy for each SLY is shown below. (D) Schematic representation of the SLY regions conserved (in light blue) or covarying (in black) between different SINV group viruses and SLY copies.
Fig 7.
Replication of CHIKV chimeric viruses with the SINV 3’UTR in mosquito and mammalian cells.
(A) Left, schematic representation of CHIKV-Caribbean virus and its 3’UTR with two SLY copies (WT) and a chimeric CHIKV- Caribbean virus with the SINV 3’UTR (chimeric virus). Right, immunofluorescence stainings of WT and chimeric viruses in mosquito C6/36 cells on days 3, 6, and 9 post-transfection and in mammalian BHK cells on days 1 and 3 post-transfection. Images correspond to one representative experiment out of two biological replicates. Data were analyzed as described in Fig 2. Viral yields in cell culture supernatants for the WT and the chimeric virus in C6/36 cells (B) and BHK cells (C). The symbols and bars depict the means ± standard deviations of the means from two independent experiments. An agarose gel with IVT RNAs used for transfection. Data were compared with a two-tailed, unpaired t-test. Bottom, a representative agarose gel of the PCR amplification of the 3’UTRs of individual chimeric virus clones after P5 in BHK cells. A DNA ladder (M) was used as the reference. The sizes of DNA bands in the ladder (in base pairs) are indicated on the right.