Fig 1.
Alphaviruses selected for the analysis and designs of trans-replication tools.
(A) Schematic representation of constructs for human cells. CMV, immediate early promoter of human cytomegalovirus; LI, leader sequence of the herpes simplex virus thymidine kinase gene with artificial intron; SV40Ter, simian virus 40 late polyadenylation region; HSPolI, a truncated promoter (residues −211 to −1) for human RNA polymerase I; MmTer, a terminator for RNA polymerase I in mice. (B). Schematic representation of constructs for Aedes albopictus cells. UBI, polyubiquitin promoter of Aedes aegypti; UL, leader sequence of Aedes aegypti polyubiquitin gene with a natural intron; AlbPolI–truncated promoter (residues −250 to −1) for Aedes albopictus RNA polymerase I; AlbTer–putative terminator for Aedes albopictus RNA polymerase I. (A, B) 5′ UTR, full length 5’ UTR of an alphavirus; 3’ UTR, truncated (last 110 residues) 3′ UTR of an alphavirus; SG—SG promoter spanning (with respect to termination codon of nsP4) from position -79 to the end of intergenic region, nsP1 N*—region encoding the N-terminal 77 to 114 amino acid residues of nsP1, depending on the virus; HDV RZ—antisense strand ribozyme of hepatitis delta virus. Red arrow indicates the location of the GDD motif in nsP4; in polymerase negative constructs this was replaced by GAA. The vector backbones are not shown and drawings are not to scale. (C) Phylogenetic tree of replicases of analysed alphaviruses. Phylogenetic tree was constructed using evolutionary analysis by Maximum Likelihood method and JTT matrix based model. The tree is drawn to scale, with branch lengths measured in the number of substitution per site. This analysis involved sequences of P1234 of indicated viruses. Evolutionary analysis was conducted using MEGA-X software.
Fig 2.
Alphavirus replicases can replicate and transcribe their templates in human cells.
(A) HEK293T cells in 12-well plates were co-transfected with matching pairs of CMV-P1234 and HSPolI-FG plasmids. At 4, 8, 12, 16, 20, 22, 24 and 48 h p.t. growth media aliquots were collected the activity of secreted Gluc measured. Means of relative luminescence units (RLU) per 1 μl of sample + standard deviation (SD) of three independent experiments are shown. (B) HEK293T cells in 96-well plates were co-transfected with matching pairs of CMV-P1234 and HSPolI-FG plasmids and, as negative control, CMV-P1234GAA, which lacks polymerase activity, instead of CMV-P1234. Cells were incubated at 37°C and lysed 18 h post transfection (p.t.); cells transfected with plasmids containing sequences from EILV were also incubated at 28°C and lysed 48 h p.t. Fluc (marker of replication, left panel) and Gluc (marker of transcription, right panel) activities produced by active replicases were normalized to the P1234GAA controls. Value obtained for P1234GAA controls was taken as 1; activities lower than that observed for P1234GAA are also shown as 1. Means + SD of three independent experiments are shown; p<0.01**; p<0.001*** (Student's unpaired t-test). (C) HEK293T cells in 12-well plates were co-transfected and incubated as described for panel A; control cells were mock-transfected. Total RNA was extracted and analysed by northern blotting. Full-length “genomic” template RNA of positive (+) and negative (-) polarity and subgenomic RNA are indicated. Note that transcripts made by human RNA polymerase I using HSPolI-FG plasmids as templates co-migrate with replicase-generated positive-strand genomic RNA and are detected by the same probe. The experiment was repeated twice with similar results; data from one experiment is shown.
Fig 3.
Alphavirus replicases can replicate and transcribe their templates in Aedes albopictus cells.
(A) C6/36 cells in 12-well plates were co-transfected with matching pairs of Ubi-P1234 and AlbPolI-FG plasmids. At 12, 24. 36, 48, 54, 66, 72 and 144 h p.t. growth media aliquots were collected the activity of secreted Gluc measured. Means of relative luminescence units (RLU) per 1 μl of sample + SD of three independent experiments are shown. (B) C6/36 cells in 96-well plates were co-transfected with matching pairs of AlbPolI-FG and either Ubi-P1234 or Ubi-P1234GAA plasmids. Cells were incubated at 28°C and lysed 48 h p.t. The Fluc (left panel) and Gluc (right panel) activities produced by P1234 replicases were normalized to the polymerase-defective P1234GAA controls as described for Fig 2. Means + SD of three independent experiments are shown. (C) C6/36 cells in 12-well plates were co-transfected as described for panel A. Total RNA was extracted and positive strand RNAs were detected using northern blotting. “Genomic” indicates the full-length template RNA. Note that transcripts made by mosquito RNA polymerase I using AlbPolI-FG plasmids as template co-migrate with replicase-generated positive-strand genomic RNAs. “Subgenomic” indicates the SG RNAs synthesized by replicases using the SG promoter. The experiment was repeated twice with similar results; data from one experiment is shown. (D) HEK293T cells (left panel) were co-transfected with HSPolI-FZsG-CHIKV and CMV-P1234-CHIKV; C6/36 cells (right panel) were co-transfected with AlbPolI-FZsG-CHIKV and Ubi-P1234-CHIKV. Control cells were transfected with plasmid expressing EGFP from a CMV promoter (HEK293T) or from a polyubiquitin promoter (C6/36). At 18 h (HEK293T) or 48 h (C6/36) p.t. cells were collected and analyzed with an Attune NxT Acoustic Focusing Cytometer. The number of ZsGreen-expressing cells is shown as a proportion of the number of EGFP expressing cells, to control for the different transfection efficiency of the two cell types. All transfections were performed in triplicate, means + SD are shown.
Fig 4.
Cross-utilization of templates of viruses belonging to the SFV complex in human cells.
HEK293T cells were co-transfected with combinations of each CMV-P1234 replicase expression plasmid or corresponding CMV-P1234GAA control and (A) HSPolI-FG-CHIKV, (B) HSPolI-FG-ONNV, (C) HSPolI-FG-RRV, (D) HSPolI-FG-SFV or (E) HSPolI-FG-MAYV. Transfected cells were incubated at 37°C and lysed 18 h p.t.; cells transfected with plasmids originating from EILV were incubated at 28°C and lysed 48 h p.t. Data represent the luciferase activity (Fluc and Gluc) from CMV-P1234 transfected cells normalized to the paired CMV-P1234GAA control cells. Value obtained for P1234GAA controls was taken as 1; activities lower than that observed for P1234GAA are also shown as 1. X-axis represents different replicases, means + SD are from three independent experiments.
Fig 5.
Cross-utilization of templates of outgroup alphaviruses in human cells.
HEK293T cells were co-transfected with combinations of each CMV-P1234 replicase expression plasmid or corresponding CMV-P1234GAA control and (A) HSPolI-FG-SINV, (B) HSPolI-FG-BFV, (C) HSPolI-FG-VEEV or (D) HSPolI-FG-EILV. Transfected cells were incubated at 37°C and lysed 18 h p.t.; cells transfected with plasmids originating from EILV were incubated at 28°C and lysed 48 h p.t. Data represent the luciferase activity (Fluc and Gluc) from CMV-P1234 transfected cells normalized to the paired CMV-P1234GAA control cells. Value obtained for P1234GAA controls was taken as 1; activities lower than that observed for P1234GAA are also shown as 1. X-axis represents different replicases, means + SD are from three independent experiments.
Fig 6.
Incompatible combinations of alphavirus replicases and templates have defects in synthesis of viral RNAs.
(A) HEK293T cells were co-transfected with combinations of HSPolI-FG template and CMV-P1234 replicase expression plasmids as indicated; in control cells CMV-P1234 was replaced with CMV-P1234GAA. Cells were lysed 18 h p.t. Data represent the luciferase activity (Fluc and Gluc) from CMV-P1234 transfected cells normalized to the paired CMV-P1234GAA control cells. Data is replotted from Fig 4 and Fig 5. Means + SD are from three independent experiments; ns, not significant, ****p<0.0001 (Student's unpaired t-test). (B) HEK293T cells were co-transfected with combinations of CMV-P1234 and HSPolI-FG plasmids as indicated. At 18 h p.t. cells were lysed and total RNAs were isolated. RNAs were analysed by northern blot as described for Fig 2B. The experiment was repeated twice with similar results; data from one experiment is shown.
Fig 7.
Cross-utilization of templates of viruses belonging to the SFV complex in Aedes albopictus C6/36 cells.
C6/36 cells were co-transfected with combinations of each Ubi-P1234 replicase expression plasmid or corresponding Ubi-P1234GAA control and (A) AlbPolI-FG-CHIKV, (B) AlbPolI-FG-ONNV, (C) AlbPolI-FG-RRV, (D) AlbPolI-FG-SFV or (E) AlbPolI-FG-MAYV. Transfected cells were incubated at 28°C and lysed 48 h p.t. Data represent the luciferase activity (Fluc and Gluc) from Ubi-P1234 transfected cells normalized to the paired Ubi-P1234GAA control cells. Value obtained for P1234GAA controls was taken as 1; activities lower than that observed for P1234GAA are also shown as 1. X-axis represents different replicases, means + SD from three independent experiments are shown.
Fig 8.
Cross-utilization of templates of outgroup alphaviruses in Aedes albopictus C6/36 cells.
C6/36 cells were co-transfected with combinations of each Ubi-P1234 replicase expression plasmid or corresponding Ubi-P1234GAA control and (A) AlbPolI-FG-SINV, (B) AlbPolI-FG-BFV, (C) AlbPolI-FG-VEEV or (D) AlbPolI-FG-EILV. Transfected cells were incubated at 28°C and lysed 48 h p.t. Data represent the luciferase activity (Fluc and Gluc) from Ubi-P1234 transfected cells normalized to the paired Ubi-P1234GAA control cells. Value obtained for P1234GAA controls was taken as 1; activities lower than that observed for P1234GAA are also shown as 1. X-axis represents different replicases, means + SD from three independent experiments are shown.
Fig 9.
Specificity for the replicase resides in the 5’- and SG regions of the template RNA.
(A) Schematic representation and explanation of names of SINV, CHIKV and RRV swapped template RNAs. 5’R—5’ region; 3’R– 3’ region, C* - 5’ region of CHIKV template containing 336nt from nsP1 encoding region; C—5’ region of CHIKV template containing 231nt from nsP1 encoding region. Other elements are the same as on Fig 1A. (B-E) U2OS cells grown in 12-well plate were co-transfected with plasmids encoding for indicated template RNAs and with (B, D) CMV-P1234-CHIKV, (C) CMV-P1234-SINV or (E) CMV-P1234-RRV. For control cells the plasmid expressing active replicase was substituted with corresponding CMV-P1234GAA plasmid. Cells were lysed 18 h p.t. Data represent the luciferase activity (Fluc and Gluc) from CMV-P1234 transfected cells normalized to the paired CMV-P1234GAA control cells. Means + SD are from three (B, C, D) or four (E) independent experiments are shown; ns, not significant, *p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001 (Student's unpaired t-test).
Fig 10.
Determinants located at the 5’ end of the template RNA are crucial for SINV replicase.
(A). Schematic representation of template RNAs. Changes introduced into the 5’ region of S-S-S template RNA are shown on left panel. Right panel shows 5’ UTR sequences of modified S-S-S and css-S-S templates. SL structures in CHIKV sequence are designated SL3 and SL47 according to [33]. Other elements and designations are the same as Figs 1A and 9A. (B). U2OS cells grown in 12-well plate were co-transfected with CMV-P1234-SINV and HSPolI-FG-SSS, HSPolI-FG-C*CC, HSPolI-FG-C*SS or derivatives of HSPolI-FG-SSS expressing templates containing indicated swaps in their 5' region. For transfection of control cells CMV-P1234GAA-SINV and HSPolI-FG-SSS were used. (C) U2OS cells grown on 12-well plate were co-transfected with CMV-P1234-SINV and HSPolI-FG-SSS, HSPolI-FG-cssSS or their derivatives expressing templates containing indicated mutations in the SL3 and/or in extreme 5’ end of the template. For transfection of control cells CMV-P1234GAA-SINV was used instead of CMV-P1234-SINV. Cells were lysed 18 h p.t. Fluc activities produced by active replicases were normalized to those measured in control cells. Means + SD of three independent experiments are shown; ** p<0.01, ***p<0.001, ****p<0.0001(Student's unpaired t-test).