FIG 2.

Effects of nsP2 linker mutants on the activity of CHIKV trans-replicase. (A) Schematic representation of the domain arrangement of CHIKV nsP2. Domains of nsP2 are colored as in Fig. 1D. Sequence alignment of the nsP2 linker regions from alphaviruses, including CHIKV, Eilat virus (EILV), Venezuelan equine encephalitis virus (VEEV), sleeping disease virus (SDV), SINV, o’nyong’nyong virus (ONNV), Ross River virus (RRV), Semliki Forest virus (SFV) and Eastern equine encephalitis virus (EEEV). (B) Schematic representation of CMV-P1234 (Ubi-P1234 has the same design except that it uses the Aedes aegypti polyubiquitin promoter) and pol I-Fluc-Gluc constructs. The trans-replication system for human cells is composed of a cytomegalovirus promoter-based plasmid (CMV-P1234) for the expression of the CHIKV replicase and a plasmid for the production of replication-competent RNA containing firefly luciferase (Fluc) and Gaussia luciferase (Gluc) reporters. CMV, immediate early promoter of human cytomegalovirus; L1, leader region of herpes simplex virus thymidine kinase gene with an artificial intron; SV40 term, simian virus 40 late polyadenylation region; polI, truncated promoter for human or Aedes albopictus RNA polymerase I. The 5′ and 3′ untranslated regions (UTRs) are from CHIKV. N77, region encoding the 77 N-terminal amino acid residues of nsP1; SG, CHIKV SG promoter; RZ, antisense strand ribozyme of hepatitis delta virus; polI term, terminator for RNA polymerase I from mouse or from Aedes albopictus. Positions of linker mutations (delQ465, delSHQ, delSHQMT, insGSG, ins6GS, and ins10GS) and NTP binding site mutations (K192A) are indicated. In this assay, the increases in expression levels of Fluc and Gluc represent the production of full-length genomic RNA and SG RNA, respectively. (C and D) Effects of introduced mutations on CHIKV replicase activity in human cells. U2O2 cells were cotransfected with CMV-P1234 or its mutants and HSPolI-Fluc-Gluc plasmids; as a negative control, CMV-P1234^GAA, which lacks polymerase activity, was used instead of CMV-P1234. Cells were incubated at 37°C and lysed 18 h posttransfection. Fluc (marker of replication) (C) and Gluc (marker of transcription) (D) activities produced by analyzed replicases were normalized to the P1234^GAA control. The value obtained for the P1234^GAA control was set as 1. The means and SD from three independent experiments are shown. *, P < 0.05; **, P < 0.01 (Student's unpaired t test). (E) Effects of introduced mutations on the transcriptional activity of CHIKV replicase in Aedes albopictus C6/36 cells. C6/36 cells were cotransfected with Ubi-P1234 or its mutants and the AlbPolI-Fluc-Gluc plasmid; as a negative control, Ubi-P1234^GAA, which lacks polymerase activity, was used instead of Ubi-P1234. Cells were incubated at 28°C and lysed 48 h posttransfection. Gluc (marker of transcription) activities produced by analyzed replicases were normalized to the P1234^GAA control. The value obtained for the P1234^GAA control was set as 1. The means and SD from three independent experiments are shown; ***, P < 0.001 (Student's unpaired t test).