G3BP/Rin-Binding Motifs Inserted into Flexible Regions of nsP2 Support RNA Replication of Chikungunya Virus - PubMed (original) (raw)

G3BP/Rin-Binding Motifs Inserted into Flexible Regions of nsP2 Support RNA Replication of Chikungunya Virus

Sainan Wang et al. J Virol. 2022.

Abstract

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus. In infected cells, its positive-sense RNA genome is translated into polyproteins that are subsequently processed into four nonstructural proteins (nsP1 to 4), the virus-encoded subunits of the RNA replicase. However, for RNA replication, interactions between nsPs and host proteins are also needed. These interactions are mostly mediated through the intrinsically disordered C-terminal hypervariable domain (HVD) in nsP3. Duplicate FGDF motifs in the HVD are required for interaction with mammalian RasGAP SH3-binding proteins (G3BPs) and their mosquito homolog Rin; these interactions are crucial for CHIKV RNA replication. In this study, we inactivated G3BP/Rin-binding motifs in the HVD and inserted peptides containing either native or inactivated G3BP/Rin-binding motifs into flexible regions of nsP1, nsP2, or nsP4. Insertion of native motifs into nsP1 or nsP2 but not into the C terminus of nsP4 activated CHIKV RNA replication in human cells in a G3BP-dependent manner. In mosquito cells, activation also resulted from the insertion of inactive motifs after residue 8 or 466 in nsP2; however, the effect was significantly larger when the inserted sequence contained native motifs. Nonetheless, CHIKV mutants harboring mutations in the HVD and containing insertions of native motifs in nsP2 were not viable in mosquito cells. In contrast, mutant genomes containing native motifs after residue 466 or 618 in nsP2 replicated in BHK-21 cells, with the latter mutant forming infectious progeny. Thus, the binding of G3BPs to nsP2 can support CHIKV RNA replication and restore the infectivity of viruses lacking G3BP-binding motifs in the HVD of nsP3. IMPORTANCE CHIKV is a reemerging alphavirus that has spread throughout more than 60 countries and is the causative agent of chikungunya fever. No approved drugs or vaccines are available for the treatment or prevention of CHIKV infection. CHIKV replication depends on the ability of its replicase proteins to interact with host cell factors, and a better understanding of host cell factor roles in viral infection will increase our understanding of CHIKV RNA replication and provide new strategies for viral infection attenuation. Here, we demonstrate that the motifs required for the binding of host G3BP/Rin proteins remain functional when transferred from their natural location in nsP3 to different replicase proteins and may enable mutant viruses to complete a full replication cycle. To our knowledge, this is the first demonstration of interaction motifs for crucial host factors being successfully transferred from one replicase protein to another subunit of alphavirus replicase.

Keywords: G3BP; RNA replication; Rin; alphavirus; chikungunya virus; nsP2; nsP3.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1

FIG 1

Insertion of WT or F3A peptide into the nsP2 region activates a CHIKV _trans_-replicase lacking G3BP/Rin-binding motifs in the HVD of nsP3 in C6/36 cells. (A) Schematic presentation of the CHIKV _trans_-replicase system. In CHIKV RNA template expression plasmids AlbPolI-Fluc-Gluc and HSPolI-Fluc-Gluc: Alb/HSPolI, truncated promoter for Aedes albopictus or human RNA polymerase I; 5′ untranslated region (UTR), CHIKV 5′ untranslated region, N77, the region encoding the 77 N-terminal amino acid residues of CHIKV nsP1; SG, CHIKV subgenomic RNA promoter; 3′ UTR, truncated CHIKV 3′ untranslated region; HDV RZ, antisense-strand ribozyme of the hepatitis delta virus; Alb/MmTer, terminator of RNA polymerase I in Aedes albopictus or mice. In replicase expression plasmids Ubi-P1234 and CMV-P1234: Ubi, full-length Aedes aegypti polyubiquitin promoter; UL, transcribed leader of polyubiquitin genes containing a naturally occurring intron; CMV, human cytomegalovirus immediate early promoter; L1, leader region in the herpes simplex virus thymidine kinase gene with an artificial intron; SV40Ter, simian virus 40 late polyadenylation region. F3A designates mutations preventing the binding of G3BP/Rin to the HVD in nsP3, and GAA designates a position with a GDD-to-GAA mutation in the catalytic site in nsP4. The arrows indicate positions where a WT or F3A peptide was inserted into nsP1, nsP2, and nsP4. Amino acid sequences of peptides are provided under the drawing; flexible linker residues are shown in italics; WT or mutant G3BP/Rin-binding motifs are underlined with WT (F) and mutant (A) residues shown in color. (B to D) Expression of CHIKV nsP1 (B), nsP2 (C), and nsP3 (D) in C6/36 cells transfected with different replicase expression plasmids. Cells were collected 48 hpt and lysed, and the obtained samples were analyzed using Western blotting. β-actin was used as the loading control. Positions of CHIKV nsP1, nsP2, nsP3, and P23 are shown; The asterisk (*) indicates mosquito protein recognized by an anti-nsP3 antibody. (E) C6/36 cells were cotransfected with AlbPolI-Fluc-Gluc and Ubi-P1234 (P1234), Ubi-P123F3A4 (P123F3A4), or its variants containing insertions encoding the WT or F3A peptide. Control cells were cotransfected with AlbPolI-Fluc-Gluc and Ubi-P1234GAA. Cells were lysed 48 hpt, and the activities of Gluc (a marker of transcription) were measured and normalized to those of the P1234GAA control (equal to 1; values below that of the control are also shown as 1). Each column represents an average based on three independent experiments; error bars represent the standard deviation. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s unpaired t test).

FIG 2

FIG 2

Insertion of WT peptide into nsP1 or nsP2 activates CHIKV _trans_-replicase lacking G3BP/Rin-binding motifs in the HVD of nsP3 in U2OS cells. (A to C) Expression of CHIKV nsP1 (A), nsP2 (B), and nsP3 (D) in U2OS cells transfected with different replicase expression plasmids. Cells were collected 18 hpt, lysed, and analyzed using Western blotting. β-actin was used as the loading control. Positions of CHIKV nsP1, nsP2, nsP3, and P23 are shown. (D) U2OS cells were cotransfected with HSPolI-Fluc-Gluc and CMV-P1234 (P1234), CMV-P123F3A4 (P123F3A4), or its variants containing an insertion encoding the WT or F3A peptide. Control cells were cotransfected with HSPolI-Fluc-Gluc and CMV-P1234GAA. Cells were lysed 18 hpt, and the activities of Fluc (a marker of replication, left panel) and Gluc (a marker of transcription, right panel) were measured and normalized to those of the P1234GAA controls (equal to 1; values below those of control are also shown as 1). Each column represents an average based on three independent experiments; error bars represent the standard deviation. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s unpaired t test).

FIG 3

FIG 3

G3BP1/Rin binds to nsP2 containing the WT peptide but not the F3A peptide. (A) U2OS cells were transfected with CMV-EGFP-nsP2, CMV-EGFP-nsP2pcWT, or CMV-EGFP-nsP2pcF3A. Cells were fixed at 24 hpt and immunostained for G3BP1 and CHIKV nsP2. Scale bar = 20 μm. (B) (left) U2OS cells were transfected with CMV-EGFP-nsP2, CMV-EGFP-HVD, CMV-EGFP-nsP2paWT, CMV-EGFP-nsP2paF3A, CMV-EGFP-nsP2pbWT, CMV-EGFP-nsP2pbF3A, CMV-EGFP-nsP2pb10GS, CMV-EGFP-nsP2pcWT, or CMV-EGFP-nsP2pcF3A or mock-transfected; (right) C6/36 cells were cotransfected with Ubi-AlbRin-V5 and Ubi-EGFP-nsP2, Ubi-EGFP-HVD, Ubi-EGFP-nsP2paWT, Ubi-EGFP-nsP2paF3A, Ubi-EGFP-nsP2pbWT, Ubi-EGFP-nsP2pbF3A, Ubi-EGFP-nsP2pb10GS, Ubi-EGFP-nsP2pcWT, or Ubi-EGFP-nsP2pcF3A. At 24 hpt, the cells were lysed, and recombinant proteins and the cellular proteins bound to them were pulled down using EGFP-binding magnetic beads. The samples obtained were analyzed by immunoblotting using antibodies against nsP3, nsP2, and G3BP1 (left) or the V5 epitope tag (right).

FIG 4

FIG 4

Overexpression of G3BP1 affects the activity of CHIKV _trans-_replicase in U2OS ΔΔ cells. U2OS ΔΔ cells were cotransfected with HSPolI-Fluc-Gluc and CMV-P1234 (P1234), CMV-P123F3A4 (P123F3A4) or its variants containing insertions encoding the WT or F3A peptide. Control cells were cotransfected with HSPolI-Fluc-Gluc and CMV-P1234GAA. (A and B) The CMV-G3BP1 plasmid was either not added (A) or added (B) to the transfection mixture. The experiment was performed and the data were collected and analyzed as described in the legend of Fig. 2D. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s unpaired t test).

FIG 5

FIG 5

The flexible linker inserted after residue 466 of CHIKV nsP2 increases the RNA replicase activity of CHIKV P123F3A4 in a G3BP-independent manner. (A to C) U2OS (A) or U2OS ΔΔ cells (B and C) were cotransfected with HSPolI-Fluc-Gluc and CMV-P1234 (P1234), CMV-P123F3A4 (P123F3A4), or its variants containing an insertion encoding the WT peptide, F3A peptide, or 10GS linker after residue 466 of nsP2. Control cells were cotransfected with HSPolI-Fluc-Gluc and CMV-P1234GAA. The CMV-G3BP1 plasmid was either not added (A and B) or added (C) to the transfection mixture. Cells were lysed 18 hpt, and the activities of Fluc (a marker of replication, left panels) and Gluc (a marker of transcription, right panels) were measured and normalized to those of the P1234GAA controls (equal to 1; values below these of the control are also shown as 1). Each column represents an average based on three independent experiments; error bars represent the standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s unpaired t test).

FIG 6

FIG 6

Insertion of the sequence encoding the WT or F3A peptide in the nsP2 region of SP6-3F3ACHIKV does not rescue replication of corresponding transcripts in C6/36 cells. (A) C6/36 cells were transfected with 5 μg of capped RNA transcripts of SP6-CHIKV, SP6-3F3ACHIKV, or their mutants harboring an insertion in the nsP2 region. The transfected cells were incubated at 28°C for 72 h, after which, P0 virus stocks were collected. The cells were harvested and lysed, and the samples obtained were subjected to SDS-PAGE with 12% gels. The CHIKV capsid protein was detected using the corresponding antibody; an antibody against β-actin was used to detect the loading control. The number of viral RNA copies in selected P0 stocks was measured using RT-qPCR and is presented below the panel as the RNA copy/mL; ND, not detectable; NA, not analyzed. (B) A total of 150 μL of each obtained P0 stock was used to infect 1 × 106 BHK-21 cells. The cells were incubated at 37°C until CPEs were observed or for 72 h, collected, and analyzed as described in panel A. The data from one of three independent reproducible experiments are shown.

FIG 7

FIG 7

Insertion of the sequence encoding the WT but not the F3A peptide in the nsP2 region of SP6-3F3ACHIKV rescues replication of corresponding transcripts in BHK-21 cells. (A) BHK-21 cells were transfected using 5 μg of capped RNA transcripts of SP6-CHIKV, SP6-3F3ACHIKV, or their mutants harboring an insertion in the nsP2 region. The transfected cells were incubated at 37°C until CPEs were observed or for 72 h, after which, P0 virus stocks were collected, and cells were harvested. Virus stocks and cell lysates were analyzed, and the data are presented as described in Fig. 6A. (B) A total of 150 μL of each obtained P0 stock was used to infect 1 × 106 BHK-21 cells. The experiment was performed, and the data are presented as described in Fig. 6B. The data from one of three independent reproducible experiments are shown.

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