Expression of Alphavirus Nonstructural Protein 2 (nsP2) in Mosquito Cells Inhibits Viral RNA Replication in Both a Protease Activity-Dependent and -Independent Manner

Abstract

Alphaviruses are positive-strand RNA viruses, mostly being mosquito-transmitted. Cells infected by an alphavirus become resistant to superinfection due to a block that occurs at the level of RNA replication. Alphavirus replication proteins, called nsP1-4, are produced from nonstructural polyprotein precursors, processed by the protease activity of nsP2. Trans-replicase systems and replicon vectors were used to study effects of nsP2 of chikungunya virus and Sindbis virus on alphavirus RNA replication in mosquito cells. Co-expressed wild-type nsP2 reduced RNA replicase activity of homologous virus; this effect was reduced but typically not abolished by mutation in the protease active site of nsP2. Mutations in the replicase polyprotein that blocked its cleavage by nsP2 reduced the negative effect of nsP2 co-expression, confirming that nsP2-mediated inhibition of RNA replicase activity is largely due to nsP2-mediated processing of the nonstructural polyprotein. Co-expression of nsP2 also suppressed the activity of replicases of heterologous alphaviruses. Thus, the presence of nsP2 inhibits formation and activity of alphavirus RNA replicase in protease activity-dependent and -independent manners. This knowledge improves our understanding about mechanisms of superinfection exclusion for alphaviruses and may aid the development of anti-alphavirus approaches.

Keywords: RNA replication; alphaviruses; mosquito; nsP2; protease; superinfection exclusion.

Figure 1

Schematic overview of used plasmids and verification of nsP2 expression in transfected C6/36 cells. (A). Trans-replicase plasmid for expression of alphavirus ns-polyprotein. Ubi—full-length Aedes aegypti polyubiquitin promoter; UL—transcribed leader of polyubiquitin gene containing naturally occurring intron; SV40Ter—SV40 late polyadenylation region. (B) Constructs expressing template RNAs for trans-replicases. AlbPolI—truncated (−250 to −1) promoter for Aedes albopictus RNA polymerase I; AlbTer—tentative terminator for Aedes albopictus RNA polymerase I. The 5′ and 3′ UTRs and SG promoter are from CHIKV, SINV, SFV, RRV, MAYV, EILV, VEEV or EEEV; nsP1N—region encoding for the N-terminal region of nsP1; HDV RZ—antisense strand ribozyme of hepatitis delta virus. (C). Constructs expressing nsP2 of CHIKV or SINV. nsP1*—region encoding for 10 C-terminal amino acid residues of nsP1; DmHSP70Ter—transcription terminator of Drosophila melanogaster hsp70 gene. (A–C). The vector backbones are not shown; drawings are not in scale. (D). C6/36 cells were transfected with pPubi-CHIKV-nsP2 (left) or pPubi-SINV-nsP2 (right). Cells were harvested at 12, 18, 24, 36 or 48 hpt and lysed in 1× Laemmli buffer. Proteins were separated using SDS-PAGE in 10% gels and transferred to PVDF membranes. nsP2 proteins were detected using anti-CHIKV and anti-SINV nsP2 antibodies, and β-actin was detected as the loading control. (E) C6/36 cells were transfected with (left panel) pPubi-CHIKV-nsP2 (WT), pPubi-CHIKV-nsP2^CA, pPubi-CHIKV-nsP2^EV, pPubi-CHIKV-nsP2^YA+EV, pPubi-CHIKV-nsP2^ALT/ERR, pPubi-CHIKV-nsP2^KR/DD; (right panel) pPubi-SINV-nsP2 (WT), pPubi-SINV-nsP2^CA, pPubi-SINV-nsP2^ND, pPubi-SINV-nsP2^ND+PQ, pPubi-SINV-nsP2^PQ or pPubi-SINV-nsP2^KR/DD. Cells were harvested at 48 hpt and analyzed as described for (D).

Figure 2

Co-expression of CHIKV and SINV nsP2 inhibits activity of trans–replicase of homologous virus. (A). Mutations introduced to ns-polyprotein of CHIKV. Mutation G534V leads to expression of P1^GV234 polyprotein with uncleavable 1/2 processing site, while mutation G1332V leads to expression of P12^GV34 polyprotein with uncleavable 2/3 processing site. (B). C6/36 cells grown at 96-well plate were co-transfected with Alb-FG-CHIKV, wt or mutant ns-polyprotein expression plasmid (Ubi-P1234-CHIKV, Ubi-P1^GV234-CHIKV, Ubi-P12^GV34-CHIKV or Ubi-P1234^GAA-CHIKV) and pPubi-CHIKV-nsP2^CA, pPubi-CHIKV-nsP2, pPubi-CHIKV-nsP2^EV, pPubi-CHIKV-nsP2^YA+EV, pPubi-CHIKV-nsP2^ALT/ERR, pPubi-CHIKV-nsP2^KR/DD or with dummy plasmid (no-nsP2 control). Cells were incubated at 28 °C and lysed 48 hpt. Data represent the luciferase activity (Fluc and Gluc) from Ubi-P1234-CHIKV (left), Ubi-P1^GV234-CHIKV (middle) and Ubi-P12^GV34-CHIKV (right) transfected cells normalized to the Ubi-P1234^GAA control cells. Value obtained for P1234^GAA control was taken as 1. (C). C6/36 cells grown at 96-well plate were co-transfected with Alb-FG-SINV, Ubi-P1234-SINV or Ubi-P1234^GAA-SINV and pPubi-SINV-nsP2^CA, pPubi-SINV-nsP2, pPubi-SINV-nsP2^ND, pPubi-SINV-nsP2^ND+PQ, pPubi-SINV-nsP2^PQ, pPubi-SINV-nsP2^KR/DD or with dummy plasmid (no-nsP2 control). The experiment was performed and data analyzed as described for panel B. (B,C). Means ± SD from three biological replicates are shown. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA test).

Figure 3

Efficiency of infection of C6/36 cells by CHIKV VRPs is reduced by expression of CHIKV or SINV nsP2 proteins. C6/36 cells grown in 12-well plates were transfected with pB-IE1.dsR (no nsP2), pPubi-CHIKV-nsP2^CA, pPubi-CHIKV-nsP2, pPubi-SINV-nsP2^CA or pPubi-SINV-nsP2. At 48 hpt, cells were infected with VRPs containing CHIKVRepl-ZsGreen replicon at a multiplicity of infection of approximately 0.4. Cells were harvested at 16 h post-infection and fixed and analyzed with an Attune NxT acoustic focusing cytometer. Y-axes: percentage of ZsGreen-positive cells (i.e., harboring replicating CHIKV replicon) from DsRed-positive cells (i.e., cells successfully transfected with nsP2 expression or control plasmid). Means ± SD from two independent experiments performed in triplicate are shown. ns, not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA test).

Figure 4

Co-expression of nsP2 of CHIKV or SINV inhibits activity of trans-replicases of heterologous alphaviruses. (A) C6/36 cells grown on 96-well plates were co-transfected with matching pairs of AlbPolI-FG and Ubi-P1234 or Ubi-P1234^GAA plasmids of alphaviruses shown at X-axes and with pPubi-CHIKV-nsP2^CA, pPubi-CHIKV-nsP2 or dummy plasmid (no-nsP2 control). Cells were incubated at 28 ^°C and lysed 48 hpt. Data represent the Gluc activity from Ubi-P1234-CHIKV transfected cells normalized to the Ubi-P1234^GAA control cells. Value obtained for P1234^GAA control was taken as 1. (B). Experiment was performed as described for panel A except that pPubi-SINV-nsP2^CA or pPubi-SINV-nsP2 plasmids were used. (A,B). Means ± SD are shown for three biological replicates. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA test).