nsP4 Is a Major Determinant of Alphavirus Replicase Activity and Template Selectivity

Abstract

Alphaviruses have positive-strand RNA genomes containing two open reading frames (ORFs). The first ORF encodes the nonstructural (ns) polyproteins P123 and P1234 that act as precursors for the subunits of the viral RNA replicase (nsP1 to nsP4). Processing of P1234 leads to the formation of a negative-strand replicase consisting of nsP4 (RNA polymerase) and P123 components. Subsequent processing of P123 results in a positive-strand replicase. The second ORF encoding the structural proteins is expressed via the synthesis of a subgenomic RNA. Alphavirus replicase is capable of using template RNAs that contain essential cis-active sequences. Here, we demonstrate that the replicases of nine alphaviruses, expressed in the form of separate P123 and nsP4 components, are active. Their activity depends on the abundance of nsP4. The match of nsP4 to its template strongly influences efficient subgenomic RNA synthesis. nsP4 of Barmah Forest virus (BFV) formed a functional replicase only with matching P123, while nsP4s of other alphaviruses were compatible also with several heterologous P123s. The P123 components of Venezuelan equine encephalitis virus and Sindbis virus (SINV) required matching nsP4s, while P123 of other viruses could form active replicases with different nsP4s. Chimeras of Semliki Forest virus, harboring the nsP4 of chikungunya virus, Ross River virus, BFV, or SINV were viable. In contrast, chimeras of SINV, harboring an nsP4 from different alphaviruses, exhibited a temperature-sensitive phenotype. These findings highlight the possibility for formation of new alphaviruses via recombination events and provide a novel approach for the development of attenuated chimeric viruses for vaccination strategies. IMPORTANCE A key element of every virus with an RNA genome is the RNA replicase. Understanding the principles of RNA replicase formation and functioning is therefore crucial for understanding and responding to the emergence of new viruses. Reconstruction of the replicases of nine alphaviruses from nsP4 and P123 polyproteins revealed that the nsP4 of the majority of alphaviruses, including the mosquito-specific Eilat virus, could form a functional replicase with P123 originating from a different virus, and the corresponding chimeric viruses were replication-competent. nsP4 also had an evident role in determining the template RNA preference and the efficiency of RNA synthesis. The revealed broad picture of the compatibility of the replicase components of alphaviruses is important for understanding the formation and functioning of the alphavirus RNA replicase and highlights the possibilities for recombination between different alphavirus species.

Keywords: RNA polymerases; RNA replication; alphavirus; genetic recombination; replicase.

FIG 1

Expression plasmids for single-, two-, and three-component replicases. (A) Schematic presentation of template RNA (top) and replicase protein expression constructs. HSPolI, a truncated promoter (residues −211 to –1) for human RNA polymerase I; 5′ UTR, full-length 5′ UTR of an alphavirus; nsP1 N*, region encoding the N-terminal 77 to 114 amino acid residues of nsP1, depending on the virus; SG, SG promoter spanning (with respect to the termination codon of nsP4) from position –79 to the end of the intergenic region; 3′ UTR, truncated (last 110 residues) 3′ UTR of an alphavirus; HDV RZ, antisense strand ribozyme of hepatitis delta virus; MmTer, a terminator for RNA polymerase I in mice; 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; Ubi, sequence encoding human ubiquitin. The red arrow indicates the location of the GDD motif in nsP4; in polymerase-negative constructs, this was replaced by GAA. The green arrow indicates the location of the catalytic Cys residue in the active site of nsP2 protease; in protease-negative constructs, this was replaced by Ala residue. The vector backbones are not shown, and drawings are not to scale. (B) Phylogenetic tree of nsP4 RNA-dependent-RNA-polymerase proteins of the analyzed alphaviruses. The phylogenetic tree was constructed using evolutionary analysis with the maximum likelihood method and JTT matrix-based model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analysis was conducted using MEGA-X software.

FIG 2

Single- and two-component replicases of alphaviruses have similar activities. (A). Schematic presentation of expressed replicase precursors. C1, single-component replicase from plasmids CMV-P1234-SINV and so on; C2, two-component replicase from plasmids CMV-P123-SINV+ CMV-ubi-nsP4-SINV and so on. (B) HEK293T cells in 96-well plates were cotransfected with matching pairs of CMV-P1234 and HSPolI-FG plasmids (C1) or with matching combinations of CMV-P123, CMV-ubi-nsP4 (in a 1:1 molar ratio), and HSPolI-FG plasmids (C2). As the negative control, CMV-P1234^GAA, which encodes polyprotein lacking RNA polymerase activity, was used instead of CMV-P1234. Cells were incubated at 37°C and lysed for 18 h p.t.; cells transfected with plasmids containing sequences from EILV were incubated at 28°C and lysed for 48 h p.t. Fluc (marker of replication) and Gluc (marker of transcription) activities produced by active replicases were normalized to the P1234^GAA controls. The value obtained for the P1234^GAA controls was taken as 1. The means ± standard deviation (SD) of three independent experiments are shown. (C) HEK293T cells in 12-well plates were cotransfected with matching combinations of CMV-P123, CMV-ubi-nsP4 (in 1:1 molar ratio), and HSPolI-FG plasmids; control cells were mock-transfected. Cells were incubated as described for panel B, after which total RNA was extracted and analyzed 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 comigrate 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

Activity of two-component replicase depends on the relative amount of nsP4. (A) HEK293T cells in 96-well plates were cotransfected with matching combinations of CMV-P123, CMV-ubi-nsP4, and HSPolI-FG plasmids of CHIKV, SINV, BFV, VEEV, and EILV. As negative controls, CMV-P1234^GAA was used instead of CMV-P123+CMV-ubi-nsP4. The amounts of CMV-P123 and HSPolI-FG were kept constant, while the CMV-ubi-nsP4 was provided at a 1:10, 2:10, 4:10, 6:10, 8:10, 1:1, 2:1, 4:1, 6:1, or 8:1 molar ratio in respect to CMV-P123. Cells were incubated and the data were collected and analyzed as described for Fig. 2B. Boosts of Fluc (replication, left panel) and Gluc (transcription, right panel) activities are shown as the means ± SD of three independent experiments. (B) HEK293T cells in 96-well plates (left) or in 24-well plates (right) were cotransfected with matching combinations of CMV-P123-CHIKV, CMV-ubi-nsP4-CHIKV, and HSPolI-FZsG-CHIKV. As the negative control, CMV-P1234^GAA-CHIKV was used instead of CMV-P123+CMV-ubi-nsP4. The amounts of CMV-P123-CHIKV and HSPolI-FG-CHIKV were kept constant, while the CMV-ubi-nsP4-CHIKV plasmid was provided at a 1:10, 2:10, 4:10, 6:10, 8:10, 1:1, 2:1, 4:1, 6:1, or 8:1 molar ratio in respect to CMV-P123-CHIKV. (Left panel) Cells were incubated and the data were collected and analyzed as described for Fig. 2B, except that only Fluc activity (replication) was measured and shown as the means ± SD of three independent experiments. (Right panel) Cells were collected at 18 h p.t. and analyzed with an Attune NxT acoustic focusing cytometer. The percentage of ZsGreen-expression from living cells and mean fluorescence intensity (MFI) in arbitrary units for ZsGreen-positive cells are shown. The means ± SD of three independent experiments are shown.

FIG 4

P123 components of viruses belonging to the SFV complex are compatible with heterologous nsP4 proteins. (A to E) HEK293T cells were cotransfected with HSPolI-FG-SINV and (A) CMV-P123-CHIKV, (B) CMV-P123-ONNV, (C) CMV-P123-SFV, (D) CMV-P123-RRV, or (E) CMV-P123-MAYV. In each transformation, one plasmid, expressing nsP4 from a virus shown on the x axes, was added in an equimolar amount to the indicated CMV-P123 plasmid. Transfected cells were incubated at 37°C and lysed for 18 h p.t.; in addition, cells transfected with CMV-ubi-nsP4-EILV were incubated at 28°C and lysed for 48 h p.t. The data represent the activity of Fluc and Gluc from CMV-ubi-nsP4-transfected cells normalized to the paired CMV-P1234^GAA control cells. Values obtained for P1234^GAA controls were taken as 1. The means ± SD from three independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant (Student’s unpaired t test).

FIG 5

P123 components of outgroup alphaviruses are generally not compatible with heterologous nsP4 proteins. (A to D). HEK293T cells were cotransfected with HSPolI-FG-SINV and (A) CMV-P123-BFV, (B) CMV-P123-EILV, (C) CMV-P123-VEEV, or (D) CMV-P123-SINV. In each transformation, one plasmid, expressing nsP4 from a virus shown on the x axes, was added in an equimolar amount to the indicated CMV-P123 plasmid. Transfected cells were incubated at 37°C and lysed for 18 h p.t.; cells transfected with CMV-ubi-nsP4-EILV were also incubated at 28°C and lysed for 48 h p.t. All cells transfected with CMV-P123-EILV were incubated at 28°C and lysed for 48 h p.t. (E and F) HEK293T cells were cotransfected with (E) CMV-P123-VEEV or (F) CMV-P123-SINV and matching pairs of plasmids expressing nsP4 and template RNA from a virus shown on the x axes. Transfected cells were incubated at 37°C and lysed for 18 h p.t.; cells transfected with CMV-ubi-nsP4-EILV and HSPolI-FG-EILV were incubated at 28°C and lysed for 48 h p.t. In all experiments, data were collected, analyzed and presented as described for Fig. 4; activities lower than those observed for negative controls are also shown as 1. The means ± SD from three independent experiments are shown.

FIG 6

Properties of chimeric SFV and SINV harboring heterologous nsP4. (A) Schematic presentation of chimeric SFV (left) and SINV (right) genomes. Swapped nsP4 regions are color-codes. The modified junction region between ns- and structural ORFs, including the codon-altered region of nsP4 (Table 1), is shown below the drawing of chimeric SFV genomes. The arrow indicates the transcription start site of the SG promoter of the virus. The red X indicates inactivation of the SG promoter by synonymous mutations introduced into the sequence encoding the C-terminal region of nsP4. (B) The lysates of the BHK-21 cells transfected with icDNA plasmids of SFV and its chimeras (left panels) or with in vitro transcripts of icDNA of SINV and its chimeras (right panels) and lysate from mock-transfected control cells were subjected to SDS-PAGE and immunoblot analysis with antibodies against the corresponding nsP2 and capsid proteins. β-actin is shown as a loading control. The top panels show lysates of transfected cells that were incubated at 37°C, while the bottom panels show lysates of transfected cells that were incubated at 28°C. The experiment was repeated twice with similar results; the data from one experiment are shown.

FIG 7

Two- and three-component replicases of SINV and CHIKV containing inactive protease are capable of RNA synthesis. (A) Schematic presentation of the replicases used. C1 shows the P1234 expressed from a single-component CMV-P1234 plasmid, C2 shows the P123 and nsP4 expressed by the two-component replicase, and C3 shows nsP1, P2^CA3 and nsP4 expressed by the three-component replicase. (B). HEK293T cells in 96-well plates were cotransfected with CMV-P12^CA3-CHIKV, CMV-ubi-nsP4-CHIKV, and HSPolI-FG-CHIKV or CMV-P12^CA3-SINV, CMV-ubi-nsP4-SINV, and HSPolI-FG-SINV plasmids. As the negative controls, CMV-P1234^GAA, which lack polymerase activity, were used. The amounts of CMV-P12^CA3 and HSPolI-FG plasmids were kept constant, while the CMV-ubi-nsP4 plasmid was provided in a 1:10, 2:10, 4:10, 6:10 8:10, 1:1, 2:1, 4:1, 6:1, or 8:1 molar ratio in respect to CMV-P12^CA3. Cells were incubated and the data were collected and analyzed as described for Fig. 2B. Boosts of Fluc (replication, left panel) and Gluc (transcription, right panel) activities are shown as the means ± SD of three independent experiments. (C). HEK293T cells in 96-well plates (left) or in 24-well plates (right) were cotransfected with matching combinations of CMV-P12^CA3-CHIKV, CMV-ubi-nsP4-CHIKV, and HSPolI-FZsG-CHIKV. As the negative control, CMV-P1234^GAA-CHIKV was used instead of CMV-P12^CA3+CMV-ubi-nsP4. This experiment was performed and the data were collected, analyzed, and presented as described for Fig. 3B. (D) U2OS cells in 12-well plates were cotransfected with single-, two-, and three-component replicases as indicated on the x axes. For two- and three-component replicases, all plasmids encoding ns-proteins were combined in equimolar amounts. As negative controls, CMV-P1234^GAA were used instead of CMV-P1234. Cells were incubated at 37°C and lysed for 18 h p.t. Fluc (replication, left panel) and Gluc (transcription, right panel) activities produced by active replicases were normalized to the P1234^GAA controls. The values obtained for the P1234^GAA controls were taken as 1. The means ± SD of three independent experiments are shown; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant (Student’s unpaired t test).

FIG 8

Template specificity of replicases of alphaviruses belonging to the SFV complex is associated with the nsP4 component. HEK293T cells in 96-well plates were cotransfected with (A) matching and heterologous combinations of CMV-P123-CHIKV or CMV-P123-RRV and CMV-ubi-nsP4-CHIKV or CMV-ubi-nsP4-RRV. Plasmids encoding template RNA were HSPolI-FG-SINV (left), HSPolI-FG-CHIKV (middle), or HSPolI-FG-RRV (right); (B) matching and heterologous combinations of CMV-P123-CHIKV or CMV-P123-SFV and CMV-ubi-nsP4-CHIKV or CMV-ubi-nsP4-SFV. Plasmids encoding template RNA were HSPolI-FG-SINV (left), HSPolI-FG-CHIKV (middle), or HSPolI-FG-SFV (right); (C) matching and heterologous combinations of CMV-P123-MAYV or CMV-P123-SFV and CMV-ubi-nsP4-MAYV or CMV-ubi-nsP4-SFV. Plasmids encoding template RNA were HSPolI-FG-SINV (left), HSPolI-FG-MAYV (middle), or HSPolI-FG-SFV (right). (A to C). CMV-P123 and CMV-ubi-nsP4 were always used in a 1:1 molar ratio. As negative controls CMV-P1234^GAA were used. Cells were incubated at 37°C and lysed for 18 h.p.t. In all experiments, data were collected, analyzed, and presented as described for Fig. 4; activities lower than those observed for negative controls are shown as 1. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant (Student’s unpaired t test).