Lack of nsP2-specific nuclear functions attenuates chikungunya virus replication both in vitro and in vivo

Abstract

Chikungunya virus (CHIKV) is an important arthritogenic human pathogen that is already circulating in both hemispheres. In the present study, we substituted VLoop, located on the surface of nsP2, by other amino acid sequences. These modifications had deleterious effects on viral nuclear functions and made CHIKV incapable of interfering with the induction of type I interferon and the antiviral response in both mouse and human cells. Importantly, the identified mutations have no significant effects on the synthesis of virus-specific RNAs and viral structural proteins. The designed mutants induced a few orders of magnitude lower viremia but remained highly immunogenic in mice. Thus, the proposed modifications of nsP2 can additionally improve the safety of the attenuated strain CHIKV 181/25. Furthermore, defined mutations in the macro domain of another nonstructural protein, nsP3, additionally reduce cytopathogenicity of nsP2 mutants in human cells, and can be potentially applied for CHIKV attenuation.

Keywords: Alphaviruses; Chikungunya virus; Cytopathic effect; Transcription inhibition; Vaccines; Viral RNA replication; Virus-host interaction; nsP2; nsP3.

FIG. 1.

CHIKV 181/25 derivatives with mutated VLoop are viable. (A) The alignment of CHIKV nsP2 fragment containing indicated mutations. Dashes indicate identical aa. The numbers correspond to aa in nsP2 of parental CHIKV 181/25. (B) RNA infectivity and viral titers at 24 h post electroporation. BHK-21 cells were electroporated with 3 μg of in vitro-synthesized RNAs of parental CHIKV 181/25 and the designed mutants. Electroporated cells were used for the infectious center assay (see Materials and Methods for details) and for generating viral stocks. Viral titers were assessed by plaque assay on BHK-21 cells. Transfection experiments were reproducibly repeated three times and the presented values are ranges from three individual experiments.

FIG. 2.

CHIKV nsP2 mutants efficiently replicate in rodent and human cells. 5x10⁵ BHK-21, Vero, NIH 3T3, HEK 293, MRC-5 and BJ-5ta cells in 6-well Costar plates were infected with CHIKV 181/25 or designed mutants at an MOI of 0.01 PFU/cell. At the indicated time points, media were replaced, and viral titers were determined by plaque assay on BHK-21 cells. The experiments were reproducibly repeated 3 times, and the results of one representative experiment are presented.

FIG. 3.

The designed CHIKV nsP2 mutants demonstrate an efficient synthesis of virus-specific RNAs and viral structural proteins. (A) 5x10^s cells in the 6-well Costar plate were infected with parental CHIKV 181/25 and designed mutants at an MOI of 20 PFU/cell. At 7 h PI, they were washed with PBS, and proteins were metabolically labeled for 30 min with [³⁵S]methionine (see Materials and Methods for details). Equal amounts of lysates were analyzed by gel electrophoresis in 10% NuPAGE gels (Invitrogen), followed by autoradiography. (B) 5x10⁵ cells in the 6-well Costar plate were infected with the indicated viruses at an MOI of 20 PFU/cell. Virus-specific RNAs were metabolically labeled between 4 and 8 h PI in complete media, supplemented with [³H]uridine (20 μCi/ml) and Actinomycin D (1 μg/ml), then RNAs were isolated and analyzed by agarose gel electrophoresis as described in Materials and Methods.

FIG. 4.

CHIKV nsP2 mutants, but not the parental CHIKV 181/25, are potent inducers of IFN-β in both murine and human cell lines. NIH 3T3 (A), MRC-5 (B), and HFF-1 (C) cells were infected at an MOI of 20 PFU/cell with nsP2 mutants and parental CHIKV 181/25. At 18 h PI, the supernatants were harvested to determine viral titers and the levels of IFN-β. n.d. indicates that the concentration of IFN-β was below the limit of detection. Similar experiments were made multiple times with consistent results. The results of one of the representative experiments are presented.

FIG. 5.

CHIKV nsP2 mutants do not form plaques on the cells competent in type I IFN induction and signaling. CHIKV nsP2 mutants and parental CHIKV 181/25 were used in plaque assay performed on the indicated cell lines. This figure displays wells with monolayers of different cells, which were infected with the same dilutions of the indicated viruses. All of the plates were stained with crystal violet after 3 days of incubation at 37°C

FIG. 6.

Infections of murine NIH 3T3 and human MRC-5 fibroblasts with CHIKV nsP2 mutants result in ISG activation. NIH 3T3 and MRC-5 cells were infected with the indicated viruses at an MOI of 20 PFU/cell, and, at 16 h PI, the induction of indicated ISGs and IFN-β was evaluated by RT-qPCR as described in Materials and Methods. The fold increase in the ISGs transcript level in virus-infected cells relative to the mock-infected cells were calculated using ΔΔCT method. The experiment was reproducibly repeated three times with similar results, and the data from one representative experiment are shown. Average values from triplicate samples are presented, but the error bars are too small to be visible at this scale.

FIG. 7.

The designed CHIKV 181/25-based nsP2 mutants remain immunogenic and protect mice against following challenge with wCHIKV. Two-to-three-week old C57BL/6 mice (n = 6/group) were infected in the left footpad with 5x10³ PFU of CHIKV 181/25 and its indicated nsP2 mutants. (A) The levels of neutralizing Abs were assessed at day 21 PI as described in Materials and Methods. Results are presented as means with standard deviations. Significances of differences among the values were determined by one-way ANOVA, followed by Dunnett’s multiple comparisons test. They are indicated by asterisks as follows: *, P<0.05; **, P<0.01; ***, P<0.001. CHIKV181/25 is compared to other groups. (B) Mice were challenged on day 25 PI with 10⁵ PFU of wCHIKV (see Materials and Methods for details) using the same route of infection. Blood samples were collected on days 1, 2, and 3, and levels of viremia were assessed by plaque assay on BHK-21 cells. Each data point represents a value from an individual mouse. The dashed line indicates the limit of detection (LOD) in the plaque assay.

FIG. 8.

Mutations in nsP2 of wCHIKV strongly affect the development of viremia in mice. (A) Two-to-three-week old C57BL/6 mice (n = 6/group) were infected in the left footpad with 5x10³ PFU of wCHIKV nsP2 mutants and parental wCHIKV. Blood samples were taken on days 1, 2 and 3 PI, and the levels of viremia were assessed by plaque assay on BHK-21 cells. The dashed line indicates the limit of detection (LOD). Results are presented as means with standard deviations and significant differences, determined by one-way ANOVA, followed by Tukey’s multiple comparisons test. They are indicated by asterisks as follows: ****, P<0.0001. wCHIKV is compared to other groups. (B) Weight change post infection with indicated viruses. Results are presented as means with standard deviations.

FIG. 9.

Additional mutations in the macro domain of nsP3 in CHIKV/NGK/GFP reduce viral cytopathogenicity in human and Vero cells. (A) The schematic presentation of the genomes of recombinant viruses. (B) 5x10⁵ cells of the indicated cell lines in 6-well Costar plates were infected with the mutants at an MOI of 20 PFU/cell. Media were replaced at the indicated time points, and cells were split upon reaching confluency. Viral titers were determined by plaque assay on BHK-21 cells. The dashed line indicates the limit of detection (LOD).

FIG. 10.

Mutations in the macro domain of nsP3 in wCHIKV/NGK variant have no negative effect on viral replication in mice. (A) The schematic presentation of the recombinant genomes. (B) Two-to-three-week old C57BL/6 mice (n = 6/group) were infected in the left footpad with 5x10³ PFU of the indicated mutants and parental wCHIKV. Blood samples were collected on days 1, 2, and 3 PI to analyze the levels of viremia. The dashed line indicates the limit of detection (LOD). Results are presented as means with standard deviations and significant differences, determined by one-way ANOVA, followed by Tukey’s multiple comparisons test. wCHIKV is compared to other groups. **** indicates P<0.0001 (C) Titers of neutralizing antibodies were evaluated on day 21 PI. Results are presented as means with standard deviations. Values are not significantly different, as determined by one-way ANOVA, followed by Dunnett’s multiple comparisons test. P>0.05.