Design of chimeric alphaviruses with a programmed, attenuated, cell type-restricted phenotype

Abstract

The Alphavirus genus in the Togaviridae family contains a number of human and animal pathogens. The importance of alphaviruses has been strongly underappreciated; however, epidemics of chikungunya virus (CHIKV), causing millions of cases of severe and often persistent arthritis in the Indian subcontinent, have raised their profile in recent years. In spite of a continuous public health threat, to date no licensed vaccines have been developed for alphavirus infections. In this study, we have applied an accumulated knowledge about the mechanism of alphavirus replication and protein function in virus-host interactions to introduce a new approach in designing attenuated alphaviruses. These variants were constructed from genes derived from different, geographically isolated viruses. The resulting viable variants encoded CHIKV envelope and, in contrast to naturally circulating viruses, lacked the important contributors to viral pathogenesis: genes encoding proteins functioning in inhibition of cellular transcription and downregulation of the cellular antiviral response. To make these viruses incapable of transmission by mosquito vectors and to differentially regulate expression of viral structural proteins, their replication was made dependent on the internal ribosome entry sites, derived from other positive-polarity RNA (RNA(+)) viruses. The rational design of the genomes was complemented by selection procedures, which adapted viruses to replication in tissue culture and produced variants which (i) demonstrated different levels of replication and production of the individual structural proteins, (ii) efficiently induced the antiviral response in infected cells, (iii) were incapable of replication in cells of mosquito origin, and (iv) efficiently replicated in Vero cells. This modular approach to genome design is applicable for the construction of other alphaviruses with a programmed, irreversibly attenuated phenotype.

Fig. 1.

Chimeric VEE/CHIKV/GFP and EEE/CHIKV/GFP viruses are capable of noncytopathic replication in cells of vertebrate origin. (A) The schematic representation of GFP-encoding chimeric and nonchimeric viruses used in the experiments. Arrows indicate positions of the subgenomic promoters. (B, C, and D) Replication of the designed viruses in the IFN-α/βR^−/− MEFs, STAT1^−/− MEFs, and NIH 3T3 cells. Cells were infected at an MOI of 20 PFU/cell, media were replaced at the indicated times, and virus titers were determined by a plaque assay on BHK-21 cells. The dashed line indicates the limits of detection. In all of the indicated cell lines, by 48 h postinfection, VEE/GFP, SINV/GFP, and SIN/CHIKV/GFP developed complete CPE, and samples were no longer harvested. (E) Samples harvested in the experiment presented in panel D were used to assess the concentration of IFN-α/β in a biological assay (see Materials and Methods for details).

Fig. 2.

Mutations in CHIKV E2 increase titers and replication rates of chimeric viruses in Vero cells. Vero cells were infected at an MOI of 5 PFU/cell, media were replaced at the indicated times, and virus titers were determined by a plaque assay on Vero cells. The introduced adaptive mutations, W64 → R and N72 → Y, are indicated.

Fig. 3.

Chimeric viruses have different effects on expression of cellular genes. The results of GeneChip analysis of mRNA expression in NIH 3T3 cells infected with the indicated chimeric viruses are shown. Cells were infected at an MOI of 20 PFU/cell. Total cellular RNAs were isolated at 16 h postinfection, and the analysis was performed as described in Materials and Methods.

Fig. 4.

Replacement of the subgenomic promoter by EMCV IRES attenuates replication of the chimeric viruses. (A) The schematic representation of the chimeric viral genomes and viral titers at 24 h post-electroporation of the in vitro-synthesized viral RNAs (see Materials and Methods for details). All of the chimeras contained the W64 → R and N72 → Y mutations in the E2-coding sequence. (B) Analysis of replication of the recombinant viruses in BHK-21 cells after electroporation of the in vitro-synthesized RNAs. Cells were electroporated with 1 μg of in vitro-synthesized RNAs, media were replaced at the indicated times, and virus titers were determined by a plaque assay on Vero cells. (C) Analysis of synthesis of virus-specific proteins in infected Vero cells. Cells were infected at an MOI of 20 PFU/cell and, at 16 h postinfection, metabolically labeled with [³⁵S]methionine for 30 min at 37°C. Equal amounts of cell lysates were analyzed by SDS-PAGE followed by autoradiography, and the same gel was also analyzed on a PhosphorImager to assess synthesis of capsid protein.

Fig. 5.

IRES-containing, chimeric virus adapts to more-efficient replication. (A) The schematic representation of chimeric alphavirus genomes having EMCV IRES cloned under the control of the subgenomic promoter and titers of the viruses recovered at 24 h postelectroporation. All of the chimeras contained the W64 → R and N72 → Y mutations in the E2-coding sequence. (B) Replication of the recombinant VEE/CHIKV/IRESe/C and VEE/CHIKV/IRESe/C1 variants with identified adaptive mutations in nsP2 in BHK-21 cells after electroporation of the in vitro-synthesized RNAs. Cells were electroporated with 1 μg of in vitro-synthesized RNAs, media were replaced at the indicated times, and virus titers were determined by a plaque assay on Vero cells. (C) Analysis of synthesis of virus-specific RNA synthesis in infected Vero cells. Cells were infected at an MOI of 20 PFU/cell and, at 4 h postinfection, metabolically labeled with [³H]uridine in the presence of ActD for 4 h at 37°C (see Materials and Methods for details). Equal amounts of RNA were analyzed by agarose electrophoresis under denaturing conditions followed by autoradiography. Fragments corresponding to genomic and subgenomic RNAs were excised from the dried gels, and the amounts of incorporated [³H]uridine were analyzed by liquid scintillation counting to determine the molar ratio of subgenomic to genomic RNAs.

Fig. 6.

EMCV, BVDV, and HCV IRESes can efficiently drive replication of chimeric viruses. (A) The schematic representation of the chimeric viral genomes and viral titers at 24 h post-electroporation of the in vitro-synthesized viral RNAs (see Materials and Methods for details). All of the chimeras contained the W64 → R and N72 → Y mutations in the E2-coding sequence. (B) Analysis of replication of the recombinant viruses in BHK-21 cells after electroporation of the in vitro-synthesized RNAs. Cells were electroporated with 1 μg of in vitro-synthesized RNAs, media were replaced at the indicated times, and virus titers were determined by a plaque assay on Vero cells. (C) Analysis of synthesis of virus-specific proteins in infected Vero cells. Cells were infected at an MOI of 20 PFU/cell and, at 16 h postinfection, metabolically labeled with [³⁵S]methionine for 30 min at 37°C. Equal amounts of cell lysates were analyzed by SDS-PAGE followed by autoradiography, and the same gel was also analyzed on a PhosphorImager to assess synthesis of capsid protein.

Fig. 7.

EMCV IRES can be used for differential regulation of expression of structural genes carried in chimeric virus genomes. (A) The schematic representation of the chimeric viral genomes and viral titers at 24 h post-electroporation of the in vitro-synthesized RNAs (see Materials and Methods for details). All of the chimeras contained the W64 → R and N72 → Y mutations in the E2-coding sequence. (B) Analysis of synthesis of virus-specific proteins in infected Vero cells. Cells were infected at an MOI of 20 PFU/cell and, at 16 h postinfection, metabolically labeled with [³⁵S]methionine for 30 min at 37°C. Equal amounts of cell lysates were analyzed by SDS-PAGE followed by autoradiography. (C) The same gel was analyzed on a PhosphorImager to assess synthesis of capsid and E1 proteins. Data were normalized on the levels of capsid and E1 synthesis detected in the VEE/CHIKV-infected cells.

Fig. 8.

The chimeric, IRES-dependent viruses demonstrate different levels of replication and gene induction in mammalian cells and are incapable of replication in the cells of mosquito origin. (A) The schematic representation of the genomes of the chimeras used in the experiments. All of the chimeras contained the W64 → R and N72 → Y mutations in the E2-coding sequence. (B) NIH 3T3 cells were infected with the indicated viruses at an MOI of 1 PFU/cell, and at 24 h postinfection, the concentrations of released IFN-β were determined by ELISA. “N.D.” indicates an IFN concentration below a detectable level (20 pg/ml). (C) The results of GeneChip analysis of mRNA expression in NIH 3T3 cells infected with the indicated chimeric viruses. Cells were infected at an MOI of 20 PFU/cell. Total cellular RNAs were isolated at 16 h postinfection, and the analysis was performed as described in Materials and Methods. (D) Vero cells were infected with the indicated viruses at an MOI of 20 PFU/cell. At the indicated time points, medium was replaced, and virus titers were determined by plaque assay on Vero cells. (E) Mosquito C7/10 cells were infected by the indicated viruses at an MOI of 20 PFU/cell. At 48 h postinfection, 100 μl of the medium was used to infect naive C7/10 cells, and virus titers were determined by plaque assay on Vero cells. “N.D.” indicates a virus concentration below a detectable level (50 PFU/ml).

Fig. 9.

Chimeric, EMCV IRES-containing viruses remain immunogenic. (A) The schematic representation of genomes of chimeric viruses VEE/CHIKV/IRESe/C1 and EEE/CHIKV/IRESe/C and vaccine strain CHIKV 181-25. (B) Six-week-old female CD1 mice were infected with 10⁵ PFU of the indicated viruses. Titers of neutralizing Abs were evaluated at days 28 and 120 using the PRNT₈₀ assay.