IRES-dependent replication of Venezuelan equine encephalitis virus makes it highly attenuated and incapable of replicating in mosquito cells

Abstract

The development of infectious cDNA for different alphaviruses opened an opportunity to explore their attenuation by extensively modifying the viral genomes, an approach that might minimize or exclude the reversion to the wild-type, pathogenic phenotype. Moreover, the genomes of such alphaviruses can be engineered to contain RNA elements that would be functional only in cells of vertebrate, but not insect, origin. In the present study, we developed a recombinant VEEV that is more attenuated than TC-83 and capable of replicating only in vertebrate cells. This phenotype was achieved by rendering the translation of the viral structural proteins, and ultimately viral replication, dependent on the internal ribosome entry site of encephalomyocarditis virus (EMCV IRES). This recombinant virus was viable, but required additional, adaptive mutations in nsP2 that strongly increased its replication rates. In spite of efficient replication in cultured vertebrate cells, the genetically modified VEEV demonstrated a highly attenuated phenotype in newborn mice, and yet induced protective immunity against VEEV infection.

Fig. 1

Replication of the recombinant, EMCV IRES-encoding, VEEV TC-83-derived viruses in BHK-21 cells. (A) Schematic representation of the designed viral genomes, infectivities of the in vitro-synthesized RNAs in the infectious center assay, virus titers at 24 h post transfection of 1 μg of the in vitro-synthesized RNAs into BHK-21 cells, and sizes of the plaques, formed by indicated viruses in BHK-21 cells at 48 h post transfection. Arrows indicate functional subgenomic promoters. Filled boxes indicate positions of EMCV IRES. (B) Alignment of the subgenomic promoter-containing fragment of the VEEV TC-83 genome and the corresponding fragment of the VEEV/mutSG/IRES. The position of the promoter is indicated by open box. The start of the subgenomic RNA in the VEEV TC-83 genome and the beginning of the EMCV IRES are indicated by arrows. The mutations, introduced into the VEEV/mutSG/IRES genome are shown in lower case letters. (C) Plaques, formed in BHK-21 cells by viruses, harvested at 24 h post transfection. (D) Replication of the viruses after transfection of 1 μg of the in vitro-synthesized RNAs into BHK-21 cells.

Fig. 2

Mutations found in the plaque-purified VEEV/mutSG/IRES variants, which demonstrated more efficient replication in BHK-21 cells, and the effect of the defined adaptive mutations on VEEV TC-83 and VEEV/mutSG/IRES replication. (A) The list of the mutations, found in the genomes of plaque isolates, compared to published sequence of VEEV TC-83 (Kinney et al., 1989). (B) The schematic representation of the VEEV TC-83 and VEEV/mutSG/IRES genomes, having either one or two of the identified mutations, and the infectivity of the in vitro-synthesized viral RNAs in the infectious center assay. Functional subgenomic promoters are indicated by arrows, and EMCV IRES by filled boxes. (C) Replication of the designed viruses in BHK-21 cells after transfection of 1 μg of the in vitro-synthesized viral genomes.

Fig. 3

Mutations identified in the nsP2 protein of VEEV/mutSG/IRES variants demonstrating a large-plaque phenotype. (A) List of the mutations identified in the genomes of the plaque-purified isolates from virus stock, harvested at 24 h post transfection of the in vitro-synthesized RNA (Orig.), and in the genomes of isolates from the stock that was additionally passaged three times in Vero cells (Pass.). (B) Localization of the defined mutations in the VEEV nsP2. The positions of currently known functional domains in alphavirus nsP2 (Russo, White, and Watowich, 2006; Strauss and Strauss, 1994) are indicated.

Fig. 4

Analysis of protein and RNA synthesis in BHK-21 cells transfected with the in vitro-synthesized recombinant viral RNAs. Cells were electroporated with 4 μg of the indicated RNAs and seeded into 35-mm dishes as indicated in Materials and Methods. (A) At 4.5 h post transfection, medium in the wells was replaced by 1 ml of αMEM supplemented with 10% FBS, ActD (1 μg/ml) and [³H]uridine (20 μCi/ml). After 4 h of incubation at 37°C, RNAs were isolated and analyzed by agarose gel electrophoresis as described in the Materials and Methods. The positions of viral genomic and subgenomic RNAs are indicated by G and SG, respectively. The VEEV/IRES-specific subgenomic RNA forms a more diffuse band than do other, subgenomic RNA-producing, viruses, because, in the gel, it co-migrates with the ribosomal 28S RNA. (B) At 12 h post transfection, proteins were metabolically labeled with [³⁵S]methionine and analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel as described in the Materials and Methods. The positions of molecular weight markers (kDa) are indicated at the left side of the gel. The positions of viral structural proteins: C, E1, E2 and p62 (the precursor of E2) are shown at the right side of the gel. Asterisks indicate the positions of cellular proteins (the heat-shock proteins), induced by replication of the IRES-encoding viruses (see text for details). Noticeable differences in capsid mobility indicate the presence of additional 4 amino acids, which were cloned into IRES-containing viral genomes (see Materials and Methods for details). (C) The same samples of the proteins were analyzed by Western blotting. Membranes were developed by VEEV-specific mouse antibody and anti-mouse IRDye 800 secondary antibody. The intensity of the capsid-specific signals was evaluated on LI-COR imager. The positions of viral structural proteins: C, E1, E2 and p62 (the precursor of E2) are shown at the right side of the gel.

Fig. 5

Passaging of the recombinant, EMCV IRES-encoding VEEV variants in C₇10 cells. (A) The schematic representation of viral genomes. Arrow indicates the position of the functional subgenomic promoter. Filled box indicates the position of EMCV IRES. (B) Titers of the recombinant viruses after passaging in C₇10 cells. Cells in 35-mm dishes were infected with 400 μl of virus samples harvested either at 24 h post transfection of BHK-21 cells with the in vitro-synthesized RNA (P1) or 48 h post infection of C₇10 cells (P2). Samples were harvested at 48 h post infection, and titers were determined by plaque assay on BHK-21 cells. Dashed line indicates the limit of detection. n.d. indicates that titer was below the detection limit. (C) Replication of the indicated viruses after transfection of 5 μg of the in vitro-synthesized RNAs into C₇10 cells. Titers were determined by plaque assay on BHK-21 cells. (D) The deletions of the IRES-specific sequence identified in the plaque-purified VEEV/IRES variants, demonstrating efficient replication in C₇10 cells. The residual EMCV IRES-specific sequences are indicated by lower case letters.

Fig. 6

Replication of VEEV/mutSG/IRES/1 and VEEV TC-83 in the NIH 3T3 cells. Cells were infected at an MOI of 10 PFU/cell. Media were replaced at the indicated time points, and virus titers were measured by plaque assay on BHK-21 cells. The same samples were used to measure IFN-α/β release in biological assays, as described in Materials and Methods. Concentrations of released IFN-α/β are presented in international units (IU) per ml.

Fig. 7

Survival of mice infected with VEEV TC-83 and VEEV/mutSG/IRES/1 viruses. Six-day-old NIH Swiss mice were inoculated i.c. with ca. 10⁶ PFU of the indicated viruses. Animals were monitored for two months. No deaths occurred after day 9 post-infection in these experiments.

Fig. 8

Survival following vaccination and challenge of adult mice. Five-to-6-week-old female NIH Swiss mice were immunized s.c. with VEEV strain TC-83 or the recombinant virus at a dose of ca. 10⁶ PFU. Three weeks after immunization, mice were challenged s.c. with ca. 10⁴ PFU of VEEV strain 3908, and mortality was recorded.