Comparative analysis of the alphavirus-based vectors expressing Rift Valley fever virus glycoproteins

Abstract

During the last decade, alphaviruses became widely used for expression of heterologous genetic information and development of recombinant vaccines against a variety of human and animal pathogens. In this study, we compared a number of vectors based on the genome of Sindbis (SINV) and Venezuelan equine encephalitis (VEEV) viruses for their ability to express the Rift Valley fever virus (RVFV) envelope glycoprotein Gn and induce a protective immune response against RVFV infection. Our results suggest that (i) application of VEEV-based expression systems appears to be advantageous, when compared to similar systems designed on the basis of the SINV genome. (ii) Alphavirus-specific E3 and E2 proteins and furin-specific cleavage sites can be used for engineering secreted forms of the proteins. (iii) Alphaviruses can be modified for expression of the large fragments of heterologous proteins on the surface of chimeric, infectious viral particles. Thus, alphavirus-based expression systems may have the potential for a broader application beyond their current use as replicons or double-subgenomic vectors.

FIG. 1

The expression cassettes designed for expression of RVFV-specific antigen from SINV and VEEV replicons. (A) The schematic representation of SINV and VEEV replicons used in the study. The arrow indicates the position of the subgenomic promoter. (B) The hydrophobicity profile (Kyte-Doolitle) of the entire polyprotein encoded by the M segment of RVFV genome, and the fragments cloned into the subgenomic RNA of SINV replicon. The positions of signal peptides and transmembrane domains are indicated by black boxes. The spGn(opt) synthetic gene, encoding exactly the same protein as does spGn, is indicated in grey color. The starting and the last a.a. of each cassette are indicated.

FIG. 2

SINV-based replicons used in the study and analysis of the expression of the RVFV-specific glycoproteins by the designed constructs. (A) The schematic representation of the replicons and the titers of the packaged replicons obtained after co-transfection of the replicon and helper RNAs into BHK-21 cells. One of multiple reproducible experiments is presented. The spGn(opt) synthetic gene is indicated in grey color (B) Replication of the tri-component genome virus, having a SINrep/spGn(opt) replicon genome and the genomes of capsid- and glycoprotein-coding helpers, in BHK-21 cells. Cells were infected at an MOI of 8 inf.u/cell, and, at 12 h post infection, the FBS-containing medium was replaced by serum-free VP-SFM medium. Samples of medium were taken at the indicated time points and titers of packaged replicons were determined as described in Materials and Methods. (C) Analysis of RVFV-specific protein expression in cells infected with SINV replicons. BHK-21 cells were infected with packaged replicons at an MOI of 20 inf.u/cells or RVFV MP12 at an MOI of 2.5 PFU/cell and incubated in complete medium at 37°C for 12 h. Equal amounts of cell lysates were analyzed by electrophoresis in SDS-10% polyacryamide gel, followed by Western blotting. Membranes were processed by mouse anti-RVFV antibodies and IRdye 800CW-labeled secondary antibodies. Images were acquired on a Odyssey Infrared Imager (LI-COR). (D) Distribution of virus-specific proteins in the cells infected with RVFV. BHK-21 cells were infected with RVFV MP12 at an MOI of 50 PFU/cell and, at 13 h post infection, stained with mouse anti-RVFV antibodies and AlexaFluor 546-labeled goat anti-mouse secondary antibodies. Panel (a) presents staining of the Triton X-100-permeabilized cells; panel (b) presents surface staining of a non-permeabilized cell. Gn and Gc develop well-distinguishable patches on the cell surface. E) Distribution of the RVFV-specific proteins in the cells infected with packaged SINV replicons that express different fragments of the polyprotein encoded by the M segment of the RVFV genome. Panels (a) and (b) present cells infected with SINrep/NSm+Gn+Gc replicon, panels (c) and (d) present cells infected with SINrep/NSm+Gn replicon, panels (e) and (f) present cells infected with SINrep/spGn, replicon, panels (g) and (h) present cells infected with SINrep/spGn(secr) replicon. Panels (a), (c), (e) and (g) represent images of the cells that were permeabilized with Triton X-100 prior to immunostaining. Panels (b), (d), (f) and (h) present images of the cells stained with antibodies without permeabilization. Images were acquired on a Zeiss LSM510 META confocal microscope using a 63X 1.4NA oil immersion planapochromal lens, as described in Materials and Methods.

FIG. 3

VEEV replicon and helpers used in the animal studies. (A) The schematic representation of the RVFV Gn-expressing VEEV replicon and two pairs of helpers encoding VEEV capsid and glycoproteins. Representative titers, obtained in one of multiple, reproducible experiments, are indicated. (B) Analysis of RVFVGn expression in cells infected with SINrep/spGn and VEErep/spGn replicons. BHK-21 cells were infected with packaged replicons at an MOI of 20 inf.u/cells and incubated in complete medium at 37°C for 12 h. Equal amounts of cell lysates were analyzed by SDS-10% polyacryamide gel electrophoresis, followed by Western blotting. Membranes were processed by mouse anti-RVFV antibodies and IRdye 800CW-labeled secondary antibodies. Images were acquired on a Odyssey Infrared Imager (LI-COR). (C) Distribution of RVFV Gn in the cells infected with packaged VEErep/spGn replicon. BHK-21 cells were infected with the packaged replicon at an MOI of 50 inf.u/cell and, at 12 h post infection, stained with mouse anti-RVFV antibodies and AlexaFluor 546 goat anti-mouse secondary antibodies. Panel (a) presents staining of the Triton X-100-permeabilized cells; panel (b) presents cell stained with antibodies without permeabilization. Images were acquired on a Zeiss LSM510 META confocal microscope using a 63X 1.4NA oil immersion planapochromal lens, as described in Materials and Methods. (C) Survival of mice immunized with 5×10⁶ inf.u of packaged VEErep/spGn replicon and challenged in 42 days with 5×10³ PFU s.c. of RVFV ZH501. The control group was injected with PBS and challenged with the same dose of RVFV.

FIG. 4

The schematic representation of the VEEV genomes encoding the RVFV Gn protein in the structural polyprotein and the genomes of the selected deletion mutants. The positions of found in-frame deletions in the Gn-coding sequence are indicated.

FIG. 5

The expression cassettes designed for expression of secreted forms of RVFV Gn and analysis of the Gn expression. (A) The hydrophobicity profile (Kyte-Doolitle) of the Gn protein. Fragments cloned into the VEEV structural polyprotein are indicated by black lines. (B) The schematic representation of the recombinant VEEV genomes, containing the insertions of the Gn-encoding sequences in the subgenomic RNA. The numbers correspond to the positions of the first and the last a.a. of the insertion in the polyprotein encoded by the RVFV M segment. The triangles indicate positions of engineered and natural furin-specific cleavage sites. Indicated viral titers and plaque size were derived from one of multiple, highly reproducible experiments. (C) Analysis of the stability of VEEV/C/spGn(opt)td⁻ during serial passaging in tissue culture. Virus was passaged three times in BHK-21 by infecting the monolayer of BHK-21 cells in 100-mm dishes with 100 μl of the stock harvested at the previous passage. For analysis of protein expression, BHK-21 cells were infected with harvested viruses and VEEV TC-83 at an MOI of 20 PFU/cell. At 16 h post infection, cells were labeled with [³⁵S]methionine, as described in Materials and Methods, and equal aliquots of proteins were analyzed by electrophoresis in SDS-10% polyacrylamide gels. The positions of VEEV-specific proteins and Gn-2A are indicated. Asterisks indicate the positions of incompletely processed products. (D) and (E) Analysis of proteins expressed by the recombinant viruses and secreted to the medium. Metabolic labeling of the proteins was performed as described in Materials and Methods. Viral particles (V) were pelleted by ultracentrifugation and the RVFV Gn proteins were isolated by immunoprecipitation. Samples were analyzed by electrophoresis in SDS-10% polyacrylamide gels, followed by autoradiography. The positions of proteins and molecular weight markers are indicated. The molecular weights of the expressed Gn protein fragments and its fusion forms, predicted based on the a.a. sequence, are indicated in the brackets. Gn239 and Gn318 contain one potential glucosylation site, Gn340-2A-E3, and Gn340-E3 have two potential sites.

FIG. 6

The expression cassettes designed for expression of RVFV Gn in viral particles and analysis of the protein expression. (A) The hydrophobicity profile (Kyte-Doolitle) of the Gn protein. Fragments cloned into VEEV structural polyprotein are indicated by black lines. (B) The schematic representation of the recombinant VEEV genomes containing the insertions of the Gn-encoding sequences in the subgenomic RNA. The numbers correspond to the positions of the first and the last a.a. of the insertion in the polyprotein encoded by the RVFV M segment. The triangles indicate positions of engineered furin-specific cleavage sites. VEEV-specific sequences are indicated in grey color. Indicated viral titers and plaque size were derived from multiple experiments. (C and D) The results of metabolic labeling of the proteins expressed by recombinant viruses. The pulse labeling (C) and pulse-chase labeling (D) with [³⁵S]methionine, followed by SDS-10% polyacrylamide gel electrophoresis, were performed at 16 h post infection as described in the Materials and Methods. Final processing products of furin protease are indicated by asterisks. (E) Distribution of Gn-E2 fusion protein in the VEEV/Gn318(opt)-infected cells. Panels (a) represent images of the cells that were permeabilized with Triton X-100 prior to immunostaining with RVFV-specific antibodies. Panels (b) present images of the cells stained with the same antibodies without permeabilization. Images were acquired on a Zeiss LSM510 META confocal microscope using a 63X 1.4NA oil immersion planapochromal lens, as described in Materials and Methods.

FIG. 7

Survival of mice immunized with the indicated recombinant viruses after s.c. challenge with 5×10³ PFU of RVFV ZH501. Mice were immunized either once (A) or twice (B) and challenged as described in the Materials and Methods.