7 and data not shown). of the proteins. iii) Alphaviruses can be revised for manifestation of the large fragments of heterologous proteins on the surface of chimeric, infectious viral particles. Thus, alphavirus-based manifestation systems may have the potential for a broader software beyond their current use as replicons or double-subgenomic vectors. from the VEEV replicons for efficient helper RNA replication and production of the structural proteins. In our earlier studies, both pairs of helpers packaged not only replicons, but also their personal RNAs and, therefore, VEEV replicon and two helper RNAs could be efficiently passaged in the form of a tri-component genome disease (Volkova, Gorchakov, and Frolov, 2006). Open Efonidipine in a separate window 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 manifestation 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 37C for 12 h. Equivalent 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 Efonidipine 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 illness, 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 5106 inf.u of packaged VEErep/spGn replicon and challenged in 42 days with 5103 PFU s.c. of RVFV ZH501. The control group was injected with PBS and challenged with the same dose of RVFV. VEEV replicon expressing RVFV spGn was packaged into viral particles (Fig. 3A) that were used for analysis of protein manifestation and immunization of mice. VEErep/spGn indicated Gn noticeably more efficiently than did SINrep/spGn (Fig. 3B), and the protein was localized not only in the ER and Golgi compartment (Fig. 3C, panel a), but was also present in high concentrations within the external membrane (Fig. 3C, panel b) Its distribution was related to that found in the cells infected with the SINrep/spGn replicon (compare Figs. 2E, panel f, and 3C, panel b). In mice, one immunization with packaged VEErep/spGn did not induce neutralizing antibodies to titers higher than those recognized in mice immunized with related SINV-based replicons (data not shown). The PRNT80 titers were constantly lower than 1:80. However, actually one immunization efficiently safeguarded animals against an ensuing illness with RVFV ZH501, a getting consistent with the idea that VEEV replicons are more immunogenic and, hence, more appropriate for development of recombinant vaccines. However, the bad feature of the VEErep/spGn replicon was in the strong effect of indicated Gn within the assembly and/or release of the viral particles. In contrast to our success with SINV replicons in terms of their passaging in cells tradition, we were incapable of passaging the Gn-expressing VEErep in cell tradition, regardless of the helpers used. Even after electroporation, the Kdr titers of the infectious particles were constantly lower than in the experiments with replicons expressing additional heterologous genes Efonidipine (data not shown). Therefore, the possible problem with the large-scale production of packaged Gn-encoding VEEV replicons should be considered in the future studies. Manifestation of RVFV Gn in VEEV structural polyprotein The above-described experiments suggested a need for additional approaches to enable the manifestation of RVFV antigens by alphaviruses and the induction of protecting immune response against RVFV illness. The rationale was to develop Gn manifestation systems.