Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs

Abstract

Since the development of infectious cDNA clones of viral RNA genomes and the means of delivery of the in vitro-synthesized RNA into cells, alphaviruses have become an attractive system for expression of heterologous genetic information. Alphaviruses replicate exclusively in the cytoplasm, and their genetic material cannot recombine with cellular DNA. Alphavirus genome-based, self-replicating RNAs (replicons) are widely used vectors for expression of heterologous proteins. Their current design relies on replacement of structural genes, encoded by subgenomic RNAs (SG RNA), with heterologous sequences of interest. The SG RNA is transcribed from a promoter located in the alphavirus-specific RNA replication intermediate and is not further amplified. In this study, we have applied the accumulated knowledge of the mechanism of alphavirus replication and promoter structures, in particular, to increase the expression level of heterologous proteins from Venezuelan equine encephalitis virus (VEEV)-based replicons. During VEEV infection, replication enzymes are produced in excess to RNA replication intermediates, and a large fraction of them are not involved in RNA synthesis. The newly designed constructs encode SG RNAs, which are not only transcribed from the SG promoter, but are additionally amplified by the previously underused VEEV replication enzymes. These replicons produce SG RNAs and encoded proteins of interest 10- to 50-fold more efficiently than those using a traditional design. A modified replicon encoding West Nile virus (WNV) premembrane and envelope proteins efficiently produced subviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.

Keywords: expression vectors; vaccines.

Fig. 1.

The schematic representation of alphavirus vectors and protein expression strategies. (A) The alphavirus genome. (B) Standard alphavirus replicons. Viral structural genes are replaced by heterologous gene of interest. (C) The newly designed VEEV DI replicons. The DI SG RNAs contain all of the promoter elements, which are normally present only in the 5′ end of VEEV genomic RNA (Fig. S1 for details). Presence of these regulatory sequences allows nsPs not only to transcribe the DI SG RNAs but also to amplify these RNA as viral genomes.

Fig. 2.

VEEV DI replicons express heterologous proteins more efficiently than those having standard design. (A) The schematic representation of VEEV replicons encoding GFP or Luc genes under control of the subgenomic promoters. (B) BHK-21 cells were infected with the indicated packaged replicons at an MOI of 20 inf.u/cell, and GFP fluorescence was evaluated by FACS analysis at 20 h postinfection. (C) Cells were infected with indicated replicons at the same MOI, harvested at 20 h postinfection, lysed in loading buffer, and samples were analyzed by SDS/PAGE, followed by Coomassie blue staining. Cm, noncytopathic VEEV capsid protein; nsP1Δ, a peptide, whose coding sequence contains a 51-nt CSE.

Fig. 3.

DI replicons demonstrate higher levels of SG RNA synthesis in the infected cells. BHK-21 cells were infected with the indicated packaged replicons at an MOI of 20 inf.u/cell. Replicon-specific RNAs were metabolically labeled with [³H]uridine in the presence of ActD and analyzed by agarose gel electrophoresis (Materials and Methods for details). Panels represent fragments of the same gel and the same X-ray film.

Fig. 4.

DI replicons demonstrate higher levels of heterologous protein synthesis, which correlates with the increase of SG RNA production. Infected cells were metabolically labeled with [³⁵S]methionine at 7 h postinfection (Materials and Methods for details). Fragments of the same gel are presented. Radioactivity in the GFP- and Luc-containing bands was measured on Storm phosphorimager. The data were normalized to the radioactivity in the GFP and Luc bands detected in the VEErep/GFP and VEErep/Luc/GFP samples, respectively.

Fig. 5.

VEErep/DI-Cm-WNV expresses high level of WNV-specific E protein, which is released from the cells in the SVP form. (A) The schematic representation of VEErep/DI-Cm-WNV replicon, encoding prM and E proteins of WNV. (B) Fractions of the concentrated samples of the media, harvested at 12 h postinfection, were used for negative staining and analyzed by EM. Bar corresponds to 50 nm. (C) SDS/PAGE analysis of WNV E protein present in the cells or released in SVPs at 12 h postinfection. Gel was stained with Coomassie blue.

Fig. 6.

Neutralizing antibody titers (50% focus reduction) and protection among 3- to 4-wk-old Swiss Webster mice inoculated with a single dose of VEErep/DI-Cm-WNV and challenged with 100 LD₅₀ of WNV NY99. Animals were bled at 19 d postimmunization and challenged at 22 d postimmunization. n/a, not applicable.