Sindbis Virus Vectors Designed To Express a Foreign Protein as a Cleavable Component of the Viral Structural Polyprotein

Abstract

Alphavirus-based expression vectors commonly use a duplicated 26S promoter to drive expression of a foreign gene. Here we describe an expression strategy in which the foreign sequences are linked to the gene encoding the 2A protease of foot-and-mouth disease virus and then inserted in frame between the capsid and E3 genes of Sindbis virus. During replication, the 2A fusion protein is synthesized as a component of the viral structural polyprotein that is then released by intramolecular cleavages mediated by the capsid and 2A proteases. Recombinant Sindbis viruses that expressed fusion proteins composed of 2A linked to the green fluorescent protein (GFP) and to the VP7 protein of bluetongue virus were constructed. Viruses engineered to express GFP and VP7 from a duplicate 26S promoter were also constructed. All four viruses expressed the transgene and grew to similar titers in cultured cells. However, the GFP/2A-and VP7/2A-expressing viruses displayed greater expression stability and were less attenuated in newborn mice than the cognate doublesubgenomic promoter-based viruses. By combining the two expression strategies, we constructed bivalent viruses that incorporated and expressed both transgenes. The bivalent viruses grew to lower titers in cultured cells and were essentially avirulent in newborn mice. Groups of mice were vaccinated with each VP7-and VP7/2A-expressing virus, and antibody responses to native VP7 were measured in an indirect enzyme-linked immunosorbent assay. Despite their genetic and phenotypic differences, all viruses induced similarly high titers of VP7-specific antibodies. These results demonstrate that 2A fusion protein-expressing alphaviruses may be particularly well suited for applications that require enduring expression of a single protein or coexpression of two alternative proteins.Alphaviruses are enveloped, single-stranded, positive-sense RNA viruses that belong to the Togaviridae virus family (Alphavirus genus). Upon entry into the host cell, the viral genome is translated into the four nonstructural proteins (nsp1 to -4) that comprise the viral transcriptase-replicase complex. The viral replicase uses the genomic RNA as a template to synthesize a complementary, full-length, negative-sense RNA. The negative-sense RNA in turn serves as a template for the synthesis of two different positive-sense RNAs. Positive-strand synthesis initiating at the 3Ј end of the negative-strand RNA results in the production of full-length genomic RNA. Positivestrand synthesis can also initiate at an internal promoter sequence (26S or subgenomic promoter) to produce a subgenomic mRNA that is colinear with approximately the 3Ј-terminal one-third of the genomic RNA. Subgenomic mRNAs are translated into the viral structural proteins (40).

Stable, High-Level Expression of Reporter Proteins from Improved Alphavirus Expression Vectors To Track Replication and Dissemination during Encephalitic and Arthritogenic Disease

“…The SINV 3=DP-nLuc virus plasmid was constructed by replacement of the corresponding fLuc region of the SINV 3=DP-fLuc with a Flag-tagged nLuc gene (nLuc-Flag). Capsid fusion reporter alphaviruses expressing reporter proteins (enhanced GFP [eGFP], mCherry, fLuc, or nLuc) as a cleavable in-frame fusion between the capsid and E3 proteins were constructed essentially as described previously (17), except that the QuikChange II XL mutagenesis kit (Agilent) was used to insert a PCR fragment at the capsid/E3 junction that encoded the first five amino acids of E3 fused in frame to reporter genes followed by the TaV 2A-like protease. The nsP3 fusion reporter alphaviruses, with the exception of EEEV, were created by introducing a unique restriction enzyme FseI site at the corresponding positions in the nsP3 protein using QuikChange mutagenesis and inserting the reporter genes (eGFP, mCherry, fLuc, or nLuc) using the unique enzyme.…”

“…These were inserted either into a traditional 3= DP vector, as an nsP3 fusion into the region described for SINV (14), or between the capsid and PE2 proteins, as an autocleaving element as described previously (17), but improved by using the highly efficient Thosea asigna virus (TaV) 2A-like protease (18). Transgene expression, stability, and virulence of the vectors in animals were then compared.…”

Oncolytic Viral Therapy for Cervical and Ovarian Cancer Cells by Sindbis Virus AR339 Strain

“…The GFP-expressing recombinant SIN (TR339-GFP/ 2A) was derived from cDNA clone pTR339-GFP/2A, which was kindly provided by Dr. Hans W. Heidner (University of Texas, San Antonio, TX; ref. 15). The cDNA clone was linearized by digestion with XhoI, and a run-off transcript was produced by using SP6 RNA polymerase.…”

Alteration of cell cycle progression by Sindbis virus infection

“…The plasmid pTR339-GFP-2A was kindly provided by Dr. Hans W. Heidner (University of Texas, San Antonio, TX; [16]). The BFP gene is substituted for the GFP gene in the pTR339-GFP-2A plasmid, to yield pTR339-BFP-2A.…”