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

Dal Young Kim, Svetlana Atasheva, Alexander J. McAuley, Jessica Plante, Elena I. Frolova, David Beasley, Ilya Frolov

Research output: Contribution to journalArticle

11 Citations (Scopus)

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 producedsubviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.

Original languageEnglish (US)
Pages (from-to)10708-10713
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume111
Issue number29
DOIs
StatePublished - Jul 22 2014

Fingerprint

Alphavirus
Replicon
RNA
Venezuelan Equine Encephalitis Viruses
Proteins
West Nile virus
Viral Envelope Proteins
Viral Genome
Viral RNA
Virus Diseases
Enzymes
Virus Replication
Neutralizing Antibodies
Genes
Immunization
Cytoplasm
Complementary DNA
Clone Cells
Genome

Keywords

  • Expression vectors
  • Vaccines

ASJC Scopus subject areas

  • General

Cite this

Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs. / Kim, Dal Young; Atasheva, Svetlana; McAuley, Alexander J.; Plante, Jessica; Frolova, Elena I.; Beasley, David; Frolov, Ilya.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 111, No. 29, 22.07.2014, p. 10708-10713.

Research output: Contribution to journalArticle

Kim, Dal Young ; Atasheva, Svetlana ; McAuley, Alexander J. ; Plante, Jessica ; Frolova, Elena I. ; Beasley, David ; Frolov, Ilya. / Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs. In: Proceedings of the National Academy of Sciences of the United States of America. 2014 ; Vol. 111, No. 29. pp. 10708-10713.
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