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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.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Kim DY,Atasheva S,McAuley AJ,Plante JA,Frolova EI,Beasley DW,Frolov I

doi

10.1073/pnas.1408677111

subject

Has Abstract

pub_date

2014-07-22 00:00:00

pages

10708-13

issue

29

eissn

0027-8424

issn

1091-6490

pii

1408677111

journal_volume

111

pub_type

杂志文章

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