Alphavirus cDNA‐based expression vectors: Effects of RNA transcription and nuclear export

Abstract

The construction of layered DNA-RNA replicons has facilitated and expanded the use of alphavirus vectors to vaccine development, construction of packaging cell lines and long-term heterologous gene expression. In these vector systems, the alphavirus replicon is under the control of a strong RNA polymerase II promoter and replicon RNA is transcribed from DNA before transport to the cytoplasm. Efficient RNA amplification catalyzed by the viral replicase results in high levels of mRNA and the recombinant protein. Recently, we developed a temperature-regulated Sindbis replicon-based DNA expression system characterized by a linear increase of expression upon decrease of the temperature from 37 degrees C to 29 degrees C. Modifications known to affect transcription and nuclear export of RNA led to a 5-fold increase in expression in BHK cells and up to over 80-fold increase in CHO cells and BF fibroblasts in transient transfection experiments. Furthermore, reducing cell proliferation resulted in a further 2- to 3-fold higher expression. While increased expression per cell was responsible for some of the enhanced production, it was primarily the number of expressing cells that made the difference in most cell lines. Further experiments indicated that a threshold amount of replicon RNA had to reach the cytoplasm in order for replication to occur. Thus, alterations that improve transcription, nuclear export and stability of the RNA had a significant impact on protein production in the pCytTS expression system and probably in other layered DNA-based viral vectors. Furthermore the results indicate that RNA replication is differentially regulated in DNA layered RNA replicons versus viral infection.

Dose-Dependent Protection against or Exacerbation of Disease by a Polylactide Glycolide Microparticle-Adsorbed, Alphavirus-Based Measles Virus DNA Vaccine in Rhesus Macaques

“…The monkeys given the high dose i.m. averaged 101 Ϯ 28 SFC/10 6 PBMCs, compared to control values of 21 Ϯ 17 SFC (P Ͻ 0.05). No H-specific IL-4 production was detected (data not shown), suggesting a predominantly type 1 T-cell cytokine response.…”

“…The RNA expressed from the DNA plasmid is self-amplifying in the cytoplasm and generates large amounts of mRNA for the protein antigen in cells in which sufficient replicon RNA reaches the cytoplasm (6). A related alphavirus replicon approach in which the RNA expressing MV H is packaged into virion particles for delivery protects monkeys from rash but not from viremia (42).…”

“…When the DNA is transcribed to mRNA, the nonstructural proteins are translated, replicate the RNA, and produce abundant subgenomic mRNAs that code for the antigen. Thus, there is amplification of the mRNAs so that large amounts of protein are synthesized (6,16,25). In addition, these vaccines activate innate immune pathways to increase the immunogenicity of the antigens encoded (30).…”

Cellular factors influencing Semliki Forest Virus vector biology

“…This is specifically the case when using the VSV-G VLP, where the VSV-G is produced by the RNA Pol II-dependent cellular machinery. 11,16 Any reduction in the transexpression of the VSV-G directly affects the titre and thereby the utilization opportunities of the produced particles.…”

“…9,10 Semliki Forest Virus (SFV) and Sindbis Virus (SV) are the main two alphaviruses from which vectors have been obtained. 11,12 In agreement with their biology, these vectors are predominantly adapted to high levels of transient transgene expression. This results from an optimized cytoplasmic genomic replication combined with the cytotoxic effect induced by a number of virally encoded proteins.…”