Alphavirus-based expression vectors: strategies and applications.

Abstract

Alphaviruses are positive-strand RNA viruses that can mediate efficent cytoplasmic gene expression in insect and vertebrate cells. Through recombinant DNA technology, the alphavirus RNA replication machinery has been engineered for high-level expression of heterologous RNAs 1 and 2). Over the past 10 years, the alphavirus RNA replication and packaging machinery has been adapted for expression of heterologous RNAs and proteins in animal cells (for reviews, see refs. 3-6). As transient expression systems, alphaviruses offer several advantages. These include (i) a broad range of susceptible host cells including those of insect, avian, and mammalian origin; (ii) high levels of cytoplasmic RNA and protein expression without splicing; and (iii) the facile construction and manipulation of recombinant RNA molecules using full-length cDNA clones from which infectious RNA transcripts can be generated by in vitro transcription. Two principal strategies are being employed for expression of heterologous sequences: (i) engineering infectious recombinant RNAs that express additional subgenomic RNAs and (ii) replacement of the structural genes to produce self-replicating RNA "replicons" that can be packaged into infectious particles using defective helper RNAs or packaging cell lines. In addition, incorporation of heterologous ligands or receptors into the virion envelope may eventually allow targeting of engineered alphavirus RNAs to specific cell types. This overview briefly discusses the background, methodology, and applications of these alphavirus vector systems, which range from high-level protein production in cell culture to the induction of protective immunity in animals. (Fig; 1). This subgenomic RNA, which can accumulate to levels approaching 106 molecules per cell, is the mRNA for translation of the structural proteins. The synthesis of minus, plus, and subgenomic RNAs is temporally regulated via proteolytic processing of nonstructural polyprotein replicase components by a virus-encoded protease residing in the C-terminal region of nsP2 (8, 9).The structural proteins are initially translated as a polyprotein (NH2-C-E3-E2-6K-E1-COOH) that is processed coand posttranslationally to produce the mature products. Cleavage at the C-E3 site is mediated by a chymotrypsin-like protease activity residing in the C-terminal portion of the C protein. E3 and E2 are initially made as a precursor (called PE2 or P62) that is processed by a furin-like activity late during release of the virus from infected cells. Envelope glycoproteins El and PE2, separated by signal peptidase cleavages, form a heterodimer that migrates through the secretory pathway to the plasma membrane. In the cytoplasm, C-protein subunits complex with the genome RNA to form a nucleocapsid that matures by budding through the plasma membrane, acquiring a lipid bilayer envelope with embedded viral glycoproteins. Infectious Alphavirus cDNA ClonesStudies on the use of alphaviruses as vectors have required the recovery of infectious replication-competent ...

Latest Developments in Gene Transfer Technology: Achievements, Perspectives, and Controversies over Therapeutic Applications

“…Preclinical tests have been carried out to characterize the gene delivery properties of vectors based on foami virus [95][96][97], lentiviruses (such as HIV-1 [98][99][100][101][102][103][104] and feline immunodeficiency virus (FIV) [105][106][107]), human cytomegalovirus (CMV) [108], Epstein-Barr virus [109], negative-strand RNA viruses (influenza virus) [110], alphaviruses [111], herpesvirus saimiri [112], hybrid adenoviral/retroviral vector systems [113,114], and hybrid alphavirus/retroviral vector [115]. Other preclinical studies are also assessing the level of vector design improvement that has been reported for a variety of gene transfer models.…”

Evaluation of recombinant alphaviruses as vectors in gene therapy

“…Theoretically, only the vector RNA with the transgene is packaged into the virion particles, which are capable of transducing target cells, but are replication-incompetent. For more information about alphaviral vector systems, see Frolov et al 21 Both Sindbis and SFV vector stocks were produced in infectious particles per ml. The main factors influencing vector production were the quality and amount of in vitro transcribed RNA and the efficiency of electroporation (data not shown).…”

Modulation of disease, T cell responses, and measles virus clearance in monkeys vaccinated with H-encoding alphavirus replicon particles

“…The nonstructural proteins are translated from the genomic RNA whereas the structural proteins are translated from the abundant subgenomic RNA. The subgenomic promoter can be engineered to express foreign genes either by duplicating it in the genome or by substituting the foreign gene for the structural proteins (22)(23)(24). In the latter replicon system, these RNAs can be used directly for vaccination (25)(26)(27) or packaged into particles by supplying the viral structural proteins, selected for delivery to specific target cells, in trans (28)(29)(30).…”