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FIG. 1. Alphavirus replication cycle. Translated regions of alphavirus genomic and subgenomic RNAs are shown as boxes with the nonstructural proteins and structural proteins (STRUCTURAL) indicated as open and lightly shaded boxes, respectively. Cis-acting sequences important for replication and transcription are shown (small, checkered boxes) as is the sequence in the nonstructural region important for encapsidation (solid box). The start site for subgenomic mRNA transcription on the (−) strand genome-length RNA template is indicated by an arrow. Translation initiation (aug) and termination signals (trm) are indicated by open triangles and solid diamonds, respectively (from ref. 4).

RNA transcription, and genome RNA packaging (2) (see Fig. 1). Since the alphaviruses are positive-strand RNA viruses, virion-associated proteins are not required for initiation of the replication cycle. Capped RNA transcripts, produced by in vitro transcription with SP6 or T7 polymerase, are typically used to transfect tissue culture cells, usually a continuous hamster kidney line (BHK) or secondary chicken embryo fibroblasts. RNA transfection is facilitated by DEAE-dextran, cationic liposomes, or electroporation. In the latter method, efficiencies can approach 100% for BHK cells (11).

Although less efficient than transfection of full-length RNAs, alphavirus replication can also be initiated by transfection of plasmid DNA (14, 15). In this case, full-length 5′-capped RNAs are transcribed in the nucleus using a polymerase II promoter and transported to the cytoplasm, the site of primary translation and RNA amplification.

Replication and Packaging-Competent Vectors

Several approaches have been taken for independent expression of heterologous genes using the alphavirus RNA replication machinery. The identification of the SIN subgenomic RNA promoter element allowed the construction of RNAs with additional subgenomic RNA promoters (Fig. 2). Recombinant RNAs containing two promoters for subgenomic mRNA synthesis are referred to as double subgenomic RNA vectors (dsSIN) (1618). Heterologous sequences, expressed via a second subgenomic mRNA, can be located either 3′ or 5′ to the structural protein genes. These vectors are both replication and packaging competent and allow the rapid recovery of high-titered infectious recombinant virus stocks usually in the range of 108–109 plaque-forming units/ml. In initial studies (16), dsSIN recombinants were engineered to express bacterial chloramphenicol acetyltransferase (CAT), a truncated form of the influenza hemagglutinin (HA), or minigenes encoding two distinct immunodominant cytotoxic T-cell (CTL) HA epitopes. Infection of murine cell lines with these recombinants resulted in the expression of 106–107 CAT polypeptides per cell and efficient sensitization of target cells for lysis by appropriate major histocompatibility complex (MHC)-restricted HA-specific CTL clones in vitro. In addition, priming of an influenza-specific T-cell response was observed after immunizing mice with dsSIN recombinants expressing either truncated HA or the immunodominant influenza CTL epitopes. This system allows the generation of high-titered recombinant virus stocks in a matter of days and has been useful for mapping and mutational analysis of class I MHC-restricted T-cell epitopes expressed via the endogenous pathway of antigen processing and presentation (19, 20). Because of packaging constraints and instability of larger inserts upon passaging, this approach is primarily useful for short (<2 kb) heterologous sequences.

In other studies, dsSIN recombinants have been used to express the Japanese encephalitis virus (21) and rubella virus (22) stuctural proteins, to deliver a single chain antibody for intracellular immunization against tick-borne encephalitis (23), to map the domain of GLUT-4, the insulin-regulatable glucose transporter, which is responsible for efficient intracellular sequestration (24), to study structure-function aspects of ras-like GTP-binding proteins involved in vesicular transport (2529), and to probe the interplay between viral and cellular genes involved in apoptotic cell death (30, 31).

Another interesting application has been for gene expression studies in mosquito cells and mosquitoes (32). Engineered dsSIN recombinants have been used to follow virus spread in whole mosquitoes (33) and to express antisense RNAs or viral proteins that are capable of specifically inhibiting replication (3436) and transmission (37) of heterologous viruses and may be useful for studies of normal mosquito gene function via antisense RNA-mediated inhibition.

Alphavirus RNA Replicons

The prototype replication-competent, but packaging-defective, alphavirus RNA replicon was developed by replacing the SIN structural genes with the CAT gene (38) (Fig. 3, upper-left section). In cells transfected with this SIN recombinant RNA, CAT is expressed rapidly and up to 108 CAT polypeptides are produced per transfected cell by 16–20 h. CAT expression could be regulated by inclusion of a ts

FIG. 2. Double subgenomic RNA vectors. Infectious alphavirus vectors that contain both the replication machinery and the structural proteins. Heterologous gene products are expressed by synthesis of a second subgenomic mRNA. For other symbols see Fig. 1. Adapted from ref. 4.

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