Generation of recombinant herpes simplex virus ampiicons.

The herpes simplex virus type 1 (HSV-1) amphcon has been developed as a novel eukaryotic expression vector, which contains an HSV-1 ori for DNA replication and a pac signal for cleaving/packaging genomes into viral capsids (1-4). As shown in Fig. 1, amplicon vector can be amplified into head-to-tail concatemers and then packaged into defective HSV-1 viral particles up to one genome size (~150 kb) in the presence of HSV-1 helper viruses. The helper viruses provide all necessary proteins and enzymes for amplicon DNA replication/packaging and for the assembly of defective amplicon viruses (1). One of the applications of this defective amplicon virus system is to transfer efficiently high copy numbers of foreign genes into a broad range of mammalian cells for high-level expression and gene therapy (4,5). We have utilized the amphcon system to produce high levels of HCV NS3/4A complexes in mammalian cells (6). Our results have demonstrated that the amplicon system provides a potential to study the expression of HCV proteins in a broadspectrum of mammalian cell lines, especially in those of human hepatocyte origin that may be biologically more relevant to HCV infection. In this chapter, methods for using the amphcon expression system will be described in three subsections: 1. Generation of high-titer defective HSV-1 amplicon virus stocks Fig. 1. Illustration of HSV-I amplicon expression system. 2. Determination of the titers of amphcon viruses. 3. Expression of HCV NS3/4A complexes using the defective amphcon viruses.