Expression of human immunodeficiency virus type 1 (HIV-1) gag, pol, and env proteins from chimeric HIV-1-poliovirus minireplicons

Abstract

Recent studies have demonstrated that genomes of poliovirus with deletions in the P1 (capsid) region contain the necessary viral information for RNA replication. To test the effects of the substitution of foreign genes on RNA replication and protein expression, chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes were constructed in which regions of the gag, pol, or env gene of HIV-1 were substituted for regions of the P1 gene in the infectious cDNA clone of type 1 Mahoney poliovirus. The HIV-1 genes were inserted between nucleotides 1174 and 2956 of the poliovirus cDNA so that the translational reading frame was maintained between the HIV-1 genes and the remaining poliovirus genes. The chimeric genomes were positioned downstream from a T7 RNA polymerase promoter and transcribed in vitro by using T7 RNA polymerase, and the RNA was transfected into HeLa cells. A Northern (RNA blot) analysis of the RNA from transfected cells demonstrated the appropriate-size RNA, corresponding to the full-length chimeric genomes, which increased over time. Immunoprecipitation with antibodies specific for poliovirus RNA polymerase or sera from AIDS patients demonstrated the expression of the poliovirus RNA polymerase and HIV-1 proteins as fusions with the poliovirus P1 protein. The expression of the HIV-1-poliovirus P1 fusion protein was dependent upon an intact RNA polymerase gene, indicating that RNA replication was required for efficient expression. A pulse-chase analysis of the protein expression from the chimeric genomes demonstrated the initial rapid proteolytic processing of the polyprotein from the chimeric genomes to give HIV-1-poliovirus P1 fusion protein in transfected cells; the HIV-1 gag-P1 and HIV-1 pol-Pl fusion proteins exhibited a greater intracellular stability than the HIV-1 env-P1 fusion protein. Finally, superinfection with wild-type poliovirus of HeLa cells which had been transfected with the chimeric genomes did not significantly affect the expression of chimeric fusion protein. The results are discussed in the context of poliovirus RNA replication and demonstrate the feasibility of using poliovirus genomes (minireplicons) as novel vectors for expression of foreign proteins.

Expression of an Antigenic Adenovirus Epitope in a Group B Coxsackievirus

“…Ample evidence demonstrates that PV can be engineered as multivalent chimeric vaccines against non-PV infectious agents such as primate and human retroviruses (2,17,19,54,59), rotaviruses (42), hepatitis B virus (68), and potentially against cancer (38,45) as well. However, it has been questioned whether intact enteroviruses can package sufficiently large proteins while simultaneously maintaining the foreign insert for sufficient time to serve as useful chimeric vaccines (48).…”

Examples of expression systems based on animal RNA viruses: Alphaviruses and influenza virus

“…In the group employing the simplest strategy, which includes the picornavirus and flavivirus families, only genome-length positive strands are synthesized. Picornaviruses have been widely used for constructing chimeric viruses expressing short heterologous epitopes [5] and have found more limited use in the production of larger proteins [1,6]. In the other group, which includes the togavirus and coronavirus families, negative strands also function as templates for high level transcription of one or more 3' co-terminal subgenomic mRNAs.…”

Chapter 1 Viruses as Model Systems in Cell Biology

“…The interaction of the virus with its cellular receptor has been studied in detail, as has the mechanism by which the virus alters host cell protein synthesis. In addition, studies in which poliovirus has been used as a vector for expression of foreign genes have been reported (Choi et al, 1991;Percy et al, 1992;Andino et al, 1993;Porter et al, 1993).…”