Poliovirus replicons that express the gag or the envelope surface protein of simian immunodeficiency virus SIV(smm) PBj14.
Poliovirus genomes encoding the complete gag or env surface gene of the simian immunodeficiency virus SIV(smm) PBj14 (SIV-PBj14) were constructed. The in vitro-transcribed RNA from these genomes, referred to as replicons, have the capacity for self-replication when transfected into tissue culture cells. Serial passage of the replicons containing the SIV-PBj14 gag or SIV-PBj14 env (SU) genes with a recombinant vaccinia virus, VV-P1, which provides P1 in trans, resulted in the encapsidation of these replicons. Infection of cells with the encapsidated replicons that encode gag, referred to as vIC-SIV-PBj14 Gag, resulted in the production of a 55-kDa protein that was released from the infected cells. Using a sucrose density-gradient analysis, the protein was found to sediment at a density consistent with that of a virus-like particle. Infection of cells with a replicon that encodes the env SU gene, referred to as vIC-SIV-PBj14 SU, resulted in the production of two SIV-PBj14 envelope-related intracellular proteins. One of these proteins had a molecular mass consistent with that of the unglycosylated SIV-PBj14 SU protein (63 kDa); the second protein had a higher molecular mass (>160 kDa). Characterization of this larger protein revealed that it was glycosylated and possibly represented a dimer of the SU protein. A pulse-chase analysis of cells infected with vIC-SIV-PBj14 SU demonstrated that a 110- to 130-kDa protein was released, which is consistent with the molecular mass of the SIV-PBj14 SU protein. The results of these studies demonstrate that poliovirus replicons can be used to express foreign proteins, including glycoproteins, which retain many of the physical features of the native protein.
Anderson
M J
MJ
Department of Microbiology, University of Alabama at Birmingham 35294, USA.
Porter
D C
DC
Fultz
P N
PN
Morrow
C D
CD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D015686:Gene Products, env / Q000737:chemistry / Q000235:genetics*; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D000882:Haplorhini; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D012093:Replicon; D015302:Simian Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics*; D017945:Theilovirus / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication
D017931:DNA Primers; D015686:Gene Products, env; D015683:Gene Products, gag