title: The nonmyristylated Pr160gag-pol polyprotein of human immunodeficiency virus type 1 interacts with Pr55gag and is incorporated into viruslike particles.
abstract: The expression of the pol gene of human immunodeficiency virus type 1 occurs via a ribosomal frameshift between the gag and pol genes. The resulting protein, a Gag-Pol polyprotein, is produced at a level 5 to 10% of that of the Gag protein. The Gag-Pol polyprotein is incorporated into virions and provides viral protease, reverse transcriptase, and integrase, which are essential for infectivity. It is generally believed that the Gag-Pol polyprotein is incorporated into virions via interaction with the Gag protein, although the details of the mechanism are unknown. To further study this problem, we have constructed a human immunodeficiency virus type 1 proviral genome which overexpresses the Gag-Pol polyprotein (Pr160gag-pol). Transfection of this proviral genome (pGPpr-) into COS-1 cells resulted in the expression of full-length Pr160gag-pol polyprotein. Although the majority of the Pr160gag-pol was confined to the cells, low levels of reverse transcriptase activity were detectable in the cell supernatants. The cotransfection of pGPpr- with a second plasmid which expresses only the Pr55gag precursor (pGAG) resulted in a significantly higher level of Pr160gag-pol in the medium of transfected cells. Sedimentation analysis using sucrose density gradients demonstrated that most Pr160gag-pol was found in fractions corresponding to the density of virion particles, indicating that the Pr160gag-pol polyprotein was released in association with a Pr55gag viruslike particle. To further characterize the requirements for the release, a mutation was constructed to express an unmyristylated Pr160gag-pol polyprotein. Coexpression with Pr55gag demonstrated that the unmyristylated Pr160gag-pol was also incorporated into virion particles. Subcellular fractionation experiments revealed that the distributions of the Pr160gag-polmyr- and Pr160gag-pol in the membrane and cytosol were similar under low- or high-ionic-strength conditions. Taken together, these results suggest that myristylation of the Pr160gag-pol polyprotein is not required for the interaction with the Pr55gag necessary for packaging into a viruslike particle.
authors.affiliation: Department of Microbiology, University of Alabama, Birmingham 35294-0007.
authors.forename: J
authors.initials: J
authors.lastname: Park
authors.forename: C D
authors.initials: CD
authors.lastname: Morrow
mesh_terms: D001483:Base Sequence; D015685:Fusion Proteins, gag-pol / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D016679:Genome, Viral; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000254:growth & development*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011533:Proviruses / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000032:analysis; D016364:Reading Frames; D014162:Transfection; D014779:Virus Replication
chemical_list: D015685:Fusion Proteins, gag-pol; D015683:Gene Products, gag; D009227:Myristic Acids; D011498:Protein Precursors; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D019814:Myristic Acid; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016333:HIV Protease