Cleavage of p15 protein in vitro by human immunodeficiency virus type 1 protease is RNA dependent.
The human immunodeficiency virus (HIV) gag polyprotein is processed by the viral protease to yield the structural proteins of the virus. One of these structural proteins, p15, and its protease cleavage products, p7 and p6, are believed to be responsible for the viral RNA binding which is prerequisite for assembly of infectious virions. To better understand potential interactions between viral RNA, p15, and the HIV protease, we have synthesized p15 in an in vitro system and studied its processing by the viral protease. Using this system, we demonstrate that p15 synthesized in vitro is properly cleaved by the HIV protease in an RNA-dependent reaction. Mutation of cysteine residues in either zinc-binding domain of the p7 portion of p15 does not alter the RNA-dependent cleavage, but mutation of three basic residues located between the zinc-binding domains blocks HIV protease susceptibility. The results support a previously unrecognized role for the interaction of RNA and nucleocapsid-containing gag precursors that may have important consequences for virus assembly.
Sheng
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Molecular Biology Department, DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0400.
Erickson-Viitanen
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003545:Cysteine; D017931:DNA Primers; D015683:Gene Products, gag / Q000096:biosynthesis*; D016333:HIV Protease / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019590:Nucleocapsid Proteins; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012367:RNA, Viral / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic; D054301:gag Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015683:Gene Products, gag; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C060295:p15 gag protein, Human immunodeficiency virus 1; D016333:HIV Protease; D003545:Cysteine