Determinants of the human immunodeficiency virus type 1 p15NC-RNA interaction that affect enhanced cleavage by the viral protease

N Sheng, S C Pettit, R J Tritch, D H Ozturk, M M Rayner, R Swanstrom, S Erickson-Viitanen
1997 Journal of Virology  
During human immunodeficiency virus type 1 (HIV-1) virion assembly, cleavage of the Gag precursor by the viral protease results in the transient appearance of a nucleocapsid-p1-p6 intermediate product designated p15NC. Utilizing the p15NC precursor protein produced with an in vitro transcription-translation system or purified after expression in Escherichia coli, we have demonstrated that RNA is required for efficient cleavage of HIV p15NC. Gel mobility shift and nitrocellulose filter binding
more » ... se filter binding experiments indicate that purified p15NC protein specifically binds its corresponding mRNA with an estimated K d of 1.5 nM. Binding was not affected by the presence or absence of zinc or EDTA. Moreover, mutagenesis of the cysteine residues within either of the two Cys-His arrays had no effect on RNA binding or on RNA-dependent cleavage by the viral protease. In contrast, decreased binding of RNA and diminished susceptibility to cleavage in vitro were observed with p15NC-containing mutations in one or more residues within the triplet of basic amino acids present in the region between the two zinc fingers. In addition, we found that 21-to 24-base DNA and RNA oligonucleotides of a particular sequence and secondary structure could substitute for p15 RNA in the enhancement of p15NC cleavage. Virus particles carrying a mutation in the triplet of NC basic residues (P3BE) show delayed cleavage of p15NC and a defect in core formation despite the eventual appearance of fully processed virion protein. These results define determinants of the p15NC-RNA interaction that lead to enhanced protease-mediated cleavage and demonstrate the importance of the triplet of basic residues in formation of the virus core.
doi:10.1128/jvi.71.8.5723-5732.1997 fatcat:kwkbnjcsnnb7flyd3o3qyjbkri