Nj. Sheng et al., DETERMINANTS OF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 P15NC-RNA INTERACTION THAT AFFECT ENHANCED CLEAVAGE BY THE VIRAL PROTEASE, Journal of virology, 71(8), 1997, pp. 5723-5732
During human immunodeficiency virus type 1 (HIV-1) virion assembly, cl
eavage of the Gag precursor by the viral protease results in the trans
ient appearance of a nucleocapsid-p1-p6 intermediate product designate
d p15NC. Utilizing the p15NC precursor protein produced with an in vit
ro transcription-translation system or purified after expression in Es
cherichia coli, we have demonstrated that RNA is required for efficien
t cleavage of HIV p15NC. Gel mobility shift and nitrocellulose fitter
binding experiments indicate that purified p15NC protein specifically
binds its corresponding mRNA with an estimated K-d of 1.5 nM. Binding,
vas 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 t
he viral protease. In contrast, decreased binding of RNA and diminishe
d susceptibility to cleavage in vitro were observed with p15NC-contain
ing mutations in one or more residues within the triplet of basic amin
o acids present in the region between the two zinc fingers. In additio
n, we found that 21- to 24-base DNA and RNA oligonucleotides of a part
icular sequence and secondary structure could substitute for p15 RNA i
n the enhancement of p15NC cleavage. Virus particles carrying a mutati
on 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 o
f the p15NC-RNA interaction that lead to enhanced protease-mediated cl
eavage and demonstrate the importance of the triplet of basic residues
in formation of the virus core.