AEGiS-11IAC: Interactions between HIV GAG p15 and viral RNA as targets for HIV therapeutics.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

Interactions between HIV GAG p15 and viral RNA as targets for HIV therapeutics.

Int Conf AIDS 1996 Jul 7-12; 11:217 (abstract no. Th.A.382)
Erickson-Viitanen S, Ozturk D, Choi HK, Sheng N, Tritch R, Petit S, Swanstrom R; The DuPont Merck Pharmaceutical Co., Wilmington, DE, USA.

Introduction/

OBJECTIVES: The nucleocapsid protein of the Human Immunodeficiency Virus plays both structural and functional roles in the assembly of mature, infectious virions. During the assembly process, nucleocapsid protein is produced from its precursor p15NC by specific cleavage mediated by the viral protease. Our objective has been to determine the interactions between the nucleocpasid precursor, viral RNA and the viral protease during the virus maturation process.

RESULTS: Using p15NC precursor protein produced in vitro or in E. coli, we have demonstrated that cleavage of HIV p15NC occurs efficiently only when viral RNA is present. Cleavage by the viral protease at other sites within the GAG protein is not RNA dependent. Gel mobility shift and nitrocellulose filter binding experiments indicate that the p15NC binds its corresponding mRNA with a Kd of 1.5 nM. This binding is not affected by the presence or absence of zinc or EDTA. Moreover, both RNA binding and RNA dependent, HIV protease-mediated cleavage either in vitro or in HIV-infected cells occur when mutant p15NC with alterations in the cysteine residues within the two Cys-His arrays are utilized. In contrast, decreased binding of RNA, and diminished susceptibility to viral protease-mediated cleavage in vitro or in infected cells were observed with a mutant p15NC in which the three basic residues present in the region between the two zinc fingers have been mutated to non-basic residues. Through deletion analysis and use of synthetic oligonucleotide mapping, we have found that specific oligonucleotides of 21-24 nucleotides that are capable of forming stable stem-loop structures can displace labeled RNA in binding assays. These small oligonucleotides can substitute for viral RNA in conferring susceptibility of gag p15NC to viral protease processing.

CONCLUSIONS: Our studies suggest the intriguing possibility that a small oligonucleotide or mimic may be able to serve as an inhibitor of these required proteolytic events in cells.

Keywords: AEGIS, RNA, Viral, Gene Products, gag, HIV Protease, Nucleocapsid Proteins, Virion, Drug Interactions, Zinc Fingers, DNA Primers, RNA, Messenger, Endopeptidases, Human, In Vitro, ICA11

960707
ThA382