Production of HIV-1(MN) Nucleocapsid Protein (p7) by Recombinant DNA Technology

Abstract

Members of the Retroviridae family of RNA viruses exhibit an extraordinarily rapid mutation rate. This rate of mutation not only enables these viruses to evade immune surveillance and develop drug resistance but has also resulted in great molecular diversity so that very few sequences are conserved between diverse members of the family. However, all lentiviral and oncoviral gag nucleocapsid (NC) proteins contain one or two copies of a highly conserved amino acid sequence: C X₂ C X₄ H X₄ C. The conserved cysteine and histidine residues coordinate zinc ions and form retroviral zinc fingers. Strict conservation of these viral zinc fingers suggests that they are absolutely required by the virus and therefore may be mutation intolerant. Mutational analysis has shown that viral zinc fingers are required for viral RNA packaging and also play an additional role in viral infectivity. The HIV-1 (NC) protein contains two retroviral zinc fingers and strict conservation and requirements for these structures suggests that they are a good target for retroviral drug therapy to combat the AIDS pandemic. Highly pure NC protein is a fundamental requirement for research and development of drugs that attack retroviral zinc fingers. To provide sufficient protein, the NC protein gene sequence was cloned into a bacterial expression vector (pMal-cᵀᴹ) and expressed as a maltose-binding protein-NC fusion protein. The vector contains a factor Xa protease recognition site so that NC can be released from the fusion protein with no vector derived sequences. RP-HPLC purification of the final product yields NC protein that is >97% pure. Recombinant NC protein was characterized by amino acid analysis, amino acid sequencing, mass spectrometry and nuclear magnetic resonance and recombinant protein is identical to native protein. Purified NC protein is a key component of the AIDS drug screening program to identify compounds that serve as anti-viral "zinc finger" drugs.