Retroviral Integrase: A Novel Target in Antiviral Drug Development and Basic In Vitro Assays with the Purified Enzyme

Abstract

Two critical events are the signature of the life cycle of retroviruses (1). The first is reverse transcription, whereby the single-stranded RNA genome of the retrovirus is copied into double-stranded DNA. The second of these events is integration, whereby this viral DNA is inserted into a chromosome of the host cell, establishing what is known as the proviral state. The proviral state is required for efficient replication of retroviruses. This crucial second event is catalyzed by the integrase enzyme. Retroviruses encode the integrase at the 3' end of the pol gene. Integrase is generated by the retroviral protease as a proteolytic cleavage product of the gag-pol fusion protein precursor, and is contained in the virus particle. During viral infection, integrase catalyzes the excision of the last two nucleotides from each 3' end of the linear viral DNA, leaving the terminal dinucleotide CA-3P-OH at these recessed 3' ends. This activity is referred to as the 3'-processing or dinucleotide cleavage. After transport to the nucleus as a nucleoprotein complex ("preintegration complex"), integrase catalyzes a DNA strand transfer reaction (3'-end joining) involving the nucleophilic attack of these ends on a host chromosome. Completion of the integration process requires removal of the two unpaired nucleotides at the 5' ends of the viral DNA and gap repair reactions that are thought to be accomplished by cellular enzymes. For recent reviews, see Andrake and Skalka (2) and Rice et al. (3).