Abstract
In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3' of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3'-OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus (ASV) and human immunodeficiency virus type 1 (HIV-1) IN proteins by use of fluorescence resonance energy transfer (FRET). The results were indicative of an IN-induced intramolecular conformational change in the viral DNA ends (cis FRET). Fraying activity is tightly coupled to the DNA binding capabilities of these enzymes, as follows: an inhibitor effective against both IN proteins was shown to block ASV IN DNA binding and end fraying, with similar dose responses; ASV IN substitutions that reduced DNA binding also reduced end fraying activity; and HIV-1 IN DNA binding and end fraying were both undetectable in the absence of a metal cofactor. Consistent with our previous results, end fraying is sequence-independent, suggesting that the DNA terminus per se is a major structural determinant for recognition. We conclude that frayed ends represent a functional intermediate in which DNA termini can be sampled for suitability for endonucleolytic processing.
Highlights
Grants AI40385, CA71515, and CA006927. 1 To whom correspondence should be addressed: 333 Cottman Ave., Philadelphia, PA 19111
Sequence identity of the generally imperfect terminal inverted repeats that are embedded in the U3 and U5 regions of the long terminal repeat (LTR) at the ends of each viral DNA can range from 16/18 for murine leukemia virus, to 12/18 for avian sarcoma virus (ASV), 11/18 for human immunodeficiency virus-1 (HIV-1), and only 5/18 for HTLV-1
In this report we describe an approach based on fluorescence resonance energy transfer (FRET) for analyses of DNA end fraying by both HIV-1 and ASV IN proteins
Summary
The first clue that properties of DNA termini may contribute to IN recognition or catalysis came from the observation that extension of the viral DNA duplex beyond the terminus reduced the efficiency of IN-mediated processing [11] The fact that such an embedded DNA tip sequence is resistant to this activity demonstrated that the structural context of the terminal sequence CA/GT is important for processing. Analysis of the products formed in the processing reaction in vitro indicate that an alternate nucleophile, the 3Ј-hydroxyl oxygen of the strand to be cleaved, can attack the internal phosphodiester bond to generate a cyclic dinucleotide product [13] Such a reaction would clearly require both fraying and distortion of the viral DNA end. We provide evidence that both proteins are capable of promoting or stabilizing frayed DNA ends and uncover additional details concerning this important activity
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