Identifying and Characterizing a Functional HIV-1 Reverse Transcriptase-binding Site on Integrase

Abstract

A key step in the replication cycle of human immunodeficiency virus, type 1 (HIV-1) 2 involves the synthesis of doublestranded DNA copies of the diploid, single-stranded RNA genome by viral reverse transcriptase (RT) within the cytoplasm of infected cells. This newly synthesized viral DNA becomes an integral component of a large nucleoprotein complex termed the preintegration complex (PIC). PIC-associated proteins include HIV-1 matrix, Vpr, RT, and integrase (IN) (1-4), as well as cellular components such as lens epitheliumderived growth factor p75 (5), barrier-to-autointegration factor (6), and the high mobility group I(Y) protein (7). The PIC traverses the nuclear envelope, and within the nucleus the viral DNA is subsequently inserted into the chromosomes of the infected host, an event specifically catalyzed by IN.HIV-1 IN and RT (a heterodimeric enzyme of 66-and 51-kDa subunits) are initially synthesized as part of the Gag-Pol polyprotein, which is subsequently processed by HIV-1 protease during viral maturation to produce active IN and RT. IN (288 amino acids; 32 kDa) comprises three domains: an N-terminal zinc-binding domain (residues 1-50), a catalytic core domain (residues 51-212), and a C-terminal domain (CTD; residues 213-288) that binds DNA nonspecifically (reviewed in Ref. 8). IN has multiple effects throughout the viral life cycle, as perturbations within the IN coding sequence of the pol gene can impact not only integration but also PIC nuclear import, proper virion maturation, and reverse transcription (9 -20).Purified HIV-1 IN and RT have been found to physically interact in co-immunoprecipitation, glutathione S-transferasebased pulldown, and surface plasmon resonance (SPR) experiments (15,18,21,22), and assays with IN deletion constructs showed the IN CTD is both necessary and sufficient for this association (18,21). The significance of this IN-RT interaction in the context of viral replication has not yet been firmly established. In vitro studies with purified IN and RT, however, have shown that HIV-1 IN can stimulate both the initiation and elongation modes of RT-catalyzed reverse transcription by enhancing RT processivity (23).To aid our ongoing studies of functional interactions between IN and RT during HIV-1 replication, we have characterized in detail the RT-binding surface on the IN CTD using nuclear magnetic resonance (NMR) spectroscopy, identifying particular IN CTD amino acids that form contacts with RT through chemical shift perturbation experiments. In addition, we have assessed kinetic parameters and binding constants for both IN CTD-RT and IN-RT interactions using SPR. This experimental approach allows us to define and quantify the IN-RT interaction to a level that is not possible through coimmunoprecipitation or other fusion protein pulldown techniques.With the intention of perturbing IN-RT interactions, we introduced an alanine substitution for lysine at position 258 (K258A) within the IN CTD and subsequently used SPR to characterize the effect of this substitution upon IN⅐RT comp...

Identification of Residues in the C-terminal Domain of HIV-1 Integrase That Mediate Binding to the Transportin-SR2 Protein

“…Within these nucleoprotein complexes, IN is known to interact with the reverse transcriptase. The interface of this interaction has been mapped to three crucial amino acids (K258A, W243E, and V250E) (55), but IN mutations outside of this interface (e.g. K186Q, R228A, R262A/R263A, R262A/K264A, K266A, and R269A) were shown to affect reverse transcription as well (51,52,56).…”

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

“…HIV-1 RT has been identified to physically interact with viral integrase by using GST pulldown assays, coimmunoprecipitation assays, dot blot assays, NMR spectroscopy analyses, and surface plasmon resonance analyses (101)(102)(103)(104)(105)(106)(107)(108). The binding of integrase to RT does not require multimeric integrase or an integrase with complete enzymatic activity (108).…”

“…Regarding the interaction domains, the C-terminal domain of integrase may interact with RT (102,104,106,107). Mutagenesis analyses also suggest that integrase mutations at the catalytic core domain (e.g., C130S) and the C-terminal domain (e.g., W243E, V250E, and K258A) could severely diminish the RT-integrase interaction, thereby impairing reverse transcription (102,106).…”

Fueling HIV-1 integrase drug design with structural insights

“…Concerning the last case, it comes as no surprise that IN was found to physically interact with RT and that this interaction seems to be essential for virus replication [35]. Wilkinson et al employed NMR and SPR analyses to map the IN side of the interaction to various residues around V250 in the IN CTD [36]. Although the druggability of this site remains to be investigated, a small molecule inhibitor of IN binding right next to it has been identified: pyridoxal-5 0 -phosphate [37].…”

“…Two viral PPIs have also received attention. First, RT has been shown to functionally interact with IN and the binding site on IN has been characterized [36]. However, druggability of this interface has not been established yet.…”