These studies have suggested that a dimeric IN could suffice to process one viral DNA end [85,86]. A number of studies have suggested that a tetramer of IN synapses Bamaluzole the two viral DNA ends into the fully functional nucleoprotein complex. for the development of antiretroviral therapies. Practical benefits of these efforts have been manifested by the recent discovery of the strand transfer inhibitors (STI) and a successful launch of Raltegravir into the clinic. Strikingly, STIs selectively bind the preformed IN-viral DNA complex rather than free protein [24], thus exemplifying the significance of detailed characterization of the functional nucleoprotein complexes. == 2. DNA Processing and Joining Reactions Catalyzed by HIV-1 IN == The integration of viral DNA into a host chromosome involves two chemical reactions. In the first step, which is called 3-processing and takes place in the cytoplasm shortly after the viral DNA is made, IN hydrolyzes a GT dinucleotide from each 3 end of the viral DNA. In the second step, IN catalyzes concerted integration of the processed viral DNA ends into chromosomal DNA. Bamaluzole The sites of attack on the two target DNA strands are separated by 5 bp for HIV-1 IN, which leads to DNA strand dissociation in the small double-stranded DNA fragment between the attachment sites. The subsequent repair of the intermediate species by cellular enzymes completes the integration reaction. In infected cells IN functions in the context of a large nucleoprotein complex termed the preintegration complex (PIC), where a number of viral and cellular proteins contribute to retroviral integration [517]. PICs can be extracted from infected cells and used for biochemical assaysin vitro[1825]. However, the amounts of these nucleoprotein complexes are not sufficient to perform atomic structural or even lower resolution biophysical analyses. Therefore, recombinant IN and model DNA substrates Bamaluzole have been employed instead to study protein-nucleic acid interactions. Typically, purified recombinant protein and short DNA substrates (21-mer dsDNA mimicking the U5 end of viral DNA) are utilized to monitor 3-processing and Bamaluzole strand transfer activities Rabbit Polyclonal to SEPT1 (Figure 1A). These reactions, however, do not yield concerted integration products and instead, result in integration of one viral DNA end into the target DNA. More recently, assays using a longer donor DNA substrate of several hundred basepairs and a second circular target DNA have been devised, which allow effective concerted integration of two viral DNA ends [2631] (seeFigure 1B). This improved experimental design has furthermore allowed isolation and characterization of critical nucleoprotein intermediates that are reminiscent of IN-viral DNA interactions in the PIC in the infected cells [27,28]. == Figure 1. == Schematic presentation of HIV-1 IN activity assaysin vitro.(A)These reactions are typically performed with purified recombinant IN and 21-mer double-stranded DNA mimicking the U5 sequence. The enzyme first removes the GT dinucleotide from the 3-terminal, and then covalently joins the recessed 3-end to the target DNA. In these reactions the U5 sequence serves as both viral and target DNA. The strand transfer products result from integration of only one viral DNA end into the target DNA, while pair-wise Bamaluzole integration products are not observed.(B)The concerted integration assays and critical nucleoprotein intermediates. Selective interaction of IN with viral DNA ends results in a highly stable nucleoprotein complex termed the stable synaptic complex (SSC). Next, IN in the context of SSC engages with the target DNA to form the strand transfer complex (STC), which carries out the concerted integration reaction. These nucleoprotein complexes are readily separated by native agarose gel electrophoresis. Deproteinization of the STC leads to the formation of the concerted integration product. The asterisks in A and B indicate the P32labeled 5-end of viral DNA. Along with the biologically relevant 3-processing and strand transfer activities, purified IN exhibits additional activitiesin vitro. The enzyme can reverse the strand transfer reaction by site selectively cleaving the integrated DNA. This reaction is called disintegration [32]. A recent report has indicated that the recombinant protein can also catalyze internal cleavage at a palindromic sequence mimicking LTR-LTR junction [33,34]. However, there is no evidence as yet that these additional catalytic activities observedin vitrocan also occur in infected cells. == 3. Structure of HIV-1 Integrase == IN consists of three distinct structural and functional domains: the N-terminal domain (NTD), the catalytic core domain (CCD) and the C-terminal domain (CTD) (Figure 2A). Each domain directly or indirectly contributes to IN-DNA interactions. The NTD, which encompasses residues 146, is linked to the CCD (residues 56186) through a linker segment of aa 4755. Another flexible loop comprised of residues 187194 connects the CCD with the CTD (residues 195288). Results of structural biology studies.