Integration of a DNA copy of the viral genome into the DNA of the host cell is an essential step in the replication cycle of HIV-1 and other retroviruses 1,2
. After infection, the virion core is uncoated to expose a nucleoprotein complex, termed the reverse transcription complex (RTC)3
that contains two copies of the viral RNA, reverse transcriptase, and integrase along with other viral and cellular proteins. Reverse transcription occurs within the RTC to generate the linear blunt ended viral DNA that is the substrate for integration. The viral DNA remains associated with viral and cellular proteins in a complex called the preintegration complex (PIC) 4
. The PIC is then transported to the nucleus where the viral DNA is integrated into the host genome by the viral integrase protein. The PIC is a poorly defined entity that contains a number of proteins in addition to integrase that may play roles in nuclear import and/or auxiliary roles in integration. PICs isolated from infected cells efficiently integrate their viral DNA in vitro5,6,7
and this system has provided a powerful tool for dissecting the integration mechanism. Analysis of the structure of the integration intermediate8,9
showed that integration proceeds in a two- step process. First, two nucleotides are cleaved from each end of the initially blunt-ended viral DNA in a reaction termed 3′ end processing. Then, in the strand transfer step, the 3′ hydroxyls of the terminal adenosine at each end of the viral DNA exposed by the 3′ processing reaction attack a pair of phosphodiester bonds in the target DNA. In the resulting integration intermediate, the 3′ ends of the viral DNA are covalently joined to the 5′ ends of the target DNA at the site of integration. The 5′ ends of the viral DNA and the 3′ ends of the target DNA are not joined in the integration intermediate which must be subsequently repaired by cellular enzymes to complete the integration process. A striking feature of the association of integrase with viral DNA in the PIC is its stability against high-ionic strength which results in dissociation of many other protein DNA complexes; PICs remain integration competent after challenge with ionic strength of greater than 0.5M NaCl10,11,12
implying that integrase remains tightly associated with the viral DNA.
HIV-1 integrase catalyzes the key DNA cutting and joining steps of integration in vitro
with simple DNA substrates that mimic the two ends of the viral DNA and either Mg2+
as a cofactor13,14,15
. Under most in vitro
reaction conditions the DNA strand transfer reaction products differ from those generated in vivo in that only a single viral DNA end is inserted into a single strand of target DNA, a reaction referred to as half-site integration. In vivo
, both ends of viral DNA insert at the site of integration, with a stagger of 5 base pairs separating the sites of integration on the two target DNA strands in the case of HIV-1. The reason behind the sloppiness of the in vitro
reaction that results in this uncoupling of insertion of pairs of viral DNA ends is unknown. Half-site integration occurs efficiently under a broad range of reaction conditions in vitro, but under such conditions highly stable complexes that mimic the in-vivo
association of integrase with viral DNA in the PIC have not been detected.
Concerted integration, also called full-site integration, of pairs of viral DNA ends can be catalyzed by HIV-1 integrase in vitro
but the reaction conditions are far more restrictive than for half-site integration16,17,18,19
. The viral DNA substrates must be much longer than the 20 base pairs which are sufficient for near maximal efficiency of half-site integration. Concerted integration also requires the presence of high concentrations of the crowding agent polyethylene glycol (PEG) in the reaction mixture and is exquisitely sensitive to both the concentration and stoichiometry of integrase and DNA substrates. Under reaction conditions that promote concerted integration, stable complexes of integrase with viral DNA are formed that mimic the association of integrase with viral DNA in the PIC20,21
. These stable synaptic complexes (SSCs) are intermediates on the integration reaction pathway and are rapidly converted into strand transfer complexes (STCs) in the presence of target DNA. In the STC, a pair of viral DNA ends is integrated into the target DNA with integrase remaining tightly associated with the integration product at the site of insertion. Although SSCs are intermediate products in the integration reaction they can be trapped by preventing DNA strand transfer20
. This can be accomplished by using a selective inhibitor of DNA strand transfer or carrying out the reaction with pre-processed viral DNA ends terminating with dideoxy adenosine and therefore lacking the 3′-OH that is the nucleophile for attacking the phophodiester bond in the target DNA. A third way to trap the SSC is to use an integrase enzyme with an active site mutation in conjunction with pre-processed viral DNA ends.
We have previously analyzed HIV-1 SSCs by gel electrophoresis in combination with crosslinking and footprinting studies and demonstrated that the viral DNA ends are stably synapsed by a tetramer of integrase that protects approximately 20 base pairs of terminal viral DNA sequence22
. Here, we have used atomic force microscopy (AFM) to directly visualize nucleoprotein complexes formed by HIV-1 integrase and DNA substrate under conditions that favor concerted DNA integration. The major species are SSCs in which, a pair of viral DNA ends are bridged by integrase. However, we also observe other species that were not detected in previous analysis by gel electrophoresis, either because they were not stable during electrophoresis or did not enter the gel. A significant fraction of the viral DNA molecules, especially at early time points of assembly, consist of a tetramer of integrase bound to a single viral DNA end. Kinetic studies suggest these are an intermediate on the pathway to the SSC. Strikingly, viral DNA ends bound to integrase dimers were essentially undetectable. The SSCs self -associate through the integrase tetramers suggesting that the integrase tetramer within the SSC is different from free tetramers of integrase in solution that do not self-associate under the same conditions. Finally, the target DNA associated with the SSCs is relaxed even though it was initially supercoiled prior to the reaction. Thus, despite being tightly associated with an integrase tetramer, the 3′ ends of the target DNA strands at the site of integration must be free to rotate before integrase is dissociated. This has implications for the repair of the viral DNA within the STC that must occur to complete the integration process.