Changes in the Mechanism of DNA Integration in Vitro Induced by Base Substitutions in the HIV-1 U5 and U3 Terminal Sequences

Abstract

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration with specially designed mini-donor DNA, a supercoiled plasmid acceptor, purified bacterial-derived HIV-1 integrase (IN), and host HMG-I(Y) protein (Hindmarsh, P., Ridky, T., Reeves, R., Andrake, M., Skalka, A. M., and Leis, J. (1999) J. Virol. 73, 2994 -3003). Integration in this system is dependent upon the mini donor DNA having IN recognition sequences at both ends and the reaction products have all of the features associated with integration of viral DNA in vivo. Using this system, we explored the relationship between the HIV-1 U3 and U5 IN recognition sequences by analyzing substrates that contain either two U3 or two U5 terminal sequences. Both substrates caused severe defects to integration but with different effects on the mechanism indicating that the U3 and the U5 sequences are both required for concerted DNA integration. We have also used the reconstituted system to compare the mechanism of integration catalyzed by HIV-1 to that of avian sarcoma virus by analyzing the effect of defined mutations introduced into U3 or U5 ends of the respective wild type DNA substrates. Despite sequence differences between avian sarcoma virus and HIV-1 IN and their recognition sequences, the consequences of analogous base pair substitutions at the same relative positions of the respective IN recognition sequences were very similar. This highlights the common mechanism of integration shared by these two different viruses.Integration of retroviral DNA is an obligatory step in viral replication. Integration is catalyzed by the viral encoded enzyme, integrase (IN), 1 which brings the ends of a linear viral DNA together and inserts them into the host chromosome in a concerted reaction (see Ref. 1 for a review). Cell proteins belonging to the HMG-I(Y) family stimulate the reaction (2, 3). The sites of integration are widely distributed in the target DNA. Short duplications of the cell DNA are introduced at the insertion site, the size of which is dictated by IN. For HIV-1 and ASV the size of the duplications are five and six base pairs, respectively. During this process, two base pairs are lost from the ends of the viral LTRs.The properties of concerted DNA integration have been reconstituted in vitro using purified HIV-1 (3, 4) or ASV (3, 5-11) IN and MgCl 2 . The donor DNAs contain only 20 base pairs for HIV-1 or 15 base pairs for ASV derived from the ends of the LTRs, respectively. These viral DNA end sequences correspond to the nearly perfect inverted repeats that define the relationships between the U3 and U5 RNA ends. The inverted repeat for RSV is 12 of 15, while that for HIV-1 is 12 of 20. A comparison of the RSV and HIV-1 IN recognition sequences indicates that they are unique. The only common feature is the presence of a conserved CA dinucleotide at positions 3 and 4 from the terminus. Short oligodeoxynucleotide duplexes representing the ends of HIV-1 U5 LTR are more efficient substrates for IN processing (12-15) and strand transf...

Recombinant Human Immunodeficiency Virus Type 1 Integrase Exhibits a Capacity for Full-Site Integration In Vitro That Is Comparable to That of Purified Preintegration Complexes from Virus-Infected Cells

“…5, lane 4). The higher activities of U5 att sites over U3 att sites by HIV-1 IN were previously demonstrated for both full-site and half-site integration products (4,5,7,10,23). Digestion of the half-site product (Fig.…”

“…A series of different purification protocols were performed on recombinant HIV-1 IN, including changing the pH (7.6 to 6.8) of the HEPES buffers with and without 10 mM MgSO 4 . As previously reported (46), ϳ2 g wet weight bacterial pellets were used for purification of HIV-1 IN under low protein concentrations.…”

“…Subsequently improvements in purified recombinant HIV-1 (46) and simian immunodeficiency virus (24) IN provided an ϳ2-fold improved efficiency for producing full-site integration products over HIV-1 lysates. Recombinant HIV-1 IN using blunt-ended DNA donor substrates (ϳ300 bp) with added HMGA1 is also capable of performing unimolecular full-site integration reactions where the two att ends from the same DNA donor are inserted into the DNA target (4,29,30). However, the primary products of the above integration reactions had one donor end either inserted into the DNA target (half-site integration) or into other donors.…”

Identification of Amino Acids in HIV-1 and Avian Sarcoma Virus Integrase Subsites Required for Specific Recognition of the Long Terminal Repeat Ends

“…This indicates that there are multiple contact points along the LTRs with various amino acids on the HIV-1 IN surface that provide the specificity for the binding and processing reactions. The 5-11-bp region of the LTRs was previously shown in several studies to be important for 3Ј processing, strand transfer, and concerted DNA integration (13,16,20). As a control, when amino acid substitutions were made in the HIV-1 IN at positions not predicted to be near the LTRs, such as S124 -125, S128 -130, S193, S197, and S200 -201, there was a significant decrease in 3Ј processing without any detectable change in specificity for the LTR substrates.…”

“…The model contained 4 potassium and 4 phosphate ions from the crystal structures. The tetramer model was solvated in 16,195 water molecules. This solvated integrase was subjected to molecular dynamics simulation for 1.0 ns on a 1-GHz Linux PC.…”