Correct Integration Mediated by Integrase–LexA Fusion Proteins Incorporated into HIV- 1  Michelle L. Holmes-Son, Samson A. Chow  Molecular Therapy  Volume 5, Issue 4, Pages 360-370 (April 2002) DOI: 10.1006/mthe.2002.0559 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 DNA constructs used to produce viruses. (A) The packaging constructs that contained the hygromycin resistance gene in place of envelope (pHXB-Hygro, pHXB-IND64V-Hygro, and pHXB-IN1–234-Hygro). The constructs are not drawn to scale. The vpr gene of HXB2 is defective because of an extra T nucleotide at position 5771. The integrase gene is magnified to show the mutations introduced into this coding region. The viral constructs are identical except that pHXB-IND64V-Hygro (b) contains an inactivating mutation in the integrase catalytic motif and pHXB-IN1–234-Hygro (c) contains three stop codons after residue 234 (denoted by asterisks). D, Aspartic acid; E, glutamic acid; V, valine. (B) The pLR2P expression constructs for inclusion of proteins in trans. Vpr (R; open boxes) is expressed at the N terminus of each fusion protein for incorporation of wild-type integrase (a), integrase–LexA (b), or integrase1–234-LexA (c) into viruses. An HIV-1 protease cleavage site (PC; closed boxes) is located between Vpr and integrase–LexA for removing Vpr from the fusion protein [13,25]. The lightly shaded boxes represent integrase (wild-type protein, 288 residues), and the darkly shaded boxes represent LexA. (C) The amphotropic MLV envelope expression construct was used for pseudotyping the envelope-deleted viruses [46]. Molecular Therapy 2002 5, 360-370DOI: (10.1006/mthe.2002.0559) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 Schematic of the protocol used to sequence viral–cellular DNA junctions of proviruses in infected cells. HeLa-CD4 cells were infected with low titers of the IND64V-mutated virus containing the integrase–LexA protein. After culturing in the presence of medium containing hygromycin for 2 weeks, resistant colonies were isolated individually and expanded. The genomic DNA was extracted and digested separately with restriction enzymes that cut specifically near either the right LTR (filled arrowheads) or left LTR (open arrowheads) of the viral DNA. Intramolecular ligation of the digested DNA was carried out, followed by inverse PCR with divergent primers (U3 end, primers 1 and 3; U5 end, primers 5 and 7) against the known viral sequence. The products of the inverse PCR were used as templates for the nested PCR using primers 2 and 4 for the U3 end-containing fragment, and primers 6 and 8 for the U5 end-containing fragment. Reactions yielding a single amplified product were subjected to DNA sequencing. Molecular Therapy 2002 5, 360-370DOI: (10.1006/mthe.2002.0559) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 Bona fide integration mediated by virion-incorporated integrase–LexA fusion proteins. Genomic DNA was prepared from individually isolated hygromycin-resistant colonies that were previously infected with the HXB-IND64V virus containing the integrase–LexA fusion protein. The integrated proviral DNA is denoted as heavy lines, and the flanking cellular DNA as thin lines. (A) Sequences of the viral–cellular DNA junctions and integrase genes of proviral DNA. The proviral sequence is in uppercase and the cellular DNA is in lowercase. The highly conserved CA and TG dinucleotides are denoted in bold. The 5-nt direct repeat that flanks the provirus is underlined and in italic. Asterisks highlight proviruses where sequence from both ends was obtained. Uppercase letters and letters in parentheses listed under IN 64 are the sequence obtained at integrase codon 64 and the amino acids for those codons, respectively; D, aspartic acid; I, isoleucine; V, valine. W4 is the provirus from infection with a virus containing wild-type integrase. ND, Not determined. (B) Cellular sequences of uninfected cells at sites corresponding to integrated proviruses. Sequence information from (A) was used to carry out an NCBI GenBank database search (clones 1, 44, and 45) or PCR of genomic DNA isolated from uninfected cells (clone 30). The 5-nt cellular sequence corresponding to the short direct repeats flanking the provirus in (A) is in italic and underlined. (C) Chromosomal location of integration sites. Sites of integration were determined using the NCBI GenBank database BLAST algorithm. NM, No match found. Molecular Therapy 2002 5, 360-370DOI: (10.1006/mthe.2002.0559) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 In vitro complementation of core-mutated integrase with N- or C-terminal truncated integrases fused to LexA. In (A) and (C), the C-terminal truncated integrase–LexA (ΔCLA) was mixed with either catalytic core mutant integrase D116N (lanes 8 and 9) or C-terminal truncated integrase (lanes 10 and 11). In (B) and (D), the N-terminal truncated integrase–LexA (ΔNLA) was mixed with the catalytic core mutant integrase. The mixture was tested for its ability to restore the 3'-end processing (A and B) and 3'-end joining (strand transfer) activities (C) and (D). The total concentration of enzymes was 105 or 210 nM. In mixing reactions, the two proteins were added at a ratio of 1:1. To the left of (A) and (C) is a schematic representing the substrates and products for the two reactions with the numbers in parentheses representing the expected lengths. Bands migrating faster than the substrate in all panels (for example, see lanes 6 and 7) are the result of nonspecific nuclease activity. Filled arrowheads denote the substrate and open arrowheads denote the 3'-end processing product. Asterisks denote the locations of the 32P-label at the 5'-end of the oligonucleotide; –, no enzyme; s.t.p., strand transfer products; wt, wild-type integrase. Molecular Therapy 2002 5, 360-370DOI: (10.1006/mthe.2002.0559) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 Incorporation of Vpr-fusion proteins into HIV-1 particles encoding a C-terminal truncated integrase. Western blot analysis was carried out with antibodies directed against integrase residues 23–34 (A), LexA (B), or immune serum against HIV-1 (C). Virions were prepared for blotting by ultracentrifugation at 120,000g for 2 hours and resuspension in lysis buffer. We separated 12 ng p24 equivalent of each virus on 12% SDS-PAGE gels and transferred these to nitrocellulose membranes for probing. The numbers in lane M are the sizes in kDa of the molecular weight standards. The numbers in parentheses are the expected molecular weight in kDa for the indicated proteins. In (C), CA and MA are HIV-1 capsid and matrix, respectively. WT, Wild-type HXB2; AC, HXB2 with an integrase 1–234 gene; R-IN, Vpr-integrase; R-IN1–234/LA, Vpr-integrase 1–234/LexA. Molecular Therapy 2002 5, 360-370DOI: (10.1006/mthe.2002.0559) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 6 Viruses with a C-terminal truncated integrase gene cannot be complemented by integrase 1–234/LexA proteins in trans. HeLa-CD4 cells infected with the indicated viruses were grown under the selection of hygromycin B. Colony formation due to stable expression of the hygromycin resistance gene is a result of integration. Viruses containing a wild-type integrase gene (WT, A) and integrase 1–234 gene (IN1-234, B) served as a positive and a negative control, respectively, for the assay. A positive control for complementation with HXB IN1–234 viruses, where the Vpr–integrase (R-IN) fusion protein was provided in trans, is in (C). Cells infected with HXB IN1–234 containing the Vprintegrase1–234-LexA fusion protein (R-IN1–234/LA) are in (D). Molecular Therapy 2002 5, 360-370DOI: (10.1006/mthe.2002.0559) Copyright © 2002 American Society for Gene Therapy Terms and Conditions