Brief Histories of Retroviral Integration Research and Associated International Conferences

Abstract

The field of retroviral integration research has a long history that started with the provirus hypothesis and subsequent discoveries of the retroviral reverse transcriptase and integrase enzymes. Because both enzymes are essential for retroviral replication, they became valued targets in the effort to discover effective compounds to inhibit HIV-1 replication. In 2007, the first integrase strand transfer inhibitor was licensed for clinical use, and subsequently approved second-generation integrase inhibitors are now commonly co-formulated with reverse transcriptase inhibitors to treat people living with HIV. International meetings specifically focused on integrase and retroviral integration research first convened in 1995, and this paper is part of the Viruses Special Issue on the 7th International Conference on Retroviral Integration, which was held in Boulder Colorado in the summer of 2023. Herein, we overview key historical developments in the field, especially as they pertain to the development of the strand transfer inhibitor drug class. Starting from the mid-1990s, research advancements are presented through the lens of the international conferences. Our overview highlights the impact that regularly scheduled, subject-specific international meetings can have on community-building and, as a result, on field-specific collaborations and scientific advancements.

Keywords: allosteric integrase inhibitor; bictegravir; cabotegravir; dolutegravir; elvitegravir; integrase; integrase strand transfer inhibitor; provirus; raltegravir; retroviral integration.

Figure 1

DNA cutting and joining steps of retroviral integration. Depicted is linear HIV-1 DNA (thin black lines) and chromosomal target DNA (thick green lines). Scissile phosphodiester bonds are shown as red vertical arrows (for 3′ processing) and small red circles (for DNA strand transfer). For simplicity, the IN enzyme was omitted from the drawing. See main text for additional details.

Figure 2

Design of a representative concerted integration activity assay. Shown to the left are a viral DNA oligonucleotide synthesized with a recessed CA_OH-3′ end and supercoiled target DNA. Supercoiled DNA in particular helps to distinguish products of concerted integration from single viral DNA end integration products, which are referred to as half-site integration. Deproteinized reaction products analyzed by agarose gel electrophoresis, schematized to the right, reveal the positions of unreacted viral and target DNA substrates in the absence of added IN (lane 1). Plasmid DNA isolation techniques invariably nick a fraction of supercoiled molecules, which migrate more slowly through the gel than does the compacted supercoiled population. INs that preferentially catalyze half-site integration, such as HIV-1, yield a predominance of single LTR-tagged circles, which comigrate under these conditions with nicked plasmid circles (lane 2) [12]. Other INs, such as those derived from Moloney murine leukemia virus (Mo-MLV) [3], avian myeloblastosis virus (AMV) [7], prototype foamy virus (PFV) [13], or mouse mammary tumor virus [14], possess robust concerted integration activities, which, after deproteination, yield reaction products that comigrate with linearized plasmid DNA (lane 3). HIV-1 IN concerted integration activity is enhanced through the use of comparatively long viral DNA substrates [9] or the IN-binding host factor lens epithelium-derived growth factor (LEDGF)/p75 [10].

Figure 3

Domain organization of two representative retroviral IN proteins. Conserved amino acid residues highlighted in the main text are shown in single-letter code. The additional lysine (K) in the CCD, which plays a key role in sequence-specific viral DNA binding, is conserved among retroviral INs and some bacterial transposase proteins but is not seen in retrotransposon INs [66]. Numbers refer to amino acid positions of domain boundaries, as well as to interdomain linker and C-terminal tail lengths. NED, N-terminal extension domain present in a subset of retroviral INs (γ-retroviruses, ε-retroviruses, and spumaretroviruses).

Figure 4

INSTI and intasome structures. (A) Structures of representative INSTIs RAL and BIC highlighting conserved heteroatoms (red arrows) and halo-benzyl sidechains. (B) Cryo-EM structure of the HIV-1 intasome [Protein Databank (PDB) accession code 6PUT] highlighting DDE catalytic triad residues (D64, D116, E152), CA end of the cleaved viral DNA strand, as well as opposing C-paring dG nucleotide. IN secondary structural elements are labelled. Q148, which can confer significant INSTI resistance when altered, is also highlighted. Spheres, calcium atoms; red and blue, oxygen and nitrogen atoms, respectively. The rightward panel affords an ~90° rotated “top view” of the leftward panel. (C) Same as in panel (B), except with BIC (magenta backbone with grey fluorines) bound (PDB code 6PUW), which displaces dA along with its 3′-OH required for IN strand transfer activity from committed positions at the IN active site. Spheres, Mg²⁺ ions. Panels (B,C) based on ref. [90].