Different Pathways Conferring Integrase Strand-Transfer Inhibitors Resistance

Abstract

Integrase Strand Transfer Inhibitors (INSTIs) are currently used as the most effective therapy in the treatment of human immunodeficiency virus (HIV) infections. Raltegravir (RAL) and Elvitegravir (EVG), the first generation of INSTIs used successfully in clinical treatment, are susceptible to the emergence of viral resistance and have a high rate of cross-resistance. To counteract these resistant mutants, second-generation INSTI drugs have been developed: Dolutegravir (DTG), Cabotegravir (CAB), and Bictegravir (BIC). However, HIV is also able to develop resistance mechanisms against the second-generation of INSTIs. This review describes the mode of action of INSTIs and then summarizes and evaluates some typical resistance mutations, such as substitution and insertion mutations. The role of unintegrated viral DNA is also discussed as a new pathway involved in conferring resistance to INSTIs. This allows us to have a more detailed understanding of HIV resistance to these inhibitors, which may contribute to the development of new INSTIs in the future.

Keywords: HIV-1; integrase; resistance; strand-transfer inhibitors; unintegrated viral DNA.

Figure 3

Mode of action of dolutegravir targeting HIV-1 integrase. This figure shows the 3D structure of the HIV-1 IN catalytic site complexed with DTG. The three coplanar oxygen atoms (in red) of DTG chelate Mg²⁺ cations (in orange) in the intasome active site constituted by the D116, D64 and E152 residues. The length and flexibility of the linker bridging the tricyclic metal-chelating core and the difluorophenyl ring are responsible for the high potency of inhibition of DTG. This 3-D structure was prepared with The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC using the PDB (protein data bank) 6VDK [53]. Red: oxygen atoms, blue: nitrogen atoms.

Figure 1

The different steps of HIV DNA integration into the host genome. (Step 1) 3’ -Processing: The integrase mediates the endonucleolytic cleavage of the two nucleotides GT to expose sticky CA-3’-OH at both 3’-ends of viral cDNA, which forms the pre-integration complex (PIC) with viral and host cofactors. (Step 2) Strand transfer: by transesterification reaction, the hydroxyl groups at the 3’-ends of the processed viral DNA are used to attack opposite strand phosphodiester bonds separated by 5 bases in the 5′ direction, which allows the integration of viral DNA (blue) into the host DNA (red). (Step 3) DNA repair mechanisms allow completion of the process yielding a duplication of host DNA flanking the provirus (light red).

Figure 2

Chemical structures of the clinically relevant INSTIs. The chelating motifs which interact with Mg²⁺ cofactors in the integrase active site are highlighted in the red square. The halobenzyl moieties, which are connected to the centralized pharmacophore by a linker group, are in the blue circle. These chemical structures have been drawn using ChemDraw software.

Figure 4

Sequences of wild-type and insert-containing human immunodeficiency virus type 2 (HIV-2) integrase (from codon 226 to 240). Alignment of amino acids sequence of the reference strain and sequences of integrase found in patients after initiating the RAL-based regimen. Insertions described by Le Hingrat and colleagues lead to a high level of RAL and EVG resistance and a moderate resistance to DTG and CAB [69].

Figure 5

Different pathways of INSTI resistance. Two main pathways involving the integration step or unintegrated viral DNA can be responsible for INSTI resistance. Substitution mutations (replacement of one amino-acid by another one) or insertional mutations (insertion of one or several amino-acids) in the integrase protein can lead to INSTI resistance. In these pathways, integration of the viral DNA can occur because the mutated integrase is resistant to INSTIs but still active to mediate integration. Integrated DNA then allows the production of new viruses bearing these resistance mutations. Blocking of integration by INSTI treatment leads to accumulation of unintegrated DNA, in particular, 2-LTR circles. In case of interruption of treatment, integration from 2-LTR circles can occur due to the specific cleavage of the LTR-LTR junction before integration after release of the INSTI compound from the IN/DNA complex. Thus, 2-LTRc accumulated in cells treated with an INSTI may constitute a reserve supply of HIV-1 genomes that could be integrated de novo and revive viral replication. Unintegrated viral DNA could also contribute to INSTI resistance by a basal production of viruses from 1-LTR circles. Furthermore, mutations in the 3′-PPT region modify the reverse transcription step. In this case, reverse transcription does not result in linear DNA but in a 1-LTR circle. One hypothesis is that the 3′-PPT mutation leads to a higher transcription efficiency leading a higher viral production compared to the production from a 1-LTR circle that does not highlight the 3′-PPT mutation. Viruses with 3′-PPT mutations are resistant to INSTIs since their replication is independent of the integration pathway. Yellow square: INSTI. Black arrow: basal transcription from 1-LTR circles coming from a WT infection. Green arrow: hypothesis of higher transcription due to the 3′-PPT mutation.