Treatment with suboptimal doses of raltegravir leads to aberrant HIV-1 integrations

Abstract

Integration of the DNA copy of the HIV-1 genome into a host chromosome is required for viral replication and is thus an important target for antiviral therapy. The HIV-encoded enzyme integrase (IN) catalyzes two essential steps: 3' processing of the viral DNA ends, followed by the strand transfer reaction, which inserts the viral DNA into host DNA. Raltegravir binds to IN and blocks the integration of the viral DNA. Using the Rous sarcoma virus-derived vector RCAS, we previously showed that mutations that cause one viral DNA end to be defective for IN-mediated integration led to abnormal integrations in which the provirus had one normal and one aberrant end, accompanied by rearrangements in the host genome. On the basis of these results, we expected that suboptimal concentrations of IN inhibitors, which could block one of the ends of viral integration, would lead to similar aberrant integrations. In contrast to the proviruses from untreated cells, which were all normal, ∼10-15% of the proviruses isolated after treatment with a suboptimal dose of raltegravir were aberrant. The aberrant integrations were similar to those seen in the RCAS experiments. Most of the aberrant proviruses had one normal end and one aberrant end and were accompanied by significant rearrangements in the host genome, including duplications, inversions, deletions and, occasionally, acquisition of sequences from other chromosomes. The rearrangements of the host DNA raise concerns that these aberrant integrations might have unintended consequences in HIV-1-infected patients who are not consistent in following a raltegravir-containing treatment regimen.

Keywords: chromosomal rearrangements; integrase inhibitors; strand transfer inhibitor.

Fig. 1.

Viral DNAs recovered from cells infected with HIV-1. Viral LTRs are shown as shaded boxes: the dark gray box represents the U3 region, R is shown by a black box, and U5 is indicated by the light gray box. (A) The structure of normal proviruses with a 5-bp duplication of the host DNA sequences (small bold arrow) flanking the provirus. Unintegrated forms of circular viral DNA include (B) 1-LTR circles formed by the homologous recombination of the LTRs, (C) 2-LTR circles formed by end-joining of the two LTRs, and (D) autointegrants formed by integration of the LTR into different regions of the viral DNA itself. *Although we did not recover any 2-LTR circles in the experiment in which viral DNAs were isolated from untreated cells, these are one of the types of unintegrated viral DNA circles typically found in cells infected with HIV-1, and DNAs of this type were isolated from RAL-treated cells.

Fig. 2.

Structure of aberrant proviruses isolated from cells treated with suboptimal concentrations of RAL. (A–L) Proviruses recovered from cells treated with RAL at IC₁₄, IC₃₀, IC₅₀, IC₆₀, and IC₇₅. Viral LTRs are shown as shaded boxes: the dark gray box represents the U3 region, R is shown by the black box, and U5 is indicated by the light gray box. Insertion of additional bases at the viral DNA ends is indicated by a solid red box. Deletion from the viral DNA is indicated by a black jagged end; deletion of the entire LTR is indicated by the jagged end stretching through the LTR. Large duplications of flanking host DNA are indicated by long bold arrows; the direction of the arrowheads indicates the orientation of the flanking host sequences. Acquisition of sequences from a different host chromosome is indicated by green DNA strands. Deletion of sequences from the host chromosome is indicated by a hashed line. In one case (K), there was a BclI restriction enzyme site in the duplicated host sequence. Digestion with BclI and subsequent self-ligation of the digested fragment, during the recovery of the integrated provirus, led to truncation of the flanking host sequences. The numbers of nucelotides that were deleted or inserted are indicated.

Fig. 3.

Structures of the aberrant proviruses isolated from cells treated with a high concentration of RAL. (A-G) Proviruses recovered from cells treated with 2 µM RAL. Viral LTRs are shown as shaded boxes. The aberrations in viral and host DNA sequences are indicated according to the scheme described in Fig. 2. In one case (E) DNA was acquired from two different chromosomes; this is indicated by green and pink DNA strands. The numbers of nucleotides that were deleted or inserted are indicated.