Suboptimal Doses of Raltegravir Cause Aberrant HIV Integrations

Raltegravir causes aberrant HIV-1 integrations. Viral DNA enters the nucleus through the nuclear pore (top). Integration of the DNA copy of the HIV-1 genome is mediated by integrase (green balls). Raltegravir (red “Xs”; and green stick diagram in the inset), bind to the active site of integrase (inset) inhibiting integration. Suboptimal concentrations of raltegravir block the integration of one end of the viral DNA, leaving the other end free (center). Host DNA repair machinery (light grey balls) binds to,

Raltegravir causes aberrant HIV-1 integrations. Viral DNA enters the nucleus through the nuclear pore (top). Integration of the DNA copy of the HIV-1 genome is mediated by integrase (green balls). Raltegravir (red “Xs”; and green stick diagram in the inset), bind to the active site of integrase (inset) inhibiting integration. Suboptimal concentrations of Raltegravir block the integration of one end of the viral DNA, leaving the other end free (center). Host DNA repair machinery (light grey balls) binds to, and mediates the insertion of this blocked end to the left or the right of the original insertion, causing duplications (lower left) or deletions (lower right) in the host genome.

When a cell is infected with HIV, a DNA copy of the HIV genome is inserted into that cell’s chromosomal DNA. This insertion reaction is carried out by the viral enzyme integrase (IN) and involves two distinct steps: removal of two nucleotides from each 3’ end of the viral DNA, followed by the strand transfer reaction, in which the viral DNA ends are inserted into the host chromosomal DNA. Integration is essential for viral replication, making it an important target for antiviral therapy. Raltegravir, and the other approved integrase inhibitor, Elvitegravir, are called integrase strand transfer inhibitors (INSTIs), because they bind to the active site of IN and block the strand transfer reaction.      

Using the Rous Sarcoma Virus derived vector RCAS, Stephen Hughes, Ph.D., and his colleagues in CCR’s HIV Drug Resistance Program previously reported that mutating one end of the viral DNA so that it is no longer a good substrate for IN leads to abnormal integrations. These abnormalities include integrated viral DNA having one normal and one aberrant end and are accompanied by rearrangements in the host genome. It is likely that IN inserts the “good” viral DNA end and that host enzymes mediate the insertion of the aberrant end.  If the concentration of an INSTI is below optimal levels, in some cases, only one of two IN-mediated strand transfer reactions would be blocked. Because this could have consequences similar to having one end of the viral DNA mutated, Hughes and colleagues set out to test whether a suboptimal dose of Raltegravir would allow one end of the viral DNA to be inserted normally by IN, causing the other end (whose insertion would be blocked by Raltegravir) to be inserted by host enzymes, leading to aberrant integrations.

The researchers infected cells with the virus, either in the absence of an INSTI, or in the presence of a range of suboptimal concentrations of Raltegravir. Based on an IC50 (the drug concentration causing 50 percent inhibition of the desired activity) of 8.5 nanomolar, they determined the effects of a range of suboptimal concentrations of Raltegravir (2.4-13.5 nanomolar). All of the proviruses (integrated viral DNAs) that were recovered from cells infected in the absence of Raltegravir were normal. On the other hand, approximately 10-15 percent of the proviruses recovered 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. There were significant rearrangements in the host genome, including duplications, inversions, deletions, and sometimes acquisition of sequences from other chromosomes. The data showed that the aberrant integrations were caused by the suboptimal doses of Raltegravir, and not simply revealed by the action of the drug. All of the INSTIs prevent viral DNA integration by a mechanism similar to Raltegravir; therefore, it is probable that all drugs in this class will have similar effects.

The researchers also tested whether high doses (>IC99) of Raltegravir would totally eliminate integration, or whether there would be some rare residual integration events. In the presence of a high dose of Raltegravir, about 1 percent of the viral DNA sequences were integrated into the host chromosomal DNA, and all of them were aberrant. Unlike the proviruses recovered from cells treated with suboptimal doses of Raltegravir, both viral DNA ends of most of the proviruses recovered from cells treated with a high concentration of Raltegravir were aberrant. These aberrant proviruses were also flanked by rearrangements in the host chromosome. This finding indicates that the viral DNA can be inserted, albeit inefficiently, into the host genome even in the presence of Raltegravir concentrations that are much higher than the IC50.

This study, describing rearrangements of the host chromosomal DNA resulting from suboptimal doses of Raltegravir, raises concerns that these aberrant integrations might have inadvertent consequences, for example the activation of proto-oncogenes or inactivation of tumor suppressor genes in HIV-infected patients who do not consistently follow a Raltegravir-containing treatment regimen.

Summary Posted: 12/2013

Reference

Varadarajan J, McWilliams MJ, and Hughes SH. Treatment with suboptimal doses of raltegravir leads to aberrant HIV-1 integrations. Proc Natl Acad Sci USA. 2013 PubMed Link