HIV type-1 (HIV-1) protease (PR), reverse transcriptase (RT) and integrase (IN) share the same precursor polyprotein and there is much evidence to suggest functional interactions between IN and RT. We aimed to elucidate whether long-term highly active antiretroviral therapy (HAART) targeting PR and RT could influence raltegravir susceptibility and the fitness of IN.
Methods
HIV-1 IN sequences from 45 heavily antiretroviral-experienced patients with longitudinal samples separated by a median of 10 years were obtained to estimate the rate of nucleotide substitution. IN recombinant viruses were generated from five selected patients. Phenotypic susceptibility to raltegravir was tested in vitro. Changes in viral replication capacity were assayed by growth kinetics and competition of intrapatient IN recombinant viruses.
Results
The amino acid substitution rate within IN was 0.06% per year during long-term antiretroviral treatment. Some substitutions had previously been associated with resistance to different IN inhibitors. Despite this, neither the early- nor late-derived IN recombinant viruses showed an increase in phenotypic susceptibility to raltegravir. Moreover, IN recombinant viruses corresponding to IN samples after 10 years of HAART had a replication capacity that was similar to or better than IN recombinant viruses from baseline samples.
Conclusions
HIV-1 IN from longitudinal samples taken from patients treated with IN inhibitor-sparing regimens showed no evidence of genotypic or phenotypic resistance to raltegravir. Additionally, long-term pressure with PR and RT inhibitors did not impair the fitness of HIV-1 IN. These data suggest that current antiretroviral regimens do not diminish the fitness of IN or influence raltegravir efficacy.
References
1.
DonehowerL.A., VarmusH.E.A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration. Proc Natl Acad Sci U S A1984; 81: 6461–6465.
2.
PommierY., JohnsonA.A., MarchandC.Integrase inhibitors to treat HIV/AIDS. Nat Rev Drug Discov2005; 4: 236–248.
3.
Asante-AppiahE., SkalkaA.M.Molecular mechanisms in retrovirus DNA integration. Antiviral Res1997; 36: 139–156.
4.
Van MaeleB., Van EylenL., PluymersW., DebyserZ.Evaluation of the activity of HIV-1 integrase over-expressed in eukaryotic cells. Biochem Biophys Res Commun2005; 327: 261–267.
5.
HombrouckA., De RijckJ., HendrixJ.Virus evolution reveals an exclusive role for LEDGF/p75 in chromosomal tethering of HIV. PLoS Pathog2007; 3: 47.
6.
BukrinskyM.I., SharovaN., McDonaldT.L., PushkarskayaT., TarpleyW.G., StevensonM.Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. Proc Natl Acad Sci U S A1993; 90: 6125–6129.
7.
EspositoD., CraigieR.Sequence specificity of viral end DNA binding by HIV-1 integrase reveals critical regions for protein-DNA interaction. EMBO J1998; 17: 5832–5843.
8.
Nymark-McMahonM.H., Beliakova-BethellN.S., DarlixJ.L., Le GriceS.F., SandmeyerS.B.Ty3 integrase is required for initiation of reverse transcription. J Virol2002; 76: 2804–2816.
9.
PadowM., LaiL., DeivanayagamC.Replication of chimeric human immunodeficiency virus type 1 (HIV-1) containing HIV-2 integrase (IN): naturally selected mutations in IN augment DNA synthesis. J Virol2003; 77: 11050–11059.
10.
HehlE.A., JoshiP., KalpanaG.V., PrasadV.R.Interaction between human immunodeficiency virus type 1 reverse transcriptase and integrase proteins. J Virol2004; 78: 5056–5067.
11.
EsteJ.A., CabreraC., ScholsD.Human immunodeficiency virus glycoprotein gp120 as the primary target for the antiviral action of AR177 (Zintevir). Mol Pharmacol1998; 53: 340–345.
12.
GrinsztejnB., NguyenB.Y., KatlamaC.Safety and efficacy of the HIV-1 integrase inhibitor raltegravir (MK-0518) in treatment-experienced patients with multidrug-resistant virus: a phase II randomised controlled trial. Lancet2007; 369: 1261–1269.
13.
CooperD., GatellJ., RockstrohJ.Results of BENCHMRK-1, a phase III study evaluating the efficacy and safety of MK-0518, a novel HIV-1 integrase inhibitor, in patients with triple-class resistant virus.14th Conference on Retroviruses and Opportunistic Infections. 25–29 February 2007, Los Angeles, CA, USA.
14.
SteigbigelR., KumarP., EronJ.Results of BENCHMRK-2, a phase III study evaluating the efficacy and safety of MK-0518, a novel HIV-1 integrase inhibitor, in patients with triple-class resistant virus.14th Conference on Retroviruses and Opportunistic Infections. 25–29 February 2007, Los Angeles, CA, USA.
15.
LaFeminaR.L., SchneiderC.L., RobbinsH.L.Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells. J Virol1992; 66: 7414–7419.
16.
HazudaD.J., FelockP., WitmerM.Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells. Science2000; 287: 646–650.
17.
FikkertV., HombrouckA., Van RemoortelB.Multiple mutations in human immunodeficiency virus-1 integrase confer resistance to the clinical trial drug S-1360. AIDS2004; 18: 2019–2028.
18.
HazudaD.J., YoungS.D., GuareJ.P.Integrase inhibitors and cellular immunity suppress retroviral replication in rhesus macaques. Science2004; 305: 528–532.
19.
HazudaD.J., MillerM.D., NguyenB.Y., ZhaoJ.Resistance to the HIV-integrase inhibitor raltegravir: analysis of protocol 005, a Phase II study in patients with triple-class resistant HIV-1 infection. Antivir Ther2007; 12Suppl 1: S10.
20.
ShimuraK., KodamaE., SakagamiY.Broad antiretroviral activity and resistance profile of the novel human immunodeficiency virus integrase inhibitor elvitegravir (JTK-303/GS-9137). J Virol2008; 82: 764–774.
21.
McCollD.J., FransenS., GuptaS.Resistance and cross-resistance to first generation integrase inhibitors: insights from a Phase II study of elvitegarvir (GS-9137). Antivir Ther2007; 12Suppl 1: S11.
22.
de OliveiraT., DeforcheK., CassolS.An automated genotyping system for analysis of HIV-1 and other microbial sequences. Bioinformatics2005; 21: 3797–3800.
23.
Los Alamos National Laboratory HIV Database Tools. (Updated 22 July 2008. Accessed 18 September 2008.) Available from www.hiv.lanl.gov.
24.
KorberB.HIV signature and sequence variation analysis. In Computational analysis of HIV molecular sequences. Edited by RodrigoA.G., & LearnG.H.. Dordrecht: Kluwer Academic Publishers2000; pp. 55–72.
25.
GibbsJ.S., RegierD.A., DesrosiersR.C.Construction and in vitro properties of HIV-1 mutants with deletions in ‘nonessential’ genes. AIDS Res Hum Retroviruses1994; 10: 343–350.
26.
WeberJ., WeberovaJ., CarobeneM.Use of a novel assay based on intact recombinant viruses expressing green (EGFP) or red (DsRed2) fluorescent proteins to examine the contribution of pol and env genes to overall HIV-1 replicative fitness. J Virol Methods2006; 136: 102–117.
27.
LiM., GaoF., MascolaJ.R.Montefiori, Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J Virol2005; 79: 10108–10125.
28.
FikkertV., Van MaeleB., VercammenJ.Development of resistance against diketo derivatives of human immunodeficiency virus type 1 by progressive accumulation of integrase mutations. J Virol2003; 77: 11459–11470.
29.
Ceccherini-SilbersteinF., Van BaelenK., ArmeniaD.Novel HIV-1 integrase mutations, found as minority quasispecies in patients naïve to integrase inhibitors, are associated with decreased susceptibility to integrase inhibitors in vitro.15th Conference on Retroviruses and Opportunistic Infections. 3–6 February 2008, Boston, MA, USA.
30.
SkinnerL.M., LamersS.L., SandersJ.C., EysterM.E., GoodenowM.M., KatzmanM.Analysis of a large collection of natural HIV-1 integrase sequences, including those from long-term nonprogressors. J Acquir Immune Defic Syndr Hum Retrovirol1998; 19: 99–110.
31.
LatailladeM., ChiarellaJ., KozalM.J.Natural polymorphism of the HIV-1 integrase gene and mutations associated with integrase inhibitor resistance. Antivir Ther2007; 12: 563–570.
32.
ReinkeR., SteffenN.R., RobinsonW.E.Natural selection results in conservation of HIV-1 integrase activity despite sequence variability. AIDS2001; 15: 823–830.
33.
KantorR., ShaferR.W., FollansbeeS.Evolution of resistance to drugs in HIV-1-infected patients failing antiretroviral therapy. AIDS2004; 18: 1503–1511.
34.
KristiansenT.B., PedersenA.G., Eugen-OlsenJ., KatzensteinT.L., LundgrenJ.D.Genetic evolution of HIV in patients remaining on a stable HAART regimen despite insufficient viral suppression. Scand J Infect Dis2005; 37: 890–901.
35.
PradoJ.G., ParkinN.T., ClotetB., RuizL., Martinez-PicadoJ.HIV type 1 fitness evolution in antiretroviral-experienced patients with sustained CD4+ T cell counts but persistent virologic failure. Clin Infect Dis2005; 41: 729–737.
36.
Van BaelenK., ClynhensM., RondelezE.Low level of baseline resistance to integrase inhibitors L731,988 and L870,810 in randomly selected subtype B and non-B HIV-1 strains. Antivir Ther2007; 12Suppl 1: S7.
37.
MaletI., DelelisO., ValantinA.Mutations associated with failure of raltegravir treatment affect integrase sensitivity to the inhibitor in vitro. Antimicrob Agents Chemother2008; 52: 1351–1358.
38.
KatzR.A., MackJ.P., MerkelG.Requirement for a conserved serine in both processing and joining activities of retroviral integrase. Proc Natl Acad Sci U S A1992; 89: 6741–6745.
39.
RenC., MayS., MilettiT., BedardJ.In vitro cross-resistance studies of five different classes of integrase inhibitors in recombinant HIV-1. Antivir Ther2007; 12Suppl 1: S3.
40.
CannonP.M., WilsonW., BylesE., KingsmanS.M., KingsmanA.J.Human immunodeficiency virus type 1 integrase: effect on viral replication of mutations at highly conserved residues. J Virol1994; 68: 4768–4775.
41.
WiskerchenM., MuesingM.A.Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells. J Virol1995; 69: 376–386.
42.
ZhengR., JenkinsT.M., CraigieR.Zinc folds the N-terminal domain of HIV-1 integrase, promotes multimerization, and enhances catalytic activity. Proc Natl Acad Sci U S A1996; 93: 13659–13664.
43.
EngelmanA., EnglundG., OrensteinJ.M., MartinM.A., CraigieR.Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J Virol1995; 69: 2729–2736.
44.
TroyerR.M., CollinsK.R., AbrahaA.Changes in human immunodeficiency virus type 1 fitness and genetic diversity during disease progression. J Virol2005; 79: 9006–9018.
45.
Ceccherini-SilbersteinF., MaletI., FabeniL.Specific mutations related to resistance to HIV-1 integrase inhibitors are associated with reverse transcriptase mutations in HAART-treated patients. Antivir Ther2007; 12Suppl 1: S6.
46.
SuzukiH., FujinoM., MatsudaM., YenH., IwantaniY., SigiuraW.Effects of protease and reverse transcriptase inhibitor-resistance mutations on integrase polymorphism in multidrug resistance cases. Antivir Ther2007; 12Suppl 1: S4.
