- Email: [email protected]
Mutation N155H in HIV-2 integrase confers high phenotypic resistance to raltegravir and impairs replication capacity
Journal of Clinical Virology 46 (2009) 173–175
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Mutation N155H in HIV-2 integrase confers high phenotypic resistance to raltegravir and impairs replication capacity María Salgado a , Carlos Toro b , Ainhoa Simón a , Carolina Garrido a , Francisco Blanco a , Vincent Soriano a , Berta Rodés a,∗ a b
Service of Infectious Diseases, Hospital Carlos III, Madrid, Spain Service of Microbiology, Hospital Carlos III, Madrid, Spain
a r t i c l e
i n f o
Article history: Received 18 March 2009 Received in revised form 9 June 2009 Accepted 18 June 2009 Keywords: HIV-2 Resistance Integrase inhibitors
a b s t r a c t Background: Raltegravir has been shown to be active against wildtype HIV-2 with a phenotypic susceptibility similar to HIV-1. Due to the recent introduction of these novel inhibitors, information on the selection of resistance mutations and its phenotypic effect in this population is scarce. Objectives: To explore in vitro the effect of raltegravir resistance in one individual with HIV-2 infection who failed raltegravir-HAART. Methods: A 20-year-old man with HIV-2 infection received a raltegravir-based HAART regimen. Drug resistance mutations were examined in the integrase gene by sequence analysis. Phenotypic analyses were performed in two HIV-2 isolates from the patient (wildtype isolate: SP-2p2-175 and mutant isolate: SP-2p2-189) and a laboratory reference strain (HIV-2 ROD). Susceptibility to raltegravir was assessed in a PBMC culture assay. Furthermore, a replicative capacity assay was performed. Results: After introduction of raltegravir, patient’s HIV-2 viremia dropped 1 log but did not reach undetectability. Genotypic analysis at month 8 with raltegravir, revealed the development of N155H resistant mutation along with other changes in the HIV-2 integrase: V72I, I84V, A153G, N160K and S163S/G. These changes resulted in a 37-fold increase in phenotypic resistance to raltegravir. Wildtype HIV-2 integrase (SP-2p2-175) had an IC50 of 21.5 nM and HIV-2 mutant virus (SP-2p2-189) showed an IC50 of 789 nM. SP-2p2-189 virus presented also lower replicative capacity in the absence of raltegravir than wildtype. Conclusion: A continued low HIV-2 viral load seems to be enough to select the N155H mutation, which despite significantly impairing viral replication, shows a level of resistance sufficient to give a selective advantage to the virus that maintains this pathway of resistance to raltegravir overtime. © 2009 Elsevier B.V. All rights reserved.
1. Background Antiretroviral therapeutic options for HIV-2 infected patients are limited compared with HIV-1 patients, therefore the use of new drugs with high efficacy over HIV-2 is essential. The recently introduced integrase inhibitors (INI), raltegravir and elvitegravir, have shown good activity against HIV-2 despite the heterogeneity of almost 40% between HIV-1 and HIV-2 integrase (IN) genes.1,2 This novel class of drugs represents a good therapeutic option for HIV2-infected patients who obtain a satisfactory immunological and virological early response.3 However data on response and development of resistance in vivo is scarce due to the small number of HIV-2 patients receiving INI. In HIV-1, raltegravir has a low genetic
∗ Corresponding author at: Department of Infectious Diseases, Hospital Carlos III, Calle Sinesio Delgado, 10, 28029 Madrid, Spain. Tel.: +34 91 453 2586; fax: +34 91 733 6614. E-mail address: [email protected] (B. Rodés). 1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2009.06.020
barrier to resistance, being N155H and Q148H/R/K the main mutations which have a significant impact on the virus susceptibility.4 Only two cases of development of INI resistance in vivo have been reported in HIV-2 which seem to share the same main mutations with HIV-1,5,6 but only one of them has shown a decrease in phenotypic susceptibility to raltegravir due to Q148R mutation.6 Herein we report the emergence of raltegravir resistance in a 20-yearold man with HIV-2 infection who had previously failed several treatments.7 He is multiresistant to all PI and NRTI and had been in immunological and virological failure for more than 6 years. 2. Patients and methods The 20-year-old patient had been diagnosed with HIV-2 in 1992. He initiated therapy with AZT in 1993 and had received multiple antiretroviral regimes over the years, including: AZT + ddC, d4T + 3TC + IDV, ddI + ABC + LPV/r, TPV + RTV + AZT + 3TC + ABC and LPV/r + SQV + d4T. The introduction of raltegravir-based HAART (ddI + darunavir/r + maraviroc) was in November 2007. After reach-
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Fig. 1. Alignement of Patient’s HIV-2 integrase amino acid sequence before (Sp-2p2-175, month 0) and after therapy with raltegravir (SP-2p2-183, month 8 to SP-2p2-192, month 17). Positions highlighted are those involved in resistance or that appear after raltegravir-based HAART. X* indicates amino acid mixture in that position.
ing a CD4 peak in year 2000 (450 cells/L), a steady CD4 decline was observed in the following years. CD4 levels have been less than 50 cells/L (5%) since 2006. HIV-2 viral loads were never undetectable; until 2004, HIV-2 plasma RNA oscillated between 5 and 2.11 log, in the last 5 years levels varied between 2–3.7 log. Plasma viremia and CD4 cell counts were recorded every three months. HIV-2 RNA was measured using Nuclisens EasyQ v1.2.1 (LLD: 100 RNA IU/ml). Drug resistance mutations were examined in the integrase as well as protease and RT regions. Phenotypic analyses were performed in two HIV-2 isolates from the patient (isolate with wildtype integrase: SP-2p2-175, and isolate with mutant integrase: SP-2p2-189) and a laboratory reference strain (HIV-2 ROD). Susceptibility to raltegravir was assessed in a peripheral blood mononuclear cells (PBMCs) culture assay.8 Briefly, HIV-2 viral isolates were used to infect phytohaemagglutinin-interleukin2 (PHA/IL-2) stimulated PBMCs from HIV-negative blood donors under different concentrations of raltegravir (0, 0.1, 0.4, 1.6, 6.4, 25.6, 102.4, 409.6, and 1638.4 nM). Viral production was measured by a p24 assay. Data was reported as IC50 and high resistance was defined as >10-fold increase in IC50 compared to wildtype. Furthermore, replicative capacity was assessed for all viruses by a growth kinetics assay.9 HIV-2 isolates were use to infect PBMCs over three hours. Infected PBMCs were then plated onto 24-well plates containing medium supplemented with IL-2. Supernatants (100 L) were collected every day. At day 3 fresh complete media was added at each well. Levels of p24 were measured in each supernatant to monitor replication kinetics.
showed the same multidrug resistance mutations in all analysed samples (PRO: V10I, V33L, S43T, I54M, V71I, I82F, I90M. RT: K65R, K70R, Q151M). Due to lack of treatment options the patient continued with raltegravir-based HAART; at month 17 all IN mutations were maintained and two new mutations were added (A33A/G and H51H/R). Neither of these additional mutations has been described in HIV-1 to be associated with the N155H resistance pathway and their potential role in resistance or stabilizing N155H mutation could not be determined. The GenBank (http://www.ncbi.nlm.nih.gov/GenBank) accession numbers for the IN sequences are FJ829072–FJ829076. The changes observed in IN resulted in a 37-fold increase in phenotypic resistance to raltegravir. The clinical HIV-2 isolate with wildtype integrase (SP2p2-175) had an IC50 of 21.5 nM compared to an IC50 of 3.33 nM for HIV-2 ROD. The HIV-2 resistant virus (SP-2p2-189) showed an IC50 of 789 nM. This confirms the relationship between the virological failure and N155H mutation, which is shown as an important mechanism of resistance to raltegravir in HIV-2, similar to HIV-1 (Fig. 2). Additionally, it has been reported that the N155H mutation causes a reduction in replicative capacity in HIV-1 strains.4 Likewise, changes in RT and IN regions during treatment with NRTIs and INI could influence the replicative capacity.10 In our study,
3. Results After introduction of raltegravir in the HAART regimen (ddI + darunavir/r + maraviroc), the patient experienced a reduction of 1 log in HIV-2 viral load but did not reach undetectable levels; viral load stayed stable at 2.7 log RNA IU/L for 8 months and then experienced a slight increase to 3.07 log RNA IU/L. CD4 T-cell counts did not decline but were low for the whole period (around 40 cell/L). Genotypic analysis at month 8 revealed the development of N155H primary resistance mutation along with additional mutations in the HIV-2 integrase: V72I, I84V, A153G, N160K and S163S/G (Fig. 1). Protease and RT regions
Fig. 2. Growth curves of HIV-2 clinical isolates with wildtype integrase (SP-2p2-175) or mutant integrase (SP-2p2-189) and HIV-2 laboratory strain ROD with wildtype integrase gene in the presence of different concentrations of raltegravir.
M. Salgado et al. / Journal of Clinical Virology 46 (2009) 173–175
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advantage to the virus that maintains this pathway of resistance to raltegravir overtime. Acknowledgements This work was partly supported by, Fundación para la Inves˜ (FIPSE), Fundación tigación y la Prevención del Sida en Espana Investigacion y Educacion en SIDA (FIES) and Red de Investigación en Sida (RIS) G03/173. References
Fig. 3. Effect of Integrase mutations in viral replicative capacity. Replication Kinetics curves for HIV-2 wildtype (SP-2P2-175) and mutant (Sp-2P2-189) viruses.
mutations in the RT did not vary in either isolate (SP-2p2-175 and SP-2p2-189); the patient maintained 65R, 70R, 75I, 115Y, 118I, 151M, 181I and 190A mutations for the whole follow-up. The development of N155H mutation in IN, however, affects considerably viral replication. The mutant virus SP-2p2-189 had an important reduction in replicative capacity compared to wildtype virus (SP-2p2-175) in the absence of raltegravir (Fig. 3). Despite impaired fitness the HIV2 virus carrying the N155H seemed to be stable overtime and the switch to the Q148R/H pathway reported in HIV-1 after a few weeks under continued drug pressure was not observed.11 Further genetic analysis also revealed that positions associated with RT-IN interaction (Ile178 in RT or G149 in IN)12 did not change during the 17 month follow-up. 4. Conclusion This is the first report showing high phenotypic resistance to raltegravir in HIV-2 due to development of N155H. Previously, this mutation has been described in a HIV-2 infected patient failing raltegravir, however the impact in resistance had not been assessed.5 A continued low HIV-2 viral load seems to be enough to select the N155H mutation, which despite significantly impairing viral replication, shows a level of resistance sufficient to give a selective
1. Roquebert B, Damond F, Collin G, Matheron S, Peytavin G, Benard A, et al. HIV-2 integrase gene polymorphism and phenotypic susceptibility of HIV-2 clinical isolates to the integrase inhibitors raltegravir and elvitegravir in vitro. J Antimicrob Chemother 2008;62:914–20. 2. Xu L, Anderson J, Ferns B, Cook P, Wildfire A, Workman J, et al. Genetic diversity of integrase (IN) sequences in antiretroviral treatment-naive and treatmentexperienced HIV type 2 patients. AIDS Res Hum Retroviruses 2008;24:1003–7. 3. Damond F, Lariven S, Roquebert B, Males S, Peytavin G, Morau G, et al. Virological and immunological response to HAART regimen containing integrase inhibitors in HIV-2-infected patients. AIDS 2008;22:665–6. 4. Hazuda DJ, Miller MD, Nguyen BY, Zhao J. Resistance to the HIV integrase inhibitor raltegravir: analysis of protocol 005, a phase II study in patients with triple-class resistant HIV-1. Antivir Ther 2007;12(Suppl. 1):S10. 5. Garrett N, Xu L, Smit E, Ferns B, El-Gadi S, Anderson J. Raltegravir treatment response in an HIV-2 infected patient: a case report. AIDS 2008;22:1091–2. 6. Roquebert B, Blum L, Collin G, Damond F, Peytavin G, Leleu J, et al. Selection of the Q148R integrase inhibitor resistance mutation in a failing raltegravir containing regimen. AIDS 2008;22:2045–6. 7. Rodes B, Sheldon J, Toro C, Jimenez V, Alvarez MA, Soriano V. Susceptibility to protease inhibitors in HIV-2 primary isolates from patients failing antiretroviral therapy. J Antimicrob Chemother 2006;57:709–13. 8. Vandamme A, Witvrouw M, Pannecouque C, Balzarini J, Van Laethem K, Schmit JC, et al. Evaluating clinical isolates for their phenotypic and genotypic resistance against anti-HIV drugs. In: Kirchington D, Schinazi R, editors. Antiviral methods and protocols. New Jersey: Humana Press; 2000. p. 223–58. 9. Reid P, MacInnes H, Cong M, Heneine W, Garcia-Lerma G. Natural resistence of human immunodeficiency virus type 2 to zidovudine. Virology 2005;336:251–64. 10. Wilkinson TA, Januszyk K, Phillips ML, Tekeste SS, Zhang M, Miller, et al. Identifying and characterizing a functional HIV-1 reverse transcriptase-binding site on integrase. J Biol Chem 2009;284:7931–9. 11. Malet I, Delelis O, Soulie C, Wirden M, Tchertanov L, Mottaz P, et al. Quasispecies variant dynamics during emergence of resistance to raltegravir in HIV-1-infected patients. J Antimicrob Chemother 2009;63:795–804. 12. Oz Gleenberg I, Goldgur Y, Hizi A. Ile178 of HIV-1 reverse transcriptase is critical for inhibiting the viral integrase. Biochem Biophys Res Commun 2007;364:48–52.
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Mutation N155H in HIV-2 integrase confers high phenotypic resistance to raltegravir and impairs replication capacity María Salgado a , Carlos Toro b , Ainhoa Simón a , Carolina Garrido a , Francisco Blanco a , Vincent Soriano a , Berta Rodés a,∗ a b
Service of Infectious Diseases, Hospital Carlos III, Madrid, Spain Service of Microbiology, Hospital Carlos III, Madrid, Spain
a r t i c l e
i n f o
Article history: Received 18 March 2009 Received in revised form 9 June 2009 Accepted 18 June 2009 Keywords: HIV-2 Resistance Integrase inhibitors
a b s t r a c t Background: Raltegravir has been shown to be active against wildtype HIV-2 with a phenotypic susceptibility similar to HIV-1. Due to the recent introduction of these novel inhibitors, information on the selection of resistance mutations and its phenotypic effect in this population is scarce. Objectives: To explore in vitro the effect of raltegravir resistance in one individual with HIV-2 infection who failed raltegravir-HAART. Methods: A 20-year-old man with HIV-2 infection received a raltegravir-based HAART regimen. Drug resistance mutations were examined in the integrase gene by sequence analysis. Phenotypic analyses were performed in two HIV-2 isolates from the patient (wildtype isolate: SP-2p2-175 and mutant isolate: SP-2p2-189) and a laboratory reference strain (HIV-2 ROD). Susceptibility to raltegravir was assessed in a PBMC culture assay. Furthermore, a replicative capacity assay was performed. Results: After introduction of raltegravir, patient’s HIV-2 viremia dropped 1 log but did not reach undetectability. Genotypic analysis at month 8 with raltegravir, revealed the development of N155H resistant mutation along with other changes in the HIV-2 integrase: V72I, I84V, A153G, N160K and S163S/G. These changes resulted in a 37-fold increase in phenotypic resistance to raltegravir. Wildtype HIV-2 integrase (SP-2p2-175) had an IC50 of 21.5 nM and HIV-2 mutant virus (SP-2p2-189) showed an IC50 of 789 nM. SP-2p2-189 virus presented also lower replicative capacity in the absence of raltegravir than wildtype. Conclusion: A continued low HIV-2 viral load seems to be enough to select the N155H mutation, which despite significantly impairing viral replication, shows a level of resistance sufficient to give a selective advantage to the virus that maintains this pathway of resistance to raltegravir overtime. © 2009 Elsevier B.V. All rights reserved.
1. Background Antiretroviral therapeutic options for HIV-2 infected patients are limited compared with HIV-1 patients, therefore the use of new drugs with high efficacy over HIV-2 is essential. The recently introduced integrase inhibitors (INI), raltegravir and elvitegravir, have shown good activity against HIV-2 despite the heterogeneity of almost 40% between HIV-1 and HIV-2 integrase (IN) genes.1,2 This novel class of drugs represents a good therapeutic option for HIV2-infected patients who obtain a satisfactory immunological and virological early response.3 However data on response and development of resistance in vivo is scarce due to the small number of HIV-2 patients receiving INI. In HIV-1, raltegravir has a low genetic
∗ Corresponding author at: Department of Infectious Diseases, Hospital Carlos III, Calle Sinesio Delgado, 10, 28029 Madrid, Spain. Tel.: +34 91 453 2586; fax: +34 91 733 6614. E-mail address: [email protected] (B. Rodés). 1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2009.06.020
barrier to resistance, being N155H and Q148H/R/K the main mutations which have a significant impact on the virus susceptibility.4 Only two cases of development of INI resistance in vivo have been reported in HIV-2 which seem to share the same main mutations with HIV-1,5,6 but only one of them has shown a decrease in phenotypic susceptibility to raltegravir due to Q148R mutation.6 Herein we report the emergence of raltegravir resistance in a 20-yearold man with HIV-2 infection who had previously failed several treatments.7 He is multiresistant to all PI and NRTI and had been in immunological and virological failure for more than 6 years. 2. Patients and methods The 20-year-old patient had been diagnosed with HIV-2 in 1992. He initiated therapy with AZT in 1993 and had received multiple antiretroviral regimes over the years, including: AZT + ddC, d4T + 3TC + IDV, ddI + ABC + LPV/r, TPV + RTV + AZT + 3TC + ABC and LPV/r + SQV + d4T. The introduction of raltegravir-based HAART (ddI + darunavir/r + maraviroc) was in November 2007. After reach-
174
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Fig. 1. Alignement of Patient’s HIV-2 integrase amino acid sequence before (Sp-2p2-175, month 0) and after therapy with raltegravir (SP-2p2-183, month 8 to SP-2p2-192, month 17). Positions highlighted are those involved in resistance or that appear after raltegravir-based HAART. X* indicates amino acid mixture in that position.
ing a CD4 peak in year 2000 (450 cells/L), a steady CD4 decline was observed in the following years. CD4 levels have been less than 50 cells/L (5%) since 2006. HIV-2 viral loads were never undetectable; until 2004, HIV-2 plasma RNA oscillated between 5 and 2.11 log, in the last 5 years levels varied between 2–3.7 log. Plasma viremia and CD4 cell counts were recorded every three months. HIV-2 RNA was measured using Nuclisens EasyQ v1.2.1 (LLD: 100 RNA IU/ml). Drug resistance mutations were examined in the integrase as well as protease and RT regions. Phenotypic analyses were performed in two HIV-2 isolates from the patient (isolate with wildtype integrase: SP-2p2-175, and isolate with mutant integrase: SP-2p2-189) and a laboratory reference strain (HIV-2 ROD). Susceptibility to raltegravir was assessed in a peripheral blood mononuclear cells (PBMCs) culture assay.8 Briefly, HIV-2 viral isolates were used to infect phytohaemagglutinin-interleukin2 (PHA/IL-2) stimulated PBMCs from HIV-negative blood donors under different concentrations of raltegravir (0, 0.1, 0.4, 1.6, 6.4, 25.6, 102.4, 409.6, and 1638.4 nM). Viral production was measured by a p24 assay. Data was reported as IC50 and high resistance was defined as >10-fold increase in IC50 compared to wildtype. Furthermore, replicative capacity was assessed for all viruses by a growth kinetics assay.9 HIV-2 isolates were use to infect PBMCs over three hours. Infected PBMCs were then plated onto 24-well plates containing medium supplemented with IL-2. Supernatants (100 L) were collected every day. At day 3 fresh complete media was added at each well. Levels of p24 were measured in each supernatant to monitor replication kinetics.
showed the same multidrug resistance mutations in all analysed samples (PRO: V10I, V33L, S43T, I54M, V71I, I82F, I90M. RT: K65R, K70R, Q151M). Due to lack of treatment options the patient continued with raltegravir-based HAART; at month 17 all IN mutations were maintained and two new mutations were added (A33A/G and H51H/R). Neither of these additional mutations has been described in HIV-1 to be associated with the N155H resistance pathway and their potential role in resistance or stabilizing N155H mutation could not be determined. The GenBank (http://www.ncbi.nlm.nih.gov/GenBank) accession numbers for the IN sequences are FJ829072–FJ829076. The changes observed in IN resulted in a 37-fold increase in phenotypic resistance to raltegravir. The clinical HIV-2 isolate with wildtype integrase (SP2p2-175) had an IC50 of 21.5 nM compared to an IC50 of 3.33 nM for HIV-2 ROD. The HIV-2 resistant virus (SP-2p2-189) showed an IC50 of 789 nM. This confirms the relationship between the virological failure and N155H mutation, which is shown as an important mechanism of resistance to raltegravir in HIV-2, similar to HIV-1 (Fig. 2). Additionally, it has been reported that the N155H mutation causes a reduction in replicative capacity in HIV-1 strains.4 Likewise, changes in RT and IN regions during treatment with NRTIs and INI could influence the replicative capacity.10 In our study,
3. Results After introduction of raltegravir in the HAART regimen (ddI + darunavir/r + maraviroc), the patient experienced a reduction of 1 log in HIV-2 viral load but did not reach undetectable levels; viral load stayed stable at 2.7 log RNA IU/L for 8 months and then experienced a slight increase to 3.07 log RNA IU/L. CD4 T-cell counts did not decline but were low for the whole period (around 40 cell/L). Genotypic analysis at month 8 revealed the development of N155H primary resistance mutation along with additional mutations in the HIV-2 integrase: V72I, I84V, A153G, N160K and S163S/G (Fig. 1). Protease and RT regions
Fig. 2. Growth curves of HIV-2 clinical isolates with wildtype integrase (SP-2p2-175) or mutant integrase (SP-2p2-189) and HIV-2 laboratory strain ROD with wildtype integrase gene in the presence of different concentrations of raltegravir.
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175
advantage to the virus that maintains this pathway of resistance to raltegravir overtime. Acknowledgements This work was partly supported by, Fundación para la Inves˜ (FIPSE), Fundación tigación y la Prevención del Sida en Espana Investigacion y Educacion en SIDA (FIES) and Red de Investigación en Sida (RIS) G03/173. References
Fig. 3. Effect of Integrase mutations in viral replicative capacity. Replication Kinetics curves for HIV-2 wildtype (SP-2P2-175) and mutant (Sp-2P2-189) viruses.
mutations in the RT did not vary in either isolate (SP-2p2-175 and SP-2p2-189); the patient maintained 65R, 70R, 75I, 115Y, 118I, 151M, 181I and 190A mutations for the whole follow-up. The development of N155H mutation in IN, however, affects considerably viral replication. The mutant virus SP-2p2-189 had an important reduction in replicative capacity compared to wildtype virus (SP-2p2-175) in the absence of raltegravir (Fig. 3). Despite impaired fitness the HIV2 virus carrying the N155H seemed to be stable overtime and the switch to the Q148R/H pathway reported in HIV-1 after a few weeks under continued drug pressure was not observed.11 Further genetic analysis also revealed that positions associated with RT-IN interaction (Ile178 in RT or G149 in IN)12 did not change during the 17 month follow-up. 4. Conclusion This is the first report showing high phenotypic resistance to raltegravir in HIV-2 due to development of N155H. Previously, this mutation has been described in a HIV-2 infected patient failing raltegravir, however the impact in resistance had not been assessed.5 A continued low HIV-2 viral load seems to be enough to select the N155H mutation, which despite significantly impairing viral replication, shows a level of resistance sufficient to give a selective
1. Roquebert B, Damond F, Collin G, Matheron S, Peytavin G, Benard A, et al. HIV-2 integrase gene polymorphism and phenotypic susceptibility of HIV-2 clinical isolates to the integrase inhibitors raltegravir and elvitegravir in vitro. J Antimicrob Chemother 2008;62:914–20. 2. Xu L, Anderson J, Ferns B, Cook P, Wildfire A, Workman J, et al. Genetic diversity of integrase (IN) sequences in antiretroviral treatment-naive and treatmentexperienced HIV type 2 patients. AIDS Res Hum Retroviruses 2008;24:1003–7. 3. Damond F, Lariven S, Roquebert B, Males S, Peytavin G, Morau G, et al. Virological and immunological response to HAART regimen containing integrase inhibitors in HIV-2-infected patients. AIDS 2008;22:665–6. 4. Hazuda DJ, Miller MD, Nguyen BY, Zhao J. Resistance to the HIV integrase inhibitor raltegravir: analysis of protocol 005, a phase II study in patients with triple-class resistant HIV-1. Antivir Ther 2007;12(Suppl. 1):S10. 5. Garrett N, Xu L, Smit E, Ferns B, El-Gadi S, Anderson J. Raltegravir treatment response in an HIV-2 infected patient: a case report. AIDS 2008;22:1091–2. 6. Roquebert B, Blum L, Collin G, Damond F, Peytavin G, Leleu J, et al. Selection of the Q148R integrase inhibitor resistance mutation in a failing raltegravir containing regimen. AIDS 2008;22:2045–6. 7. Rodes B, Sheldon J, Toro C, Jimenez V, Alvarez MA, Soriano V. Susceptibility to protease inhibitors in HIV-2 primary isolates from patients failing antiretroviral therapy. J Antimicrob Chemother 2006;57:709–13. 8. Vandamme A, Witvrouw M, Pannecouque C, Balzarini J, Van Laethem K, Schmit JC, et al. Evaluating clinical isolates for their phenotypic and genotypic resistance against anti-HIV drugs. In: Kirchington D, Schinazi R, editors. Antiviral methods and protocols. New Jersey: Humana Press; 2000. p. 223–58. 9. Reid P, MacInnes H, Cong M, Heneine W, Garcia-Lerma G. Natural resistence of human immunodeficiency virus type 2 to zidovudine. Virology 2005;336:251–64. 10. Wilkinson TA, Januszyk K, Phillips ML, Tekeste SS, Zhang M, Miller, et al. Identifying and characterizing a functional HIV-1 reverse transcriptase-binding site on integrase. J Biol Chem 2009;284:7931–9. 11. Malet I, Delelis O, Soulie C, Wirden M, Tchertanov L, Mottaz P, et al. Quasispecies variant dynamics during emergence of resistance to raltegravir in HIV-1-infected patients. J Antimicrob Chemother 2009;63:795–804. 12. Oz Gleenberg I, Goldgur Y, Hizi A. Ile178 of HIV-1 reverse transcriptase is critical for inhibiting the viral integrase. Biochem Biophys Res Commun 2007;364:48–52.