The organization of HIV-1 and HIV-2 genomes is similar despite them having 50–60% differences at nucleotide level and proteins with different molecular weights . Such differences may be correlated with varied susceptibility to antiretrovirals, as observed by the natural resistance of HIV-2 to nonnucleoside reverse transcriptase inhibitors and to the fusion inhibitor, enfuvirtide. Naturally decreased susceptibility to some protease inhibitors has been described [2–4].
About 50% of the HIV-2 positions in the protease-coding region have been described as polymorphic when compared with HIV-1, and most are located at positions 14, 17, 40, 41, and 70 in HIV-2 group A, with the most frequent variable residue at position 14 . HIV-2 protease polymorphisms have been observed at positions 10, 36, 46, 54, 71, 73, 77, and 90, which are known in HIV-1 to be major or minor mutations associated with drug resistance to protease inhibitors. Other studies have described polymorphisms at positions 10, 14, 17, 20, 32, 33, 36, 40, 41, 46, 47, 63, 70, 71, 73, 77, 82, and 93 [12–16].
Currently licensed protease inhibitors have been developed to fit the HIV-1 active protease site; differences in the sequences of HIV-2 protease could have a role in the effectiveness of such molecules on HIV-2 [12,15,16]. Depending on the inhibitor, the affinity of protease inhibitors could be 10–100 times lower for HIV-2 compared with HIV-1 . Various studies have compared the efficacy of various protease inhibitors: lopinavir, saquinavir, and darunavir are the most effective protease inhibitors in HIV-2, both in vivo and in vitro[2,18]. HIV-2 protease has been described as having a cleavage efficiency similar to that of HIV-1 .
Each genome is translated into Gag and Gag–Pol polyprotein precursors, given the different structural proteins: matrix (MA), capsid (CA), spacer 1 (p2), nucleocapsid (NC), spacer 2 (p1), p6, and the viral enzymes. In HIV-2, as in HIV-1, ribosomal-1 frameshifting, mediated by a downstream signal, is required to synthesize the Gag and Gag–Pol polyprotein precursors (Fig. 1). The downstream signal is composed of a heptameric X XXY YYZ consensus slippery sequence (U UUU UUA) and a downstream secondary RNA structure, which causes the ribosome to pause at the NC/p1 junction [5,6]. The downstream secondary RNA structure is a ‘stem loop’ or hairpin in HIV, which acts as a thermodynamic barrier to translation [7,8] (Fig. 1).
Frameshifting is a controlled event, allowing a correct Gag : Gag–Pol ratio for optimal virus production [6,9–11]. Protease interacts with the seven residues around the gag and gag–pol cleavage site and cleaves the Gag and Gag–Pol precursors, which enables viral structural proteins and enzymes to be liberated.
In HIV-1 infection, the presence of gag and gag–pol cleavage site mutations may [20–27] increase with the number of protease inhibitors resistance mutations [28,29] in protease inhibitors resistance mutations-experienced patients compared with antiretroviral-naive patients [21,26,27,30]. These mutations may drive the resistance mechanism to become a compensatory mechanism, which increases the activity of the mutant protease [20,21,26,30–36]. These mutations might also increase the affinity of the precursors for the mutant protease, enhancing the cleavage of Gag and Gag–Pol precursors [20,23,31,37–41]. This allows partial recovery of virus-replicative capacity compared with the level of viruses that have mutant protease and no cleavage site mutations [22,28,30,42,43]. The structure of the HIV-2 gag cleavage site has been described as being similar to those of HIV-1 [1,44,45], especially the MA/CA gag cleavage site [1,19], in contrast to the sequence of the gag p2/NC cleavage site, which shares few nucleotide similarities between the two viruses  (Fig. 1).
The aim of this exploratory in-vivo study was to compare, according to virological outcome, the baseline mutations in protease coding, gag cleavage site and gag–pol frameshift regions of protease inhibitor-naive and protease inhibitor-experienced HIV-2-infected patients receiving a lopinavir (LPV) containing regimen.
Materials and methods
In this prospective study, we focused on 46 HIV-2 group A-infected patients enrolled in the Agence Nationale de Recherches sur le SIDA et les Hepatites virales (ANRS) CO5 cohort, receiving LPV and nucleoside transcriptase reverse inhibitors. Briefly, this cohort initiated in 1994 is a national prospective study that is still ongoing in 124 French clinical centers. The inclusion criteria into the cohort are only having HIV-2 infection, being aged 18 years or more, being a resident in France for at least 1 year, and informed written consent. About 800 patients are included in the cohort.
Baseline was defined as the initiation of a regimen that included LPV and having a HIV-2 RNA plasma viral load below 100 copies/ml. Virological response was defined as a HIV-2 RNA plasma viral load less than 100 copies/ml at month 3. We selected this early endpoint (month 3) to capture the most sensitive and potent part of the regimen, and to limit any consequences caused by treatment modifications and to reduce the risk of missing data.
Preparation of viral cDNA
Viral RNA was extracted from plasma (1 ml) with an automated nucleic acid extractor (Magnapure) using the Total NA large-volume program (Roche Diagnostics, GmbH, Mannheim, Germany), and was then stored at −80°C. The protease-coding region was amplified as previously described .
To prepare gag, gag–pol cleavage site, and gag–pol frameshift cDNA, 10 μl of RNA was used for reverse transcription–PCR (Titan one-tube reverse transcription–PCR kit; Boehringer, Manheim, Germany) with the forward and reverse primers GAGOGS1 (5′-GTGGGGATGGGCGCGAGAAACT-3′) and GAGOGAS1 (5′-TCTACTGGCTGACCCTCAATG-3′), respectively, in 10 μl of 5X reverse transcription–PCR buffer, 2.5 μl of dithiothreitol solution, 4 μl of deoxynucleoside triphosphate (dNTP) mix, and 2 μl each of 10 μmol/l primers, 1 μl of RNase inhibitor, 1 μl of Titan enzyme mix, and 17.5 μl of PCR grade water. The cycling parameters were as follows: 50°C for 30 min; 94°C for 2 min; 10 cycles at 94°C for 30 s, 55°C for 30 s, and 68°C for 2 min; 25 cycles at 94°C for 30 s, 55°C for 30 s, and 68°C for 2 min, with cycle elongation of an additional 5 s for each cycle; and a final step at 68°C for 7 min. reverse transcription–PCR products were stored at −20°C.
In order to obtain the 5′ region of gag, 5 μl of cDNA was amplified by nested PCR using the forward primer CV2TAQ5′ (5′-GCGAGAAACTCCGTCTTGAGAG-3′) and the reverse primer GAGOGAS1 (5′-GCCTTCTGAGAGTGCCTGAAATCC-3′). To obtain the 3′ region of gag, 5 μl of cDNA was amplified by nested PCR using the forward primer GAGOGS1INVRAC5′ (5′-CCAGGATTTCAGGCACTCTCAGAC-3′) and the reverse primer 2AGOGAS2 (5′-TGGCTGACCCTCAATGTATG-3′) in 5 μl of 10X BM-Taq DNA polymerase kit buffer (Roche Diagnostic), 0.4 μl of dNTP (100 μmol/l each; Roche Diagnostic), 2.5 μl each of 10 μmol/l primers, 0.2 μl of BM-Taq DNA polymerase-kit enzyme (Roche Diagnostic), and 35 μl of PCR grade water (Eurobio, les Ulis, France). The cycling parameters were as follows: 94°C for 2 min; 35 cycles at 94°C for 30 s, 55°C for 30 s, and 68°C for 1.5 min; and a final step at 68°C for 7 min.
The protease-coding region and reverse transcriptase were sequenced directly from PCR products as previously described .
For gag, gag–pol cleavage site, and gag–pol frameshift, population sequencing was performed using a BigDye Terminator version 1.1 cycle sequencing kit (Applied Biosystems, Paris, France), with CV2TAQ5′ and GAGOGAS1 primers for gag cleavage site MA/CA and with the forward primer LUGAG1 (5′-TAAARCAGGGACCAAARGA-3′) and the reverse primer 2AGOGAS2 for gag cleavage site CA/p2, p2/NC, NC/p1, p1/p6gag, p1/p15pol. Sequencing was performed with an ABI Prism 3130 sequencer (Applied Biosystems, Foster City, California, USA). Nucleotide sequences were aligned and differences in amino acid sequence relative to the wild-type HIV-2 group A ROD reference strain were noted. All substitutions observed in gag cleavage site and protease were noted. The HIV-2 gag, the protease-coding region and the reverse transcriptase sequences were deposited in GenBANK with the following accession numbers: JQ446324–JQ446366 and JX508650–JX508707.
The HIV-2 group was determined by analyzing the protease-coding region and reverse transcriptase. Nucleotide sequences were compared with reference sequences of known HIV-2 groups contained in GenBank (http://www.ncbi.nlm.nih.gov/retroviruses). A phylogenetic tree was generated using HIV-2 gag sequences, added in Supplemental Digital Content, http://links.lww.com/QAD/A260.
Determination of hairpin free energy
RNA folding and stability of the hairpin structure of the gag–pol frameshift region were determined by measuring free energy in accordance with Turner's rules [CombFold, RNAsoft (http://www.rnasoft.ca/cgi-bin/RNAsoft/CombFold/combfold.pl)]. The sequence analyzed was between nucleotides 1849 and 1893 with respect to the whole HIV-2 group A ROD genome (http://www.hiv.lanl.gov/content/sequence/HIV/COMPENDIUM/2010/hiv2dna.pdf).
Baseline characteristics were described as their median and interquartile ranges (IQR) for continuous variables and as frequencies for binary variables.
Associations between virological outcome at month 3 and continuous variables (baseline HIV-2 RNA viral load, baseline number of mutations in protease-coding region, and baseline number of mutations in gag cleavage site) were analyzed using Wilcoxon's rank sum test.
Associations between virological outcome at month 3 and binary variables [frequency of baseline amino acids for the protease-coding region, cleavage site in the gag open-reading frame (ORF) and p1/p15pol in the gag–pol ORF, sequences compared with the HIV-2 group A ROD reference strain] were analyzed using Fisher's exact tests.
Baseline RNA folding and stability were compared according to virological response at month 3, using Wilcoxon's rank sum test.
The association between number of mutations in the protease-coding region and the number of mutations in the gag cleavage site was analyzed using Wilcoxon's rank sum test. Associations between the number of mutations in the protease-coding region and particular mutations in gag cleavage site were analyzed using Fisher's exact test.
Data analyses were conducted using SAS, version 9.1 (SAS Institute Inc., Cary, North Carolina, USA). The statistical significance was set at P less than 0.05.
In this study, 46 HIV-2-infected patients, enrolled in the ANRS CO5 HIV-2 cohort and receiving a LPV-based regimen were included: 17 protease inhibitor-naive and 29 protease inhibitor-experienced patients. Baseline median HIV-2 RNA viral load was 2136 copies/ml (IQR = 813–10640) (Table 1). Baseline median CD4 cell count was 176 cells/μl (IQR = 137–238). Among the 29 protease inhibitor-experienced patients, the median number of baseline protease inhibitors received previously before the regimen was three (IQR = 1–5), a virological response was observed in 67% of patients, including 14 (82%) protease inhibitor-naive and 17 (59%) protease inhibitor-experienced patients.
In all patients, virological failure at month 3 was associated with higher baseline viral load (median = 6765 versus 1098 copies/ml, P = 0.02).
Baseline protease, gag cleavage site, and gag–pol frameshift signal genotypic analyses
At baseline, sequences of the protease-coding region (Table 2) and sequences of the 5′ region of gag, which allowed us to obtain the MA/CA cleavage site sequence in gag ORF (Table 3), were available for the 46 patients. CA/p2, p2/NC, NC/p1, p1/p6gag in the gag ORF, and p1/p15pol in the gag–pol ORF sequences were obtained from the 3′ region of gag in 43 patients, including 14 of 17 (82%) protease inhibitor-naive and 29 (100%) protease inhibitor-experienced patients (Table 3).
Protease inhibitor-naive patients
The presence at baseline of the Y14H mutation in the protease-coding region was associated with virological failure (n = 2/12 with virological response versus n = 3/3 patients with virological failure , P = 0.022) (Table 2a).
The baseline T435A (NC/p15pol) cleavage site mutation in gag–pol ORF (n = 1/11 patients with virological response versus n = 3/3 patients with virological failure) and the baseline V447M (p1/p6gag) cleavage site mutation in gag ORF were both associated with virological failure at month 3 (n = 0/11 patients with virological response versus n = 2/3 patients with virological failure, P = 0.033) (Table 3a).
Overall, T435A and V447M cleavage site mutations were both associated with the presence of the Y14H mutation in the protease-coding region (P = 0.018 and P = 0.039, respectively).
Protease inhibitor-experienced patients
The baseline I82F mutation within the protease-coding region was associated with virological failure (n = 0/17 patients with virological response versus n = 3/11 patients with virological failure, P = 0.050) (Table 2b). The baseline D427E (NC/p1) cleavage site mutation in gag ORF was associated with virological response (n = 15/17 versus n = 5/12, P = 0.014). The baseline A430V (NC/p1) cleavage site mutation in gag ORF was associated with virological failure (n = 3/17 patients with virological response versus n = 7/12 patients with virological failure, P = 0.046) (Table 3b). The A430V cleavage site mutation was associated with an increased number of mutations in the protease-coding region (median = 10 versus 7, P = 0.008), in particular with the I54M and N68G mutations (P = 0.028 and P = 0.045, respectively).
Comparisons between protease inhibitor-naive and protease inhibitor-experienced patients
At baseline, no difference in the number of cleavage site mutations was seen between protease inhibitor-naive and protease inhibitor-experienced patients.
A higher number of protease-coding region mutations was observed in protease inhibitor-experienced compared to protease inhibitor-naive patients (median = 8 versus 5, P = 0.003). Mutations at positions 54, 62, 71, 90, and 92 were more frequent in protease inhibitor-experienced patients compared with protease inhibitor-naive patients (P = 0.008, P = 0.036, P = 0.002, P = 0.001, and P = 0.031, respectively) (Fig. 2).
Free energy of the hairpin gag–pol frameshift signal at baseline
The measurement of baseline hairpin free energy was performed using hairpin gag–pol frameshift signal sequences in 43 patients: 14 protease inhibitor-naive and 29 protease inhibitor-experienced patients. In protease inhibitor-naive patients, the baseline median hairpin free energy was −21.5 kcal/mol in patients with virological response at month 3 and was −21.6 kcal/mol in patients with virological failure at month 3. In protease inhibitor-experienced patients, the baseline median hairpin free energy was −22.0 kcal/mol in patients with virological response at month 3 and was −21.9 kcal/mol in patients with virological failure at month 3. No association between baseline hairpin free energy and virological outcome was evidenced in protease inhibitor-experienced patients (Table 4).
In our study, evaluating the protease-coding region and gag viral populations in HIV-2-infected patients receiving LPV-containing regimen, we have shown an association between the level of baseline HIV-2 RNA viral load and the occurrence of virological failure at month 3. Moreover, at inclusion, a significantly higher number of protease-coding region mutations was found in protease inhibitor-experienced compared with protease inhibitor-naive patients. Although no difference was observed in the total number of baseline gag cleavage site mutations between protease inhibitor-naive and protease inhibitor-experienced patients, the presence of mutations at positions 54, 62, 71, 90, and 92 in the protease-coding region was more frequent in protease inhibitor-experienced patients compared with protease inhibitor-naive patients. Furthermore, different specific gag cleavage site and protease-coding region mutations were involved in the virological outcome of these two groups of patients.
Regarding the protease-coding region, in protease inhibitor-experienced patients, selection of I82F , as observed in HIV-1, was associated with protease inhibitor resistance  and with high phenotypic resistance to LPV . Studies that have evaluated the selection of protease mutations in HIV-2-infected protease inhibitor-treated patients show that resistance mutations appeared at the same positions as those selected by HIV-1, sometimes with different amino acids: V10I, I36V, I46V, G48R, I54L/M, V71A/I, I82F/L, L90M [12,15,16]. The selection of I82F and I54M in the HIV-2 protease-coding region might cause cross-resistance to different protease inhibitors and high-level resistance to LPV . Variability at residue 14 was most frequently observed in both groups: 71% in group A and 52% in group B, with a majority of the HIV-2 group A strain harboring a Y14H mutation , as was observed in our study.
In the absence of studies analyzing the impact of gag HIV-2 cleavage site mutations on virological outcome, we assessed the impact of such mutations with regard to the documented impact of HIV-1 gag cleavage site mutations on virological outcome of a protease inhibitor-based regimen.
In HIV-1, several studies report that the p2/NC cleavage site has a large genetic polymorphism [47,48]. In the MONARK trial wherein a first-line antiretroviral induction treatment of LPV given as a monotherapy was evaluated in HIV-1-infected patients, a negative impact, of the number of baseline gag cleavage site mutations at the p2/p7 site was observed on virological outcome at week 96 . Indeed, the presence of more than two mutations in p2/NC cleavage site was significantly associated with virological failure . In contrast to HIV-1, in our study, p2/NC was the more conserved gag cleavage site, as described in complete genome database alignments (http://www.hiv.lanl.gov/content/index). We did not demonstrate any impact of the number of substitutions in this gag cleavage site on virological outcome.
In the HIV-2 NC/p1 gag cleavage site, baseline D427E was associated with improved early virological response to the LPV-containing regimen. This has been previously described in HIV-1-infected patients in whom some gag cleavage site mutations were associated with virological response [50,51]. Position 427 in HIV-2 group A corresponds to position 428 in HIV-1. Residue 427 is located far from the substrate-binding subsite at the P5 position in NC/p1 cleavage site, and might not directly alter specific interactions with the protease enzyme but might modify the conformation of the substrate region in the polyprotein, probably favoring the cleavage step . In contrast to what we found with D427E in HIV-2, baseline E428G in HIV-1 was associated with decreased virological response to darunavir at week 24 in antiretroviral-experienced patients .
In HIV-2 NC/p1 gag cleavage site, position 430 in HIV-2 group A corresponds to position 431 in HIV-1. In the literature, A431V in HIV-1 has only been found in protease inhibitor-experienced patients [20,21,26,27,31,36,48,53]. A431V is the most frequent gag cleavage site mutation associated with protease-coding region mutations . It has been reported that its frequency increases in parallel with the number of protease mutations , showing specific associations with M46I/L, I54V, or V82A/F/T protease mutations [28,39,47,48]. The A431V mutation, selected in patients receiving protease inhibitor and experiencing virological failure [27,29,32,54], increased resistance level to LPV, nelfinavir, indinavir, and fosamprenavir [39,53] has been associated, in vitro, with decreased sensitivity to amprenavir, saquinavir, ritonavir, nelfinavir, lopinavir, and atazanavir . No information is available for darunavir, which has not been tested. However, we have recently described an association between baseline A431V and virological response with a darunavir-containing regimen in HIV-1 protease inhibitor-experienced patients . These results might be because the protease inhibitor regimens are different. Mutated NC/p1 cleavage site, when it harbors A431V, should be, in vitro, a better substrate for the mutated protease. It should increase the specificity for protease  and result in increased Gag precursor cleavage [30,37,42,55], thus, allowing partial recovery of viral replicative capacity . This mutation could create additional contacts with protease  and increase Van der Waals forces [37,40].
In HIV-2 p1/p6pol gag–pol cleavage site, position 435 in HIV-2 group A corresponds to position 436 in HIV-1, which has not been associated with virological outcome in HIV-1.
In HIV-2 p1/p6gag gag cleavage site, position 447 corresponds to position 450 in HIV-1. Even though position 450 in HIV-1 is rarely mutated, this residue interacts, via hydrogen bonds, with the residue at position 30 of the protease-coding region , described to be associated with resistance to nelfinavir. Mutations in p1/p6gag cleavage site might increase the replicative capacity of viruses with a mutated protease [28,43].
A limitation of our study is that the bulk-sequencing technique only allowed us to detect polymorphisms that were present in more than 20–25% of the viral population, thus, minor sequence variants remained undetected. Moreover, population sequencing prevented us from ascertaining whether the resistance mutations were linked. Further clonal analyses might be helpful to determine whether these different mutations are harbored by the same viral genome.
In our study, we found no association between baseline free energy and virological outcome with a protease inhibitor-containing regimen. Concerning HIV-1, it has been shown that a decrease in free energy in vitro was correlated with poor efficiency in changing gag–pol ORF . An absence of HIV-1 viral replication was observed for reductions in free energy between 35 and 60% [57,58]. Furthermore, Knops et al. reported a significant increase in frameshift efficiency when HIV-1 L449F (p1/p6gag) and I437V (p7/p1) were associated with gag polymorphism. A decrease in free energy of the RNA secondary structure of the gag–pol frameshift signal, which induced signal instability, and thus a reduction in enzyme production could be thwarted by an increased affinity of substrates for protease. In our study and others, no association was found between free energy and virological outcome in HIV-1-infected patients receiving a protease inhibitor-containing regimen [49–51].
In conclusion, we have demonstrated, for the first time, in vivo, an impact of baseline gag mutations on virological outcome in HIV-2-infected patients, receiving a LPV-containing regimen. The mutations between protease inhibitor-naive and protease inhibitor-experienced patients were distinct, suggesting different evolutionary pathways according to the protease genotypic background. Further investigations are needed to clarify the clinical relevance of such mutations in a large cohort of HIV-2-infected patients receiving different protease inhibitor regimens.
We thank Audrey Lagès for her technical skills.
Authors contributions: F.D., F.B.V., S.M., G.C., and D.D. contributed to the study's concept. L.L. performed genotypic tests. A.V. and A.B. contributed to the statistical analyses. C.C., L.L., F.D., and D.D. contributed to the analyses and interpretation of data. L.L., C.C., F.D., F.B.V., B.V., and D.D. contributed to writing the article. All authors contributed to critically reviewing the article.
Members of the ANRS 127 study group, ANRS, Paris, France.
Scientific committee – P. Yeni, R. Landman, F. Brun-Vezinet, D. Descamps, G. Peytavin, M. Bentata, C. Piketty, J.P. Aboulker, C. Capitant, C. Chazallon, M.J. Commoy, Y. Bennai, B. Hadacek, D. Merah.
Participating clinical departments – Groupe Hospitalier Bichat, Paris (R. Landman, G. Fraqueiro); Groupe Hospitalier Pitié-Salpétrière, Paris (C. Katlama, M. Pauchard); Hôpital Tenon, Paris (G. Pialoux, C. Fontaine); Centre Hospitalier de la Région Annecienne, Annecy (C. Michon, M. Bensalem); Centre hospitalier départemental, La Roche sur Yon (P. Perre, I. Suaud); Hôpital Saint-Louis, Paris (J.M. Molina, A. Rachline); Hôpital de Bicêtre, Le Kremlin-Bicêtre (C. Goujard, M. Môle); Hôpital Pontchaillou – CHRU, Rennes (C. Arvieux, M. Ratajczak); Hôpital Avicenne, Bobigny (M. Bentata, F. Touam); Hôpital Saint-Antoine, Paris (P.M. Girard, J.L. Lagneau); CHU Hôtel Dieu, Nantes (F. Raffi, H. Hue); Hôpital Foch, Suresnes (D. Zucman); Hôpital Raymond Poincaré-Vidal, Garches (P. de Truchis, H. Berthe); Hôpital Henri Mondor, Créteil (Y. Levy, C. Jung); Hôpital Européen Georges Pompidou, Paris (C. Piketty); CHU Hôpital Gui de Chauliac, Montpellier (J. Reynes, M. Vidal); Hôpital Saint-Louis, Paris (D. Sereni); Hôpital Louis Mourier, Colombes (M. Bloch).
Data and Safety Monitoring Board: M.L. Chaix, P. Flandre, Y. Yazdanpanah.
Coordinating Centre: INSERM SC10, Villejuif J.P. Aboulker, C. Capitant, N. Leturque, E. Netzer, V. Foubert, C. Chazallon (statistics), S. Izard, A. Arulananthan (data management).
This work was presented at the International Workshop on HIV&Hepatitis Virus Drug Resistance And Curative Strategies, 7–11 June 2011, Los Cabos, Mexico (abstract no. 5).
This research received funding from the Agence Nationale de Recherches sur le SIDA et les Hépatites virales (ANRS), the European Aids Treatment Network (NEAT, WP6) (grant 037570), and the European Community's Seventh Framework Programme (FP7/2007–2013) under the project ‘Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)’ (grant 223131).
Conflicts of interest
There are no conflicts of interest.
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