HIV-2, the second causative agent of AIDS, has been classified into seven phylogenetic subtypes [1–3] identified mainly in West Africa . Subtype A is prevalent in the majority of countries , whereas a predominance of subtype B was suggested in Ivory Coast . For the remaining subtypes, C–G, only single viral strains have been reported. The spread of HIV-2 to Europe, Asia, and the Americas occurs slowly but continually. Population migration, international travel, historical socio-economic connections between European countries and former colonies, and expatriates returning to countries after living and working in HIV-2-endemic regions are considered responsible for spreading HIV-2 worldwide [4,6–8]. The majority of HIV-2 infections in Europe and North America have been identified among African immigrants or nationals having sex with people who have lived in West Africa [4,9–14]. A noteworthy exception is Portugal, where more than 60% of new HIV-2 infections were not linked directly to African contacts , indicating that HIV-2 is spreading among the indigenous population . Since Portugal has a high number of HIV-2 infections, up to 4.5% of this country's AIDS cases, Portugal might serve as a source for this infection in Europe [9,16]. Likewise, the spread of HIV-2 in India is steadily increasing, especially among injecting drug users . The simultaneous presence of HIV-1 and HIV-2 in certain regions leads to mixed HIV-1/HIV-2 infections [18–20].
The clinical management of HIV-1 infections has improved significantly with combinations of antiretroviral (ARV) drugs, including protease inhibitors (PI) . In contrast, there is no proven ARV drug therapy for HIV-2. HIV-2 isolates are resistant to at least some non-nucleoside reverse transcriptase inhibitors . Recently, HIV-1 PI have been used to treat HIV-2-infected persons [23–25] based on the assumption that these drugs are equally active on HIV-2 protease (PR). However, little is know about mutations associated with PI resistance in HIV-2 and whether they are similar to those seen in HIV-1. In this report, we present the first large-scale description and genetic characterization of HIV-2 PR sequences of two major subtypes, A and B, isolated from ARV-naive individuals as a baseline for comparison when following HIV-2-infected persons after drug treatments.
Materials and methods
From 1986 to1999, peripheral blood mononuclear cells (PBMC) were collected from 76 HIV-2-infected drug-naive individuals living in Africa (n = 65; Ivory Coast, 50; Nigeria, 13; Guinea Bissau, two), Asia (Lebanon, one), Europe (Portugal, seven), and the Americas (Brazil, two; USA, one). All specimens were a part of HIV-2 genetic variant studies approved by the Institutional Review Board [5,7,26]. Seventy-four percent of the specimens harbored only HIV-2, and 26% were from HIV-1/HIV-2 dual infections.
Amplification, sequencing, and phylogenetic analysis
HIV-2 DNA was extracted and used for nested PCR amplification of PR with outside DP20-forward and DP21-reverse and nested DP26a-forward and DP27-reverse primers . To minimize false-positive PCR results, disposable laboratory gowns and gloves were used, pre- and post-PCR materials were processed in separate rooms, and UV irradiation was used to treat PCR chemicals and tubes. Negative controls consisting of uninfected viral DNA and water were used in each batch of PCR. When investigated samples were closely related (< 3%), they were reevaluated using additional vials of patient PBMC. PCR products were directly sequenced using DP26a-forward and DP27-reverse-nested PCR primers and resolved on an automated DNA sequencer ABI model 377 (Applied Biosystems, Foster City, California, USA). The PR sequences were aligned by the CLUSTALW multiple sequence alignment program and used for phylogenetic analysis . HIV-1MN (M17449) was used as outgroup. GenBank accession numbers of sequences presented in this study are: L08459, L08461, L08462, L08465, L08466, AF026 912; AF073844-73874; AY102844-2856; AY343991-344017.
Amino acid analysis
The aligned DNA sequences were translated to amino acids using the Genetic Data Environment package . The first and the last seven amino acids of PR genes are parts of the 5′ and 3′ sequence primers, respectively, and were excluded from analysis. Conservative and non-conservative substitutions were estimated using the Meyers and Miller model , which identified the nine amino acid groups: A, S, T, C; D, E; N, Q; R, K; I, L, M, V; F, Y, W; G; P; and H. Information on major and minor mutations associated with HIV-1 PI resistance was obtained from the latest literature .
Phylogenetic analysis of PR genes indicated that 52 (68%) of the 76 sequences were of subtype A viruses and 24 (32%) were of subtype B (Fig. 1). Of subtype A, 56% (29/52) were from Ivory Coast, 21% (11/52) were from Nigeria, 4% (2/52) were from each of Guinea Bissau and Brazil, 13% (7/52) were from Portugal, and 2% (1/52) were from the USA. Of subtype B, 88% (21/24) were from Ivory Coast, 8% (2/24) were from Nigeria, and 4% (1/24) were from Lebanon.
Genetic distance analysis revealed that some PR sequences of subtype A were closely related. Two specimens IC310059 and IC310060 collected in 1988 from patients living in Ivory Coast showed similarity of 99.7%  and were closely (98%) related to specimen US-NJ (HIV-1888) collected in 1989 in New Jersey from an African immigrant. Information on relationships between patients harboring these strains and their sexual partners was not available. Similarly, 11 strains collected in 1998 from unrelated individuals living in 10 Nigerian states show close relationships (range, 97.2%–99.6%). These findings are unlikely to be result of contamination in the laboratory because similar data were obtained when independent PCR amplifications were done on different days using the original and the second vial of patient PBMC. Also, contamination during blood collection was rather unlikely because a disposable Vacutainer system was used to obtain each blood sample. Interestingly, PR gene sequences IC310059 and IC310060 revealed 100% relationships with ROD strain found in Senegal in 1985 whereas our Nigerian sequences formed a strong (98%) monophyletic cluster with the reference sequence of NIHZ strain (data not shown) identified in 1986 in Guinea Bissau. These results indicated that the observed close relationships between our PR sequences and the reference sequences, which were retrieved from GenBank and used as references of subtype A in phylogenetic analysis, may reflect a common origin. As changes in the conserved PR gene are very small over time (< 1% over 4 years in HIV-1; unpublished data), this viral region may be useful to study the origin, evolution, and dissemination of HIV subtype worldwide as we previously suggested .
To compare amino acid genetic diversity between HIV-2 strains, we aligned HIV-2 PR sequences of subtypes A and B with subtype A reference HIV-2ROD (Fig. 2). This analysis showed minimal amino acid variation within domains of PR that are functionally important in HIV-1, such as the enzyme active region (residues 21–33), the top of the flap (residues 47–56), and the second loop of the β sheet (residues 78–88) . In contrast, there was a broad diversity in other areas of the PR enzyme. Overall, 54% (54/99) of amino acid codons had at least one change in this HIV-2 sampling. A higher variation (43%) was observed for subtype A than subtype B (32%) strains, but this difference was not significant (P = 0.42). Also, PR subtype A differed from subtype B at five amino acid positions (12, 43, 67, 91, and 92) that most likely reflect HIV-2 PR subtype signature patterns similarly to patterns of HIV-1 PR subtype B and non-B viruses .
To evaluate differences between PR of HIV-2 and HIV-1, our HIV-2 and HIV-1  consensus amino acid sequences were aligned (Fig. 3). This analysis showed dissimilarities at 46 positions, counting 35 conservative and 65 non-conservative substitutions. Of these 46 changes, 36 (78%) occurred in HIV-2 at positions covering the variable domains of HIV-1 PR. The remaining 10 (22%) changes were located at positions that overlap the functionally important HIV-1 PR regions: codons 22, 31-33 at the active domain; codons 47, 55, and 56 at the top of the flap; and codons 79, 82, and 85 at the second loop of the β sheet. Overall, this analysis revealed > 45% differences at the protein level between HIV-2 and HIV-1, data similar to results in other reports .
We examined HIV-2 sequences for the presence of major (D30N, M46I, G48V, I50V, V82A/F/S/T/V, I84V, and L90M) and minor (L10I/V/F/R, K20R/M, L24I, V32I, L33F, M36I, M46L, I47V, I54V, L63P, A71V/T, G73S, V77I, and N88D/S) substitutions, which are associated with PI resistance in HIV-1 subtype B to saquinavir, ritonavir, indinavir, nelfinavir, and amprenavir . This analysis revealed such substitutions at eight positions of HIV-2, 10←V/I, 30←N, 32←I, 36←I, 46←I, 47←V, 71←V, and 77←I (Fig. 2). Two major substitutions, 30N and 46I, were identified in this sampling but their frequency differed. While 30N was present in only one strain (1.3%, 1/76) of HIV-2 subtype B, substitution 46I was in the majority (89%, 68/76) of strains, including 83% (43/52) of subtype A and 100% of subtype B (Fig. 4).
The frequency of minor mutations at codons 10, 32, 36, 47, and 71 was 100% for substitutions 10V/I, 32I, and 47V; 99% (75/76) for 36I; and 89% (68/76) for 71V (Fig. 4). In contrast, 77I substitution was in only one HIV-2 of subtype A from Ivory Coast. Moreover, at least 96% of HIV-2 strains harbored substitutions not seen in HIV-1 at PI-conferring resistance codons such as 20←V, 33←V, 63←E, 73←A, V77←T, and 82←I (Fig. 2). Overall, this analysis indicated that 12 of the 20 codons associated with PI resistance in HIV-1 were occupied in HIV-2 by PI major and minor mutations as well as substitutions not observed in HIV-1.
The majority of HIV-2 strains (89%, 68/76) harbored five multiple PI resistance-associated mutation patterns comprising both the major 46I and different minor mutations. The most prevalent pattern was 10V/I, 32I, 36I, 46I, 47V, 71V (76%, 58/76), followed by 10V, 32I, 36I, 46I, 47V (9%, 7/76) and by 10V, 32I, 36I, 46I, 47V, 71V, 77I; 10V, 32I, 46I, 47V, 71V; and 10V, 30N, 32I, 36I, 46I, 47V, 71V (1.3%, 1/76, for each). The remaining 11% (8/76) strains had patterns comprising minor mutations only: 10V, 32I, 36I, 47V, 71V (9%, 7/76) and 10V, 32I, 36I, 47V (1.3%). Taken together, this analysis documented that all 76 HIV-2 strains carried numerous mutations associated with PI resistance in HIV-1 that range from four to eight substitutions per strain and that 46I substitutions, a major resistance mutation in HIV-1, was present in 89% of HIV-2 sequences.
As our data showed that HIV-2 PR sequences harbored different PI-multiple mutational patterns, we further analyzed their distributions according to country of specimen collection. We found that HIV-2 strains from different geographic regions harbored similar multiple-mutation patterns that are associated with resistance to PI in HIV-1. For example, the most common pattern comprising major (46I) and five minor mutations (10V/I, 32I, 36I, 47V, and 71V) was found in 38 of 50 HIV-2 from Ivory Coast, in all 13 Nigerian specimens, in one of the two samples from Guinea Bissau, and in six of 11 samples collected outside Africa including one strain from the USA, one of two strains from Brazil, and four of seven strains from Portugal. Likewise, less frequent patterns with or without 46I major mutation were in HIV-2 from Ivory Coast, Guinea Bissau, Lebanon, Portugal, and Brazil.
This study provides the most comprehensive data to date on genetic diversity of HIV-2 PR genes among HIV-2-infected individuals in Ivory Coast, Nigeria, Guinea Bissau, Lebanon, Portugal, Brazil, and the USA. Our data revealed a similar range of amino acid variation within PR genes for both HIV-2 subtype A and B regardless of geographic region and confirmed differences in more than 45% of amino acid codons when compared with HIV-1 PR. We also showed that amino acid changes in HIV-2 were located at positions that overlap the functionally important domains in HIV-1 PR; such substitutions may be critical when designing PI for HIV-1 subtype B. It seems that previously reported differences between HIV-1 and HIV-2 PR enzymes with respect to substrate specificity and affinity to pseudopeptidil inhibitors  could be due to dissimilarities at the protein level as presented here. Furthermore, 89% of all HIV-2 strains harbored both the major and at least four minor mutations associated with PI resistance in HIV-1, while the remaining 11% had multiple minor mutations only. The high frequency of PI-associated substitutions indicates natural polymorphisms occurring in PI-naive individuals infected with HIV-2 subtypes A and B.
Comparative analysis of amino acids at 20 codons, which are linked to PI resistance in HIV-1, with those positions in HIV-2 PR sequences, provided several observations. First, the major PI resistance substitution 46I, which is associated with indinavir resistance in HIV-1, was identified in 89% of the HIV-2 sequences, whereas is rarely (< 1%) observed in global drug-naive collections of HIV-1 ([32,33]; http://hivdb.stanford.edu). Second, PI minor substitutions 10V or 10I, 32I, 47V, and 71V, which contribute to indinavir, ritonavir, saquinavir, nelfinavir or amprenavir resistance, were present either in all or in the majority (> 85%) of HIV-2 sequences, whereas these mutations are infrequent in HIV-1. Only the frequency of substitution 36I, which is associated with ritonavir and nelfinavir resistance, was similar in HIV-2 (99%) and HIV-1 non-B (83%) strains. Third, all HIV-2 PR sequences carried multiple PI resistance-associated mutations, of which 89% of strains had also the major 46I substitution that predominantly occurred in combination with five minor mutations (10V/I, 32I, 36I, 47V, 71V). In contrast, the majority (85%) of HIV-1 strains harbored only a single minor substitution (e.g., 36I, 63P or 77I) followed by about 25% dual or triple mutations (e.g., 63P, 77I or 36I, 63P or 10V/I, 36I, 63P) . Recently published data indicate that as few as four PI mutations can be associated with a high-level resistance for HIV-1 .
Genetic characterization of viral PR sequences pointed out the distinctions in amino acid compositions between HIV-1 and HIV-2 and raises the question of whether PI, which were developed for treatment of HIV-1 subtype B infections through the specific inhibition of PR activity of these viruses , would also be effective in the treatment of HIV-2-infected individuals. A recent in vitro phenotypic study (; http://www.mediscover.net) showed that PI indinavir, ritonavir, and saquinavir inhibited equally HIV-2 subtype A (ROD) and subtype B (ECHO) reference strains and HIV-1 wild-type, although both HIV-2 strains harbor the major 46I mutation and four minor substitutions (10V, 32I, 36I, 71V) conferring HIV-1 resistance . Thus, these results suggest that there are other compensatory mutations in HIV-2 ROD and ECHO that make the PR enzyme susceptible to PI despite the presence of HIV-1 PI mutations. In contrast, susceptibility to nelfinavir was reduced by 3.0-fold and susceptibility to amprenavir was reduced by 4.3 and 9.0-fold for ROD and ECHO strains, respectively. These reductions were not caused as in HIV-1 by the major mutations 30N, 90M, 50V or 84V, which are absent in both HIV-2 strains. It is not clear, however, if multiple minor mutations (10V, 32V, 36I, 46I, 71V) in these HIV-2 strains caused resistance to nelfinavir and amprenavir. Therefore, these data indicate that substitutions associated with resistance to PI in HIV-2 might differ from those in HIV-1.
The data on antiretroviral treatment of HIV-2 infections in humans are very limited and often difficult to interpret, mainly because of variations in protocols used in clinical approaches, differences in timing of therapy initiation (patients with different stages of disease ranging from asymptomatic to AIDS), and limitations in diagnostic and clinical markers monitoring the response to drug therapies [23–25]. Also, interpretation of clinical results might be obscured by treatment of patients infected with both HIV-1 and HIV-2 strains, whose numbers have increased in West Africa and India [38,39]. For example, some patients failed to respond to ARV therapies, demonstrated by no decrease in viral loads during highly active ARV treatment including the PI nelfinavir  or by development of resistance to the reverse transcriptase inhibitor lamuvidine and the PI saquinavir . In contrast, other studies have shown a fall in viral load and an increase in CD4 cell counts after regimens with nelfinavir  and indinavir [24,40]. Long-term evaluation of the impact of HIV-1 PI on progression of disease in HIV-2-infected patients needs to be conducted in closely monitored clinical trials.
Data on genotypic characterization of PI resistance mutations in HIV-2 PR sequences isolated from PI-treated patients are also limited. Analyses of four patients treated for at least 6 months with ARV showed that three individuals treated with ritonavir or indinavir had the same substitutions at 20 codons within PR sequences as in reference drug-naive subtype A ROD, NIHZ, and ISY strains . In the remaining one patient treated with indinavir, nelfinavir, and saquinavir different substitutions were at positions 54 (M) and 82 (F) of PR, but it is unclear whether such changes occurred during treatment or if they were originally present in the HIV-2 strain of this patient. As it is uncertain if amino acid mutations associated with PI resistance in HIV-2 are similar to those seen in HIV-1, evaluation of genetic diversity of PR sequences from drug-treated HIV-2-infected patients should include all amino acid positions and not only those at 20 codons. The HIV-2 database for drug-naive subtype A and subtype B PR sequences reported here may be useful in pre-selection of HIV-2 PI resistance mutations in PI-treated patients.
In summary, our data provide new information on HIV-2 genetic variation associated with PI resistance in HIV-1. The identification of multiple PI-associated substitutions in all HIV-2 subtype A and subtype B strains documents differences between HIV-2 and HIV-1 PR genes. These data may have important implications for the choice of ARV therapy. Our HIV-2 PR sequence database from drug-naive persons may be useful in phenotypic and clinical studies for determining pathways for the development of PI resistance during PI treatment.
The authors gratefully acknowledge K. Distel for excellent editorial assistance.
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