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. 2002 Mar;40(3):837–845. doi: 10.1128/JCM.40.3.837-845.2002

Human Immunodeficiency Virus Type 1 Group M Protease in Cameroon: Genetic Diversity and Protease Inhibitor Mutational Features

Peter N Fonjungo 1, Eitel N Mpoudi 2, Judith N Torimiro 2, George A Alemnji 2, Laura T Eno 2, Esther J Lyonga 2, John N Nkengasong 3,4, Renu B Lal 5, Mark Rayfield 1, Marcia L Kalish 1, Thomas M Folks 1, Danuta Pieniazek 1,*
PMCID: PMC120267  PMID: 11880402

Abstract

To establish a baseline for monitoring resistance to protease inhibitors (PIs) and examining the efficacy of their use among persons in Cameroon infected with human immunodeficiency virus type 1 (HIV-1), we analyzed genetic variability and PI resistance-associated substitutions in PCR-amplified protease (PR) sequences in strains isolated from 110 HIV-1-infected, drug-naïve Cameroonians. Of the 110 strains, 85 were classified into six HIV-1 PR subtypes, A (n = 1), B (n = 1), F (n = 4), G (n = 7), H (n = 1), and J (n = 7), and a circulating recombinant form, CRF02-AG (n = 64). PR genes from the remaining 25 (23%) specimens were unclassifiable, whereas 2% (7 of 301) unclassifiable PR sequences were reported for a global collection. Two major PI resistance-associated mutations, 20M and 24I, were detected in strains from only two specimens, whereas secondary mutations were found in strains from all samples except one strain of subtype B and two strains of CRF02-AG. The secondary mutations showed the typical PI resistance-associated pattern for non-subtype B viruses in both classifiable and unclassifiable PR genes, with 36I being the predominant (99%) mutation, followed by 63P (18%), 20R (15%), 77I (13%), and 10I or 10V (11%). Of these mutations, dual and triple PI resistance-associated substitutions were found in 38% of all the Cameroonian strains. Compared with classifiable PR sequences, unclassifiable sequences had significantly more dual and triple substitutions (64% versus 30%; P = 0.004). Phenotypic and clinical evaluations are needed to estimate whether PI resistance during antiretroviral drug treatment occurs more rapidly in individuals infected with HIV-1 strains harboring multiple PI resistance-associated substitutions. This information may be important for determination of appropriate drug therapies for HIV-1-infected persons in Cameroon, where more than one-third of HIV-1 strains were found to carry dual and triple minor PI resistance-associated mutations.

Of the estimated 36 million persons infected with human immunodeficiency virus (HIV) type 1 (HIV-1) worldwide, more than two-thirds reside in sub-Saharan Africa, although the population of this region makes up only 10% of the world's population. While an effective vaccine remains the best long-term strategy to control the AIDS pandemic, a combination of antiretroviral drugs, including protease inhibitors (PIs) that target the viral protease (PR), provides significant clinical benefit for HIV-1-infected persons (21).

Genetic characterization and phylogenetic analysis of the HIV-1 strains infecting persons throughout the world have shown that HIV-1 can be divided into three distinctive genetic groups, designated M (major), N (new or non-M, non-O), and O (outlier). Group M viruses comprise nine “pure” subtypes (subtypes A, B, C, D, F, G, H, J, and K) and at least six recombinant circulating forms (CRFs), which are primarily responsible for the global HIV-1 infection and AIDS epidemic (16, 24, 29). Most HIV-1 strains infecting persons in the Americas, Europe, and Australia belong to subtype B, whereas the majority of the remaining viral subtypes are found among persons in sub-Saharan Africa. This diversity within HIV may present a challenge for the global management of HIV infections since the available antiretroviral drugs were developed and primarily tested by using subtype B isolates. Recent data indicate that viral subtype may influence the effectiveness of antiretroviral treatment. For example, HIV-1 group O and HIV-2 are naturally resistant to nonnucleoside reverse transcriptase inhibitors (4, 23, 27). Also, some subtype F strains of HIV-1 group M are less susceptible to nonnucleoside reverse transcriptase inhibitors (2).

Previous data have documented significantly more naturally occurring PI resistance-associated minor (secondary) substitutions that contribute to drug resistance in HIV-1 non-subtype B strains than in subtype B strains collected from patients who have never received PIs (26). Moreover, the patterns of these substitutions were found to be different in subtype B strains compared to those in strains of subtypes A, C, D, F, G, and H and CRF01-AE. In addition, some differences in the frequencies of substitutions associated with resistance have been observed between HIV-1 non-subtype B strains from West and East Africa (26). It has been postulated that preexisting minor mutations could reduce the effectiveness of PI treatment through a more rapid progression to a resistant phenotype (8, 13, 34). Recent in vitro analyses of the basic biochemical properties of the HIV-1 PR revealed that in the presence of PIs such as indinavir, saquinavir, ritonavir, and nelfinavir, PRs from subtypes A and C consistently performed their catalytic functions better than PRs from subtype B (35). These results point to a greater biochemical fitness of the subtype A and C PRs in the presence of existing inhibitors.

However, the few in vitro studies reporting the susceptibilities of clinical HIV-1 isolates of different subtypes to PIs have shown either a lack of or only small differences in susceptibilities between subtypes (5, 22, 30, 39). Nevertheless, preliminary clinical data suggest that the secondary mutations 36I (the most predominant substitution in non-subtype B strains) and 10I or 10V are associated with faster decreases in drug sensitivity during treatment (C. F. Perno, A. D'Arminio-Monforte, A. Cozzi-Lepri, C. Balotta, F. Forbici, A. Bertoli, P. Pezzotti, G. Facchi, L. Monno, G. Angarano, P. Bottura, V. Vullo, A. Cargnel, M. Capobianchi, G. Ipollito, and M. Moroni, Abstr. 7th Conf. Retrovir. Opportunistic Infect., abstr. 728, 2000). Overall, these data indicate that naturally occurring PI resistance-associated mutations may differ globally because of the viral PR genetic background. Thus, drug therapies for HIV-1-infected persons may need to be adjusted to local viral PR gene profiles.

With the recent introduction of antiretroviral drugs in developing countries, including Africa (1, 39; www.nature.com/nm/biomedical_news/west.htm), there is a need to establish baselines for the PR sequences of non-subtype B HIV-1 strains from PI-naive individuals in different areas. In this report, we describe the genetic diversity of HIV-1 PR sequences and the presence of naturally occurring mutations linked to PI resistance in treatment-naïve, HIV-1-infected persons living in Cameroon, where all recognized HIV-1 group M subtypes and at least two CRFs, CRF01-AE and CRF02-AG, as well as group N and O viruses are present.

MATERIALS AND METHODS

Study population and sample collection.

Between September and November 1998, plasma specimens were collected from 110 HIV-1-positive individuals who consecutively attended the Dermatological/AIDS/STD clinic in the military hospital in Yaoundé, Cameroon (Hôpital Militaire de Yaoundé). None of these individuals had received any antiretroviral treatment. Antibodies to HIV were detected by enzyme-linked immunosorbent assay with HIV-1 and HIV-2 enzyme immunoassay kits (Murex or Genetic Systems Corporation, Redmond, Wash.), both of which are based on whole-virus lysate. All individuals provided informed consent. Blood samples were taken, and plasma was stored at −20°C.

RNA extraction, PCR, sequencing, and genetic analyses.

Total viral RNA was extracted from 200 μl of plasma with a QIAamp viral RNA kit (Qiagen, Valencia, Calif.), and nested PCR amplifications of the PR gene were performed with outside primers DP10-forward and DP11-reverse and inside primers DP16-forward and DP17-reverse as described previously (14). After purification with a PCR purification kit (Qiagen), PCR products were directly sequenced with both forward-DP16 and reverse-DP17 nested PCR primers and were resolved on an automated DNA sequencer (ABI model 377; Applied Biosystems, Foster City, Calif.). The derived nucleotide sequences of the PR region were aligned by use of the CLUSTALW multiple-sequence alignment program with both our laboratory markers and reference strains of groups M, N, and O pooled from the HIV-1 GenBank (http://hiv-web.lanl.gov/MAP/hivmap.html). Phylogenetic analysis was performed by the neighbor-joining method, with the nucleotide distance calculated by Kimura's two-parameter approach, included in the PHYLIP package (version 3.5c) (9), with and without bootstrapping. An HIV-2 Rod sequence was used as an outgroup. To avoid the influences of other sequences on the bootstrap value, subtype assignments were performed separately for each sequence. The stability of the tree topology was tested by pruning, which consists of removing one sequence from an alignment and rerunning the phylogenetic analysis. For recombination analysis of DNA sequences, SimPlot software was used (S. Ray, SimPlot for Windows, version 2.5, http://www.med.jhu.edu/deptmed/sray/download, 1999). This program calculates and plots the percent identities of the query sequence with reference sequences from group M viruses in a sliding-window manner along the alignment with the optimal step size.

Amino acid analysis.

The aligned DNA sequences were translated to amino acids by use of the Genetic Data Environment package (31). The parts of the amino acid sequences covering the nested primer regions (amino acids 1 to 7 and 93 to 99) were removed. Conservative and nonconservative substitutions were estimated by use of the model of Myers and Miller (18), which identified the following nonconservative amino acid groups: A, S, T, and C; D and E; N and Q; R and K; I, L, M, and V; F, Y, and W; G; P; and H. Information on major and accessory mutations associated with PI resistance was obtained from the literature.

Nucleotide sequence accession numbers.

The sequences presented in this report have been deposited in GenBank under accession numbers AY051198 to AY051280, AF252132, and AF252133 for PRs from strains that could be classified into subtypes and AF406707 to AF406731 for PRs from unclassifiable strains.

RESULTS

Phylogenetic subtypes and genetic diversity of DNA sequences.

For analysis of phylogenetic subtypes and the genetic diversity of the DNA sequences, viral subtypes were assigned by phylogenetic analysis of the PR gene. Our previous studies indicated that genetic variation within the 297-bp PR genes, which were sequenced from samples collected worldwide, allowed clear classification of HIV-1 strains into group M subtype A, B, C, D, F, G, H, J, and K viruses as well as group O viruses, similar to the subtype assignments based on gag or env sequences from nonrecombinant viruses (7, 10, 15, 20, 25, 28, 38, 40). Direct analysis of the PR sequence eliminates the confusion that arises from discordant classification of viruses based on other gene regions, which may occur as a result of recombination. Phylogenetic analysis of the PCR-amplified PR sequences showed that 85 of the 110 sequences could be classified unambiguously into distinct HIV-1 group M subtypes (Fig. 1a). Of these 85 sequences, 1 was subsubtype A2, 1 was subtype B, 4 were subsubtype F2, 7 were subtype G, 1 was subtype H, 7 were subtype J, and 64 were CRF02-AG.

FIG. 1.

FIG. 1.

FIG. 1.

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Phylogenetic relationships among Cameroonian HIV-1 group M PR sequences: classifiable (a) and unclassifiable (b). The reference strains are preceded by their subtype or CRF identity (in boldface), and sequences reported here are preceded by the prefix 98CM, which indicates the year and the country (Cameroon) of specimen collection. The numbers at the nodes indicate the percentages of the bootstrap value. Asterisks indicate nodes with sequences that decreased the bootstrap value when the sequences were run together, from the range to 76 to 98% to 19% for CRF02-AG (∗) and from the range of 69 to 73% to 52% for subtype G (∗∗). The scale bar indicates an evolutionary distance of 0.010 and 0.1 nucleotide per site for the sequences in panels a and b, respectively.

The remaining 25 sequences did not cluster with any known HIV-1 group M pure subtypes or CRFs or with group N or O viruses (Fig. 1b). For description purposes, this group of unclassifiable PR sequences was further categorized. Sequences showing a 99% relationship (CM220, CM153, CM178, and CM104) were called U-1. The cluster comprising CM201, CM215, CM235, CM216, CM117, CM93, CM133, and CM225, which was supported by a bootstrap value 58%, was called U-2; and the group of sequences comprising CM234, CM132, CM175, CM209, CM11, CM221, CM45, CM238, CM230, and CM231, which had a bootstrap value of <50%, was named U-3. The remaining three unrelated sequences were categorized as U-3a (CM184) and U-other (CM265 and CM113). An evaluation of the stability of the tree by pruning analysis revealed that the U-3 and U-3a sequences decreased the bootstrap value of the cluster consisting of subtype B and subtype D reference strains from 88 to 53%. These results suggested the presence of common elements in the U-3 and U3a sequences and viruses of the B and D subtypes. In contrast, evaluation of the remaining unclassifiable sequences did not affect the bootstrap values assigned to any viral subtype.

Sliding-window bootscan analysis of unclassifiable sequences showed clear recombinant patterns in some PR sequences of group U-1, with the subtype G sequence at the 5′ end and the subtype J sequence at the 3′ end (Fig. 2). The remaining unclassifiable sequences revealed nucleotide changes across the PR gene. However, it is unclear if these changes in the PR gene sequence resulted from recombination or the accumulation of point mutations.

FIG. 2.

FIG. 2.

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SimPlot analysis of Cameroonian unclassifiable PR sequence 98CM153 showing the recombination between subtypes G and J. The bootscan analysis was performed against reference strains from clades A (strain SE7253), B (strain HXB2), C (strain ET2220), D (strain NDK), F1 (strain MP411), F2 (strain MP257), G (strain NG083), H (strain V1991), J (strain SE7022), and K (strain EQTB11C).

Variability of amino acid sequences.

Since a large number (25 of 110 [23%]) of DNA sequences were phylogenetically unclassifiable, we next examined their impacts on both the variable and the conserved regions of the PR amino acid sequence by comparison with the PR amino acid sequences of classifiable strains (Fig. 3). The amino acid sequence of each strain was aligned with a PR subtype B consensus sequence. Also, for the purposes of this analysis, a non-subtype B consensus sequence, which has been established by the use of 187 global PR sequences of subtypes A, C, D, A/E, F, G, and H (26), was included. This analysis revealed minimal amino acid diversity within essential regions of the PR, including the sequence around the active site (amino acids 21 to 33), the top of the flap (amino acids 47 to 56), and the second loop of the β sheet (amino acids 78 to 88), among both unclassifiable and classifiable viruses. In contrast, variability was present in the regions outside of the functional areas of the enzyme. The combined variation at the amino acid level was 37% (37 of 99 amino acid positions showed at least one change) for the sequences of all viruses from Cameroonians, including 35% variability for strains to which a subtype was assigned and 26% for unclassifiable strains, but this difference was not significant. These changes involved similar ranges of conservative and nonconservative substitutions for strains with classifiable sequences (59 and 41%, respectively) and strains with unclassifiable sequences (62 and 42%, respectively). Overall, this analysis revealed similar ranges of diversities within conservative and variable regions of PR genes for both classifiable and unclassifiable strains, suggesting that these PRs have similar biological properties.

FIG. 3.

FIG. 3.

FIG. 3.

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Amino acid polymorphisms of HIV-1 PR sequences isolated from PI-naïve Cameroonian patients infected with classifiable HIV-1 subtype or subsubtype A2, B, F2, G, H, or J or CRF02-AG and unclassifiable viruses (U-1, U-2, U-3 or U-3a, or U-other). Below each amino acid position, amino acids are listed with the number of strains that possessed the particular substitution. When the number does not total the number of sequences analyzed, the difference reflects the consensus subtype B amino acid sequence. Dots indicate homology; downward-pointing arrows indicate positions associated with drug resistance. The functional domains of PR are shaded.

Furthermore, we compared variations at each amino acid position of unclassifiable and classifiable sequences of Cameroonian strains (Fig. 3). The amino acid substitutions in unclassifiable sequences of groups U-2, U-3, U-3a, and U-other were similar to those in classifiable sequences. These findings indicate that nucleotide changes did not cause alterations at the amino acid level. In contrast, unclassifiable U-1 sequences showed differences at four positions: 17G→E, 34E→K, 61Q→N, and 62I→M. Such substitutions either were not represented (34K) or rarely observed (17E, 1.6%; 62 M, 0.3%) among PR sequences from 301 strains collected worldwide or, like 61N, were found selectively in subtype F strains (26). Despite these differences, viruses of groups U-1, U-2, and U-other with unclassifiable PR sequences could clearly be sorted into non-subtype B viruses on the basis of their amino acid signature patterns, with characteristic polymorphisms at positions 13V, 36I, 41K, 69K, and 89 M (Fig. 3). In contrast, unclassifiable U-3 or U-3a sequences revealed the mixed amino acid pattern consistent with subtype B sequences at positions 13I and 89L and with non-subtype B sequences at positions 36I and 41K. In general, these data indicated a broad diversity among unclassifiable PR sequences, as indicated by phylogenetic DNA analysis and protein evaluation.

PI resistance-associated mutations.

To examine the frequency of major (20M; 24I; 30N; 48V; 50V; 82A, 82F, or 82T; 84V; and 90 M) and secondary (10I, 10V, or 10R; 20R; 32I; 33F; 36I; 46I or 46L; 63P; 71V or 71T; 73S; 77I; and 88D) PI resistance-associated substitutions within the PR region, we analyzed the sequences at these 19 amino acid sites, which are associated in vivo with HIV-1 resistance to saquinavir, ritonavir, indinavir, nelfinavir, and amprenavir in subtype B viruses (3, 12). Our data revealed the presence of such substitutions at six positions (10→I or V, 20→R or M, 24→I, 36→I, 63→P, and 77→I). Only two major substitutions, 20M and 24I, were identified in HIV-1 subtype H and CRF02-AG strains, respectively. In contrast, secondary amino acid mutations (10I or 10V, 20R, 36I, 63P, and 77I) were found in 107 of the 110 (97%) viral sequences (Fig. 3). Of these changes, 62% (68 of 110) were represented by single amino acid mutations, whereas the remaining 38% (42 of 110) harbored dual (28%) and triple (10%) PI resistance-associated mutations.

Comparative analysis of PI resistance-associated mutations between classifiable and unclassifiable PR sequences provided several findings. First, similar percentages of classifiable (96%; 82 of 85) and unclassifiable (100%; 25 of 25) sequences carried at least one accessory substitution associated with drug resistance. Second, 36I (96%; 82 of 85) was the predominant PI resistance-associated substitution among classifiable sequences, followed by 10I or 10V (13%; 11 of 85), 20R or 20M (9%; 8 of 85), 63P (8%; 7 of 85), 77I (7%; 6 of 85), and 24I (<1%; 1 of 85) (Fig. 4). The frequencies of 36I (100%; 25 of 25) and 10I (8%, 2 of 25) were similar between unclassifiable and classifiable PR sequences, while the prevalence of substitutions 20R (20%; 5 of 25), 63P (28%; 7 of 25), and 77I (20%) differed. Additionally, these three secondary mutations were not equally distributed among unclassifiable sequences, with the majority of mutations being 63P (5 of 7 strains) in group U-3 or U-3a, 77I (4 of 5 strains) in group U-1, and 20R in groups U-2 (3 of 8 strains) and U-3 or U-3a (2 of 11 strains) (Fig. 3).

FIG. 4.

FIG. 4.

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Distribution patterns of HIV-1 PR substitutions associated with PI resistance in classifiable and unclassifiable strains that were identified in Cameroonian individuals not treated with antiretroviral drugs. For comparison, the patterns of global subtype B and non-subtype B strains are included.

Although the sample size of unclassifiable sequences was not large, the frequency of PI resistance-associated substitutions at position 63P was greater in unclassifiable PR sequences than in classifiable PR sequences (P = 0.016; Fisher's exact test). In the remaining PR residues, the differences between classifiable and unclassifiable sequences were not statistically significant: for 10I or 10V, P = 0.729; for 20M or 20R, P = 0.167; and for 77I, P = 0.121. Overall, the PI mutation patterns of classifiable PR sequences of subtypes A2, F2, G, H, J, and CRF02-AG in the Cameroonian samples reflect the patterns in the global collection of non-subtype B viruses. In contrast, the unclassifiable sequences had a tendency to share the PI resistance-associated mutational patterns of both non-subtype B (36I) and subtype B (63P) viral strains.

The frequencies of dual and triple PI resistance-associated substitutions were also significantly higher among unclassifiable viruses (64%; 16 of 25) than among classifiable strains (30%; 26 of 85) (P = 0.004). The multi-PI resistance-associated patterns consisted of combinations of the observed predominant substitutions and other minor PI resistance-associated mutations. The patterns for classifiable strains were 36I and 10I or 10V (10%); 36I and 63P (8%); 10V or 10I, 20R, and 36I (6%); 10V, 36I, and 77I (3%); 36I and 29R or 10M (2%); 36I and 77I (2%); 10I, 36I, and 77I (1%); and 10V, 20R, 36I, and 77I (1%). Interestingly, triple mutation patterns were more often present in subtypes F2 (4 of 4 strains) and J (3 of 7 strains) than in CRF02-AG (2 of 64 strains). The patterns of unclassifiable strains included 36I and 63P (20%); 36I and 77I (8%); 36I and 20R (8%); 36I and 10I (4%); 36I, 63P, and 10V (4%); and 36I, 63P, and 20R (1%).

In addition, at position 20, which is the key position for substitutions K20→R or M (which are associated with drug resistance), lysine (K) was replaced with isoleucine (I) in 82% (70 of 85) of subtype-classifiable PR sequences, including 63 of 64 CRF02-AG, all 7 subtype G, and 1 of 7 subtype J sequences. In contrast, such replacement was observed in only 20% (5 of 25) of unclassifiable sequences of groups U-1 and U-other (P < 0.001). A high prevalence of the 20I substitution was previously observed in HIV-1 strains from West Africa (36), but the biological consequence of this is not known.

DISCUSSION

This study provides the most comprehensive data to date on the genetic diversity of HIV-1 PR genes among HIV-1-infected persons in Cameroon. We showed that the range of genetic diversity within the PR region of HIV-1 was significantly broader in strains from Cameroon than in those from the global collection, which included strains from Uganda, Thailand, Ivory Coast, Lebanon, Puerto Rico, Spain, the United States, Romania, Japan, South Africa, Kenya, France, Nigeria, Argentina, and China. Moreover, these genetic differences in DNA had an influence on PI resistance-associated amino acid mutational features.

Our data not only documented the presence of six HIV-1 PR subtypes or subsubtypes (A2, B, F2, G, H, and J) and one CRF (CRF02-AG) but also identified viral strains harboring phylogenetically unclassifiable PR genes. The high frequency (23%; 25 of 110) of unclassifiable Cameroonian PR sequences was in contrast to the low frequency (2%; 7 of 301) of such sequences previously reported for the global PR collection comprising 114 subtype B sequences and 187 non-subtype B sequences (26). However, there are important differences between the sources of sequences from the global set and those from this study. All but 4 of the 301 global sequences were collected in countries where HIV-1 was recently introduced or in which only a limited number of HIV-1 subtypes cocirculate. In contrast, the specimens described in this report were exclusively collected from persons in Cameroon, a country from which nine HIV-1 subtypes (A, B, C, D, F, G, H, J, and K) as well as viruses with recombinant genomes have been reported (10, 17, 19, 32, 33). Molecular epidemiological data indicate that this central region of Africa has had a long-standing HIV and AIDS epidemic (37).

In contrast to previous studies, in which 98% of the global sequences were unambiguously assigned to phylogenetic PR subtypes, our recent study documented that only 77% of the specimens collected in Cameroon could be subtyped. These results may indicate that the unclassifiable Cameroonian PR sequences represent “older,” and thus more divergent, strains and/or intersubtype recombinants rather than strains with unresolved phylogenies because only a short part of the genome was analyzed. Our findings of recombinant sequences comprising the G and J subtypes in some unclassifiable PR genes further support this possibility. Furthermore, our observation was in agreement with recent data from Vergne et al. (36), who showed recombination between subtypes A, G, D, and K in the pol region among 15 of 17 unclassifiable sequences collected in Central Africa.

It is noteworthy, however, that the unclassifiable PR sequences in our study had a remarkable degree of conservation within three highly conserved regions: the active site, the top of the flap, and the second loop of the β sheet. These data are consistent with the findings for classifiable sequences from this study and from previous reports (6, 11). These results also indicate that the biological function of PR, which is crucial in HIV-1 replication, was preserved in unclassifiable PR genes. Indeed, the large number of infections caused by viruses with phylogenetically unclassifiable PRs and the close relationships between some of these sequences suggest transmission of such variants (Fig. 1b). Overall, these results indicate that HIV-1 strains with unclassifiable PR genes, which are responsible for more than one-fifth of infections, are an integral part of the HIV infection and AIDS epidemic in Cameroon and may potentially spread to other geographic regions.

To gain a better insight into the role of distinct Cameroonian HIV-1 strains in the contribution of PR amino acid mutations that are linked to PI resistance, we estimated such substitutions in viral strains harboring both classifiable and unclassifiable PR genes. Several conclusions can be drawn from this analysis. First, 97% of the total PR sequences carried PI resistance-associated substitutions. Since all sequences were isolated from HIV-1-infected, drug-naïve individuals, the high prevalence of PI resistance-associated mutations represents natural polymorphisms. Second, only two major mutations, 20M and 24I, were found in two strains, which were subtype H and CRF02-AG, respectively. These results are in agreement with findings from other studies that show that the prevalence of major PI mutations circulating in HIV-1-infected, drug-naïve populations worldwide is very low (26, 36). Third, the patterns of PI resistance-associated substitutions in the Cameroonian sequences with classifiable PRs (subtypes A2, F2, G, H, J, and K and CRF02-AG) and unclassifiable PRs were similar to the pattern previously established for non-subtype B viruses of subtypes A, C, D, F, G, and H and CRF01-AE (26), with the 36I substitution being the predominant PI resistance-associated mutation (36). Fourth, dual and triple PI resistance-associated substitutions were present in 38% of the Cameroonian HIV-1 strains. This observation is in contrast to previous findings for the global HIV-1 collection, which indicated that 27% of non-subtype B viruses and 23% of subtype B viruses harbor multiple PI resistance-associated mutations (26). Furthermore, our data documented the fact that the Cameroonian unclassifiable PR sequences contributed considerably to this >10% difference because they carried significantly more dual and triple substitutions than the classifiable PR sequences did (64 versus 30%; P < 0.004). These findings suggest that the use of PIs in Cameroon may result in the rapid emergence of resistant strains. Phenotypic and clinical studies are needed to estimate if these viruses evolve to drug-resistant strains more rapidly than strains that lack these mutations.

In summary, the identification of a large number of viruses with unclassifiable PR gene sequence subtypes indicates the importance of infections with such strains in the HIV infection and AIDS epidemic in Cameroon. Detection of a significantly larger number of dual and triple substitutions associated with PI resistance according to subtype demonstrates the differences between classifiable and unclassifiable PR sequences in Cameroon and further highlights the need for a systematic genetic evaluation of HIV-1 PRs worldwide. The significance of multiple PI resistance-associated mutation patterns warrants further analysis of biological and clinical characteristics. Finally, the development of a database on the genetic diversity of Cameroonian HIV-1 PRs is fundamental for the monitoring of resistance to PI in this geographic region.

Acknowledgments

We are grateful to Timothy Green for the statistical analysis, and we thank Clement Zeh for helpful advice and assistance with the SimPlot program. We gratefully acknowledge Harold Jaffe for scientific review of the manuscript and Robin Moseley for excellent editorial assistance.

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