Human immunodeficiency virus type 1 group m protease in cameroon: genetic diversity and protease inhibitor mutational features

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.

FIG. 1.

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.

FIG. 1.

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.

FIG. 2.

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).

FIG. 3.

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.

FIG. 3.

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.

FIG. 4.

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.