Abstract
Although human immunodeficiency virus type 1 (HIV-1) infection causes serious health problems in Indonesia, information in regard to drug resistance is limited. We performed a genotypic study on HIV-1 integrase derived from drug-naive individuals in Surabaya, Indonesia. Sequencing analysis revealed that no primary mutations associated with drug resistance to integrase inhibitors were detected; however, secondary mutations, V72I, L74I/M, V165I, V201I, I203M, and S230N, were detected in more than 5% of samples. In addition, V201I was conserved among all samples. Most integrase genes were classified into CRF01_AE genes. Interestingly, 40% of the CRF01_AE genes had an unusual insertion in the C-terminus of integrase. These mutations and insertions were considered natural polymorphisms since these mutations coincided with previous reports, and integrase inhibitors have not been used in Indonesia. Our results indicated that further studies may be required to assess the impact of these mutations on integrase inhibitors prior to their introduction into Indonesia.
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HIV-1 is subdivided into four groups, M (major), O (outlying), N (new or non-M, non-O), and P, each the result of a separate transfer of virus from chimpanzees or gorillas into humans. The majority of the HIV-1 pandemic has been caused by group M viruses. The viruses in group M are further classified into subtypes and circulating recombinant forms (CRFs), which are prevalent in specific geographic regions. While subtype B of HIV-1 is the predominant subtype in the Americas, Europe, and Australia, there is a growing epidemic of non-B subtypes and CRFs in Africa and Asia. 3 ARV drugs have been designed against subtype B viruses, but are believed to retain their activity against other subtypes; however, only limited data are presently available as to how viral diversity among different subtypes and CRFs affects drug susceptibility and resistance. CRF01_AE is one of the major CRFs of HIV-1 dominating the global epidemic, and is prevalent throughout Southeast Asia. 3 Among Southeast Asian countries, the annual incidence rate of HIV infection has declined in many countries, whereas it has continuously increased in Indonesia. 4 Although research into HIV-1 in Indonesia is important, little research has been conducted on drug resistance. In this study, we performed a genotypic study on the integrase genes derived from drug-naive individuals residing in Surabaya, Indonesia.
Peripheral blood samples were collected from female commercial sex workers (CSW), injecting drug users (IDU), and patients who were diagnosed with HIV infection at Airlangga University Teaching Hospital in 2012–2013. Fifty-five samples, consisting of 22 from CSW, 18 from IDU, and 15 samples from Airlangga University Teaching Hospital, were subjected to this study with approval from the institutional ethics committees of Kobe University Graduate School of Medicine and the Institute of Tropical Disease, Airlangga University. Plasma was isolated from peripheral blood samples by centrifugation for 10 min at 2,000 rpm. In addition, peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Histopaque 1077 (Sigma-Aldrich, St. Louis, MO). RNA and DNA were extracted from plasma and PBMCs using the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany) and GenElute Mammalian Genomic DNA Miniprep kit (Sigma-Aldrich), respectively.
Viral RNA was reverse transcribed into cDNA using the SuperScript III First-Stand Synthesis kit (Invitrogen, Carlsbad, CA) with the reverse primer, K-env-R1, 5′-CCAATCAGGGAAGAAGCCTTG-3′ [corresponding to nucleotide (nt) 9158 to 9138 of an HIV-1 subtype B reference strain, pNL4-3 (GenBank accession number AF324493)]. The HIV-1 integrase gene was then amplified by nested PCR using Ex Taq (Takara Bio, Shiga, Japan) and primer sets, as follows. The primers, Int-outer-S1, 5′-GTCTACCTGTCATGGGTACC-3′ (nt 4140 to 4159) and Int-Outer-AS1, 5′-GTGGGATATGTACTTCTGAACTTAC-3′ (nt 5215 to 5191), were used for first PCR, and the primers, Int-Inner-S, 5′-GCACACAAAGGAATTGGAGGAAATGAAC-3′ (nt 4161 to 4188) and Int-Inner-AS, 5′-GGATGCTGGTTTCATAGTGATGTCTATAAAACC-3′ (nt 5185 to 5153), were used for nested PCR. If the viral gene fragment failed to be amplified, primer sets were replaced as follows. The primers, DRIN01, 5′-CAGACTCACAATATGCATTAGG-3′ (nt 4039 to 4060) and DRIN02, 5′-CCTGTATGCAGACCCCAATATG-3′ (nt 5264 to 5243), were used for first PCR, and the primers, DRIN05, 5′-CTGGCATGGGTACCAGCACACAA-3′ (nt 4146 to 4168) and DRIN04, 5′-TAGTGGGATGTGTACTTCTGAAC-3′ (nt 5217 to 5195), were used for nested PCR. If amplification failed again even after changing primers, DNA extracted from PBMCs was used for nested PCR instead of cDNA generated from viral RNA.
To examine the major viral population in a sample, PCR products amplified at the end-point dilution of DNA templates were subjected to sequencing analysis. Sequencing analysis of the PCR-amplified HIV-1 integrase gene was then carried out using the Big Dye Terminator v1.1 Cycle Sequencing kit with an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA), and data were assembled using Genetyx ver.10 software (Genetyx, Tokyo, Japan). The deduced amino acid sequences were then aligned with the sequence of pNL4-3, using the Clustal W algorithm and manual editing.
We successfully examined the nucleotide sequences of 43 PCR-amplified integrase genes, consisting of 21 from CSW, 16 from IDU, and 6 samples from Airlangga University Teaching Hospital, by direct sequencing. Phylogenetic tree analysis using the neighbor-joining method indicated that 40 and 3 integrase genes were classified into CRF01_AE and subtype B genes, respectively. The nucleotide sequences of the integrase genes have been deposited in the GenBank database under accession numbers KF735917–KF735959. The deduced amino acid sequences of 40 CRF01_AE and three subtype B integrase genes were translated and studied for amino acid substitutions by comparing them with pNL4-3 integrase. Many amino acid substitutions were found in CRF01_AE integrase; more than 80% of samples contained amino acid substitutions, A21T, V31I, T112V, V113I, T124A, T125A, G134N, I135V, I151V, D167E, V201I, V234I, and S283G (Table 1). Of these mutations, V113I, T124A, I135V, I151V, and V201I were detected in three, two, two, three, and three of three subtype B integrase, respectively. The analyses using QuickAlign (
Frequency of amino acid substitutions was determined by counting manually. Amino acid substitutions detected in more than 80% of samples are shown. Mutations were ordered by the frequency of appearance.
Next, we examined the appearance of primary and secondary mutations associated with drug resistance to integrase inhibitors, RAL, EGV, dolutegravir, MK-2048, S-1360, L-708906, L-187810, and other integrase inhibitors (clinical and in vitro data), including H51Y, H55Y, T66A/I/K/L, L68I/V, V72I, L74A/I/M/S, E92A/Q, Q95K, T97A, T112I, H114Y, G118R, S119G/R, F121Y, T125K, A128T, E138A/K, G140A/C/S, Y143R/C/H, Q146K/P, S147G, Q148H/K/R, V151I, S153A/Y, M154I/N, N155H/S, K156N, E157Q, K160D/N, G163K/R, V165I, R166S, E170A, V201I, I203M, T206S, S230N/R, D232N, V249I, R263K, and C280Y. 1,2,5 –10 The results showed that although no primary mutations were detected, secondary mutations associated with resistance to RAL, EGV, and/or other inhibitors, V72I, L74I/M, V165I, V201I, I203M, and S230N were detected in more than 5% of samples (Table 2). In particular, V201I was conserved in all samples. The analysis using QuickAlign revealed that V201I was found in >92% of subtypes A, C, and D, CRF01_AE, and CRF02_AG integrase, while it was found in 41.3% of subtype B integrase on the HIV sequence database, suggesting that the amino acid substitution V201I frequently appears not only in CRF01_AE viruses, but also in other major HIV-1 subtypes and CRFs. In addition, other secondary mutations, E138K, V151I, and M154I, were detected in a few samples (Table 2).
Drug resistance-associated mutations were detected manually, and the frequency of mutations was calculated. Mutations were ordered by the frequency of appearance.
Previous genotypic studies on HIV-1 integrase revealed that several secondary mutations associated with resistance to integrase inhibitors, including V72I, L74I, E92Q, T97A, V151I, M154I/L, E157Q, V165I, V201I, I203M, T206S, and S230N, were frequently detected in many subtypes and CRFs of HIV-1 derived from drug-naive patients in many countries, including Thailand, Cambodia, Vietnam, and the United States. 9 –13 In these reports, drug resistance-associated mutations appeared due to natural polymorphisms at amino acid positions 72, 74, 97, 112, 119,125, 128, 138, 151, 153, 154, 155, 156, 157, 163, 165, 201, 203, 206, and 230 of HIV-1 integrase, coinciding with our results. 9 –13 In addition to these secondary mutations, 40% (16/40) of the CRF01_AE in this study possessed an unusual tetrapeptide insertion (QNME/A) in the C-terminus of the integrase. The C-terminal region of HIV-1 integrase is involved in the dimerization and DNA binding of the enzyme 14 ; therefore, it possibly affects the efficiencies of integrase activity and integrase inhibitors. This insertion was previously reported in subtype C as a natural polymorphism. 14
Considering this information and the fact that integrase inhibitors have not yet been introduced in Indonesia, all secondary mutations and the insertion detected in this study might be due to natural polymorphisms. Further studies are necessary to clarify the impact of these mutations and/or insertions on the susceptibility of integrase inhibitors prior to their introduction into Indonesia. Finally, it was suggested that RT inhibitors, which are currently used in Indonesia, affect the polymorphism of the integrase gene 15 ; therefore, we consider that further surveillance is necessary to identify drug resistance-associated integrase mutations with a larger population of both drug-naive and drug-treated Indonesian patients.
Sequence Data
Nucleotide sequences are available under GenBank accession numbers KF735917–KF735959.
Footnotes
Acknowledgments
This study was supported by a Grant-in-Aid from the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) program from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The article was proofread by the Medical English Service (Kyoto, Japan).
Author Disclosure Statement
No competing financial interests exist.