Natural Polymorphisms of Human Immunodeficiency Virus Type 1 Integrase and Inherent Susceptibilities to a Panel of Integrase Inhibitors

214

306

Integrase (IN) strand transfer inhibitors (INSTIs) have been developed to inhibit the ability of HIV-1 integrase to irreversibly link the reverse-transcribed viral DNA to the host genome. INSTIs have proven their high efficiency in inhibiting viral replication in vitro and in patients. However, first-generation INSTIs have only a modest genetic barrier to resistance, allowing the virus to escape these powerful drugs through several resistance pathways. Second-generation INSTIs, such as dolutegravir (DTG, S/GSK1349572), have been reported to have a higher resistance barrier, and no novel drug resistance mutation has yet been described for this drug. Therefore, we performed in vitro selection experiments with DTG using viruses of subtypes B, C, and A/G and showed that the most common mutation to emerge was R263K. Further analysis by site-directed mutagenesis showed that R263K does confer low-level resistance to DTG and decreased integration in cell culture without altering reverse transcription. Biochemical cell-free assays performed with purified IN enzyme containing R263K confirmed the absence of major resistance against DTG and showed a slight decrease in 3= processing and strand transfer activities compared to the wild type. Structural modeling suggested and in vitro IN-DNA binding assays show that the R263K mutation affects IN-DNA interactions.T he high mutation rate of HIV-1 reverse transcriptase (RT) allows the virus to escape pressure through adaptive mutations that include drug resistance mutations that limit the effectiveness of antiretroviral drugs (5,31,66,69,70). The use of multiple drugs in combination can hamper this process by restraining viral replication, limiting the emergence of resistant strains. The addition of integrase inhibitors to the arsenal of drugs against HIV-1 is important since these inhibitors are active against viruses resistant to other drug classes (16,49,63).The HIV-1 integrase enzyme catalyzes two reactions. The first is 3= processing, which consists of cleavage of a dinucleotide at both 3= ends of the reverse-transcribed linear viral DNA and results in the exposure of reactive hydroxyl groups. The second step termed "strand transfer" is carried out through a nucleophilic attack by exposed 3= hydroxyl groups on host genomic DNA (26, 47). Even though 3= processing may be a suitable therapeutic target, the integrase inhibitors developed so far are integrase strand transfer inhibitors (INSTIs) that preferentially inhibit strand transfer while only modestly affecting 3= processing (18,24,26). Raltegravir (RAL) was the first INSTI to be approved for therapy in 2007 (64) and is safe and efficient in both treatment-naïve and treatment-experienced subjects (11,17,23,35,49,62,63). Elvitegravir (EVG) is another INSTI currently in advanced clinical trials (10,12,77).Although first-generation INSTIs strongly inhibit HIV-1 replication, they possess only a modest genetic barrier to resistance. Three main resistance pathways have been identified for RAL, involving initial mutations of the N1...

Section: Discussionmentioning

confidence: 99%

Characterization of the R263K Mutation in HIV-1 Integrase That Confers Low-Level Resistance to the Second-Generation Integrase Strand Transfer Inhibitor Dolutegravir

Quashie

Mésplède

Han

et al. 2012

214

306

“…All mutant proviral clones were verified by DNA sequence analysis of the entire XbaI/SacII recombinant joint. Wild-type and mutant XbaI/SacII inserts were also reconstructed into pR7/3(X/S)Bsd (52,70), an env Ϫ proviral derivative in which the amino terminus of nef is replaced with bsd, a gene conferring resistance to the microbial antibiotic blasticidin S. Single-step infectious, vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped viral stocks were prepared by cotransfection of the pR7/3(X/S)Bsd variants with pCI-VSV-G, a VSV-G expression vector.…”

Section: Methodsmentioning

confidence: 99%

Sequential Deletion of the Integrase (Gag-Pol) Carboxyl Terminus Reveals Distinct Phenotypic Classes of Defective HIV-1

Mohammed

Topper

Muesing

2011

A requisite step in the life cycle of human immunodeficiency virus type 1 (HIV-1) is the insertion of the viral genome into that of the host cell, a process catalyzed by the 288-amino-acid (32-kDa) viral integrase (IN). IN recognizes and cleaves the ends of reverse-transcribed viral DNA and directs its insertion into the chromosomal DNA of the target cell. IN function, however, is not limited to integration, as the protein is required for other aspects of viral replication, including assembly, virion maturation, and reverse transcription. Previous studies demonstrated that IN is comprised of three domains: the N-terminal domain (NTD), catalytic core domain (CCD), and C-terminal domain (CTD). Whereas the CCD is mainly responsible for providing the structural framework for catalysis, the roles of the other two domains remain enigmatic. This study aimed to elucidate the primary and subsidiary roles that the CTD has in protein function. To this end, we generated and tested a nested set of IN C-terminal deletion mutants in measurable assays of virologic function. We discovered that removal of up to 15 residues (IN 273) resulted in incremental diminution of enzymatic function and infectivity and that removal of the next three residues resulted in a loss of infectivity. However, replication competency was surprisingly reestablished with one further truncation, corresponding to IN 269 and coinciding with partial restoration of integration activity, but it was lost permanently for all truncations extending N terminal to this position. Our analyses of these replication-competent and -incompetent truncation mutants suggest potential roles for the IN CTD in precursor protein processing, reverse transcription, integration, and IN multimerization.The defining hallmarks of retroviruses are reverse transcription of the viral genomic information as encoded in polyadenylated RNA and the subsequent integration of the copied DNA genome into that of a host cell. The latter is an essential and irreversible event which is mediated by the catalytic activities of the viral integrase protein (IN), the recent target of successful chemotherapeutic intervention against HIV-1 infection (1). HIV-1 IN is a 288-amino-acid, 32-kDa protein that is cleaved from the C terminus of the Gag-Pol polyprotein (Pr160 Gag-Pol ) via viral proteolytic activity. The biochemical mechanisms that lead to retroviral integration, which have been extensively studied in vitro, are defined by two catalytically related and sequentially dependent steps (18) which may be distinguished by their respective sensitivities to current inhibitors of IN function (39). Following the completion of reverse transcription of the viral RNA into its DNA copy, IN removes two nucleotides from the 3Ј end of each strand of the viral DNA. This step, termed 3Ј processing, generates a chemically reactive 3Ј-hydroxyl group (CA OH -3Ј) at the 3Ј ends of the DNA molecule, effectively activating the termini for the subsequent reaction (strand transfer). This enzymatic step is the target of all I...

“…Primary RAL resistance mutations confer changes in the IN coding region at amino acid positions 143, 148, and 155 (4). Additional resistance mutations resulting in amino acid substitutions V72I, L74I or L74M, E92Q, T97A, F121Y, E138K, G140S or G140A, V151I, E157Q, G163R, I203M, and S230R have also been described (D. Cooper and B. Nguyen, presented at the 14th Conference on Retroviruses and Opportunistic Infections, Los Angeles, CA, 25 to 28 February 2007) (19,21). Recent studies have reported that the primary resistance mutations N155H and Q148R, Q148H, or Q148K represent mutually exclusive and nonoverlapping pathways (8,20).…”

mentioning

confidence: 99%

Substitutions at Amino Acid Positions 143, 148, and 155 of HIV-1 Integrase Define Distinct Genetic Barriers to Raltegravir Resistance In Vivo

Fransen¹,

Gupta²,

Frantzell³

et al. 2012

Mutations at amino acids 143, 148, and 155 in HIV-1 integrase (IN) define primary resistance pathways in subjects failing raltegravir (RAL)-containing treatments. Although each pathway appears to be genetically distinct, shifts in the predominant resistant virus population have been reported under continued drug pressure. To better understand this dynamic, we characterized the RAL susceptibility of 200 resistant viruses, and we performed sequential clonal analysis for selected cases. Patient viruses containing Y143R, Q148R, or Q148H mutations consistently exhibited larger reductions in RAL susceptibility than patient viruses containing N155H mutations. Sequential analyses of virus populations from three subjects revealed temporal shifts in subpopulations representing N155H, Y143R, or Q148H escape pathways. Evaluation of molecular clones isolated from different time points demonstrated that Y143R and Q148H variants exhibited larger reductions in RAL susceptibility and higher IN-mediated replication capacity (RC) than N155H variants within the same subject. Furthermore, shifts from the N155H pathway to either the Q148R or H pathway or the Y143R pathway were dependent on the amino acid substitution at position 148 and the secondary mutations in Y143R-or Q148R-or H-containing variants and correlated with reductions in RAL susceptibility and restorations in RC. Our observations in patient viruses were confirmed by analyzing site-directed mutations. In summary, viruses that acquire mutations defining the 143 or 148 escape pathways are less susceptible to RAL and exhibit greater RC than viruses containing 155 pathway mutations. These selective pressures result in the displacement of N155H variants by 143 or 148 variants under continued drug exposure.