Resistance Mutations outside the Integrase Coding Region Have an Effect on Human Immunodeficiency Virus Replicative Fitness but Do Not Affect Its Susceptibility to Integrase Strand Transfer Inhibitors

Abstract

Most studies describing phenotypic resistance to integrase strand transfer inhibitors have analyzed viruses carrying only patient-derived HIV-1 integrase genes (INT-recombinant viruses). However, to date, many of the patients on INSTI-based treatment regimes, such as raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) are infected with multidrug-resistant HIV-1 strains. Here we analyzed the effect of drug resistance mutations in Gag (p2/NCp7/p1/p6), protease (PR), reverse transcriptase (RT), and integrase (IN) coding regions on susceptibility to INSTIs and viral replicative fitness using a novel HIV-1 phenotyping assay. Initial characterization based on site-directed mutant INSTI-resistant viruses confirmed the effect of a series of INSTI mutations on reduced susceptibility to EVG and RAL and viral replicative fitness (0.6% to 99% relative to the HIV-1NL4-3 control). Two sets of recombinant viruses containing a 3,428-bp gag-p2/NCp7/p1/p6/pol-PR/RT/IN (p2-INT) or a 1,088 bp integrase (INT) patient-derived fragment were constructed from plasma samples obtained from 27 virologic failure patients participating in a 48-week dose-ranging study of elvitegravir, GS-US-183-0105. A strong correlation was observed when susceptibility to EVG and RAL was assayed using p2-INT- vs. INT-recombinant viruses (Pearson coefficient correlation 0.869 and 0.918, P<0.0001 for EVG and RAL, respectively), demonstrating that mutations in the protease and RT have limited effect on susceptibility to these INSTIs. On the other hand, the replicative fitness of viruses harboring drug resistance mutations in PR, RT, and IN was generally impaired compared to viruses carrying only INSTI-resistance mutations. Thus, in the absence of drug pressure, drug resistance mutations in the PR and RT contribute to decrease the replicative fitness of the virus already impaired by mutations in the integrase. The use of recombinant viruses containing most or all HIV-1 regions targeted by antiretroviral drugs might be essential to understand the collective effect of epistatic interactions in multidrug-resistant viruses.

Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

“…5A). As expected, all drug resistance mutations identified by population sequencing were also detected by deep sequencing, while an additional 1,073 drug resistance mutations (337 and 736 in the Seville and Madrid cohorts, respectively) were detected by deep sequencing only (i.e., 511 in the protease, 1,015 in the RT, and 97 in the integrase region) (13,68). This plasmid preparation contained the pol gene from the patient and the env gene from the CXCR4-tropic HIV-1 NL4-3 strain, i.e., 08-180 pol/NL43-(X4)env.…”

“…S2 in the supplemental material, all mutations were detected and quantified at the expected proportions, including those at a frequency of 5% of the total population. Next, in order to quantify more accurately the analytical sensitivity of the assay, we mixed DNA from a plasmid containing a patient-derived multidrug-resistant gag-p2/NCp7/p1/p6/pol-PR/ RT/IN fragment in the X4 HIV-1 NL4-3 backbone (08-180) (68) with DNA from a plasmid containing the genome of the wild-type HIV-1 NL4-3 virus carrying the env gene from the R5 HIV-1 YU2 virus (69). Plasmid DNA was quantified and dilutions were used to prepare eight mixtures containing the X4 multidrug-resistant 08-180 plasmid at 0%, 0.1%, 1%, 2%, 3%, 5%, 10%, and 100% concentrations at a final concentration of 0.1 ng/ml.…”

“…The plasma HIV-1 RNA (viral) load was determined (Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0; Roche) and dilutions were used to prepare four mixtures containing the X4 multidrug-resistant 08-194 virus at 0%, 1%, 5%, and 100% concentrations in a final viral load of 100,000 copies/ml. This was the average viral load in plasma samples obtained from highly antiretroviral-experienced patients, usually carrying multidrug-resistant viruses, from recent previous studies in our laboratory (13,68). The viral RNA was purified, reverse transcription-PCR amplified, bar coded in quadruplicate, and deeply sequenced as described in Materials and Methods.…”

“…(68), along with a pNL4-3 plasmid containing the env gene from the R5 HIV-1 YU2 virus (69). Five plasmid mixtures containing drug resistance mutations in the HIV-1 pol gene, i.e., K65R (5%) plus wild type (95%), K103N (5%) plus wild type (95%), K101E (5%) plus E138K (5%) plus wild type (90%), K101E plus E138K (10%) plus wild type (90%), and M184V (RT) plus E92Q (IN) (10%) plus wild type (90%), were obtained from Gilead Sciences, Inc. (Foster City, CA).…”

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

“…This observation is in agreement with viral fitness changes in which RT drug-resistant mutations (K103N and Y181C) rescue the replicative fitness of viruses harboring integrase drug-resistant mutations under the selective pressure of raltegravir (557). In the absence of drug pressure, protease and RT drug-resistant mutations may decrease the replica- tive fitness of viruses harboring integrase mutations (567). A list of INI-resistant mutations outside the integrase coding region has yet to be fully described.…”