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

Jan Weber,Damian J Mccoll,Justine D Rose,Ana C Vazquez,Nicolas Margot,Michael D Miller,Dane Winner,Miguel E Quiñones-Mateu,Cecilio López-Galíndez

doi:10.1371/journal.pone.0065631

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.

Highlights

Productive infection with human immunodeficiency virus type 1 (HIV-1) requires three key steps in the replication of the virus, i.e., reverse transcription of viral genomic RNA into viral cDNA by the viral reverse transcriptase (RT), integration of viral cDNA into host cell genome using the viral integrase (IN), and cleavage of newly synthesized viral polypeptide by the viral protease (PR) into individual viral proteins during new virion assembly [1]
EVG can select several INSTI resistance mutations in vitro [12,13,14] but E92Q, Q148R/H/K, and N155H mutations have been the most common EVG resistance mutations identified in vivo, with other mutations emerging during clinical studies, e.g., T66I/A/K, S147G, etc
We first tested our system by constructing p2-INTrecombinant viruses carrying these and other single, dual, or triple INSTI resistance mutations introduced by site-directed mutagenesis [15]

Summary

Introduction

Productive infection with human immunodeficiency virus type 1 (HIV-1) requires three key steps in the replication of the virus, i.e., reverse transcription of viral genomic RNA into viral cDNA by the viral reverse transcriptase (RT), integration of viral cDNA into host cell genome using the viral integrase (IN), and cleavage of newly synthesized viral polypeptide by the viral protease (PR) into individual viral proteins during new virion assembly [1]. All three steps were initially considered as targets for antiretroviral drugs, HIV-1 integrase was the last viral enzyme to emerge as a clinically validated alternative to block HIV-1 replication [2]. Since the late 1990s, several different chemical scaffolds have been studied for their ability to inhibit HIV-1 integration [3,4], leading to the approval of the first HIV-1 integrase strand transfer GROUP Patient E92Q E92Q+L68V/I E92Q+N155H

Methods

Results

Conclusion