Assessment of the susceptibility of mutant HIV-1 to antiviral agents.
Traditional phenotypic assays used to assess the susceptibility of mutant human immunodeficiency virus type-1 (HIV-1) obtained from infected patients or from resistance selection to antiviral agents in cell culture are rather tedious and time consuming. To improve the efficiency of this process, a novel method was developed in which mutant viruses are captured with magnetic nano-beads and used to infect gag-GFP reporter cells to evaluate the extent of resistance conferred by the mutant viruses against antiviral agents. The optimal timing for measuring the inhibitory potencies of antiviral agents was found to be day 3 post-infection for integrase strand transfer inhibitors and protease inhibitors and day 4 for non-nucleoside reverse transcriptase inhibitors. Comparable EC(50) values were obtained when bead-captured breakthrough virus from in vitro resistance selection experiments and its matched site-directed mutagenesis virus were tested side by side in this assay. This assay protocol was also employed to evaluate the inhibitor susceptibility of breakthrough viruses collected from resistance selections that were conducted in the presence of increasing concentrations of an HIV-1 protease inhibitor. Taken together, these findings suggest that a rapid, sensitive, non-invasive, and homogeneous phenotypic assay has been developed for assessing the antiviral agent susceptibility of mutant viruses that emerge from in vitro resistance selection studies.
Wang
Ying-Jie
YJ
Department of Antiviral Research, Merck Research Laboratories, West Point, PA 19486, USA.
McKenna
Philip M
PM
Hrin
Renee
R
Felock
Peter
P
Lu
Meiqing
M
Jones
Kristen G
KG
Coburn
Craig A
CA
Grobler
Jay A
JA
Hazuda
Daria J
DJ
Miller
Michael D
MD
Lai
Ming-Tain
MT
D019380:Anti-HIV Agents / Q000494:pharmacology*; D001681:Biological Assay; D002460:Cell Line; D024921:Drug Resistance, Multiple, Viral / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D008863:Microspheres; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D014162:Transfection; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics
D019380:Anti-HIV Agents; D054301:gag Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins