title: A sensitive real-time PCR based assay to estimate the impact of amino acid substitutions on the competitive replication fitness of human immunodeficiency virus type 1 in cell culture.
abstract: Fixation of mutations in human immunodeficiency virus type 1 (HIV-1), such as those conferring drug resistance and immune escape, can result in a change in replication fitness. To assess these changes, a real-time TaqMan PCR detection assay and statistical methods for data analysis were developed to estimate sensitively relative viral fitness in competitive viral replication experiments in cell culture. Chimeric viruses with the gene of interest in an HIV-1NL4-3 backbone were constructed in two forms, vifA (native vif gene in NL4-3) and vifB (vif gene with six synonymous nucleotide differences from vifA). Subsequently, mutations of interest were introduced into the chimeric viruses in NL4-3VifA backbones, and the mutants were competed against the chimera with the isogenic viral sequence in the NL4-3VifB backbone in cell culture. In order to assess subtle fitness differences, culture supernatants were sampled longitudinally, and the viruses differentially quantified using vifA- and vifB-specific primers in real-time PCR assays. Based on an exponential net growth model, the growth rate of each virus was determined and the fitness cost of the mutation(s) distinguishing the two viruses represented as the net growth rate difference between the mutant and the native variants. Using this assay, the fitness impact of eight amino acid substitutions was quantitated at highly conserved sites in HIV-1 Gag and Env.
authors.affiliation: Department of Microbiology, University of Washington, Seattle, WA 98195, USA. yiliu197@uw.edu
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.forename: Sarah
authors.initials: S
authors.lastname: Holte
authors.forename: Ushnal
authors.initials: U
authors.lastname: Rao
authors.forename: Jan
authors.initials: J
authors.lastname: McClure
authors.forename: Philip
authors.initials: P
authors.lastname: Konopa
authors.forename: J Victor
authors.initials: JV
authors.lastname: Swain
authors.forename: Erinn
authors.initials: E
authors.lastname: Lanxon-Cookson
authors.forename: Moon
authors.initials: M
authors.lastname: Kim
authors.forename: Lennie
authors.initials: L
authors.lastname: Chen
authors.forename: James I
authors.initials: JI
authors.lastname: Mullins
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D016655:HIV Core Protein p24 / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D009154:Mutation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014779:Virus Replication; D054299:env Gene Products, Human Immunodeficiency Virus / Q000235:genetics; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics; D054320:vif Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
chemical_list: D016655:HIV Core Protein p24; D054299:env Gene Products, Human Immunodeficiency Virus; D054301:gag Gene Products, Human Immunodeficiency Virus; C514799:p24 protein, Human Immunodeficiency Virus Type 1; D054320:vif Gene Products, Human Immunodeficiency Virus