title: Pairwise growth competition assay for determining the replication fitness of human immunodeficiency viruses.
abstract: In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.
authors.affiliation: Department of Microbiology, University of Washington.
authors.forename: Siriphan
authors.initials: S
authors.lastname: Manocheewa
authors.affiliation: Department of Microbiology, University of Washington.
authors.forename: Erinn C
authors.initials: EC
authors.lastname: Lanxon-Cookson
authors.affiliation: Department of Microbiology, University of Washington.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Microbiology, University of Washington.
authors.forename: J Victor
authors.initials: JV
authors.lastname: Swain
authors.affiliation: Department of Microbiology, University of Washington.
authors.forename: Jan
authors.initials: J
authors.lastname: McClure
authors.affiliation: Department of Microbiology, University of Washington.
authors.forename: Ushnal
authors.initials: U
authors.lastname: Rao
authors.affiliation: Department of Microbiology, University of Washington.
authors.forename: Brandon
authors.initials: B
authors.lastname: Maust
authors.affiliation: Department of Microbiology, University of Washington.
authors.forename: Wenjie
authors.initials: W
authors.lastname: Deng
authors.affiliation: Department of Microbiology, University of Washington.
authors.forename: Justine E
authors.initials: JE
authors.lastname: Sunshine
authors.affiliation: Department of Microbiology, University of Washington.
authors.forename: Moon
authors.initials: M
authors.lastname: Kim
authors.affiliation: U.S Military HIV Research Program, Walter Reed Army Institute of Research; Henry M. Jackson Foundation.
authors.forename: Morgane
authors.initials: M
authors.lastname: Rolland
authors.affiliation: Department of Microbiology, University of Washington; Departments of Medicine and Laboratory Medicine, University of Washington; jmullins@u.washington.edu.
authors.forename: James I
authors.initials: JI
authors.lastname: Mullins
mesh_terms: D004261:DNA Replication / Q000235:genetics / Q000502:physiology; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D016655:HIV Core Protein p24 / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D009154:Mutation; D014779:Virus Replication / Q000235:genetics / Q000502:physiology*
chemical_list: D004279:DNA, Viral; D016655:HIV Core Protein p24