title: In vitro functional assessment of natural HIV-1 group M Vpu sequences using a universal priming approach.
abstract: The HIV-1 accessory protein Vpu exhibits high inter- and intra- subtype genetic diversity that may influence Vpu function and possibly contribute to HIV-1 pathogenesis. However, scalable methods to evaluate genotype/phenotype relationships in natural Vpu sequences are limited, particularly those expressing the protein in CD4+ T-cells, the natural target of HIV-1 infection. A major impediment to assay scalability is the extensive genetic diversity within, and immediately upstream of, Vpu's initial 5' coding region, which has necessitated the design of oligonucleotide primers specific for each individual HIV-1 isolate (or subtype). To address this, we developed two universal forward primers, located in relatively conserved regions 38 and 90 bases upstream of Vpu, and a single universal reverse primer downstream of Vpu, which are predicted to cover the vast majority of global HIV-1 group M sequence diversity. We show that inclusion of up to 90 upstream bases of HIV-1 genomic sequence does not significantly influence in vitro Vpu expression or function when a Rev/Rev Response Element (RRE)-dependent expression system is used. We further assess the function of four diverse HIV-1 Vpu sequences, revealing reproducible and significant differences between them. Our approach represents a scalable option to measure the in vitro function of genetically diverse natural Vpu isolates in a CD4+ T-cell line.
authors.affiliation: Faculty of Health Sciences, Simon Fraser University, Burnaby, BC, Canada.
authors.forename: Asa
authors.initials: A
authors.lastname: Rahimi
authors.affiliation: Department of Molecular Biology and Biochemistry, Faculty of Science, Simon Fraser University, Burnaby, BC, Canada.
authors.forename: Gursev
authors.initials: G
authors.lastname: Anmole
authors.affiliation: Centre for Research in Infectious Diseases, National Institute of Respiratory Diseases, Mexico City, Mexico.
authors.forename: Maribel
authors.initials: M
authors.lastname: Soto-Nava
authors.affiliation: Centre for Research in Infectious Diseases, National Institute of Respiratory Diseases, Mexico City, Mexico.
authors.forename: Tania
authors.initials: T
authors.lastname: Escamilla-Gomez
authors.affiliation: Department of Molecular Biology and Biochemistry, Faculty of Science, Simon Fraser University, Burnaby, BC, Canada.
authors.forename: Tristan
authors.initials: T
authors.lastname: Markle
authors.affiliation: Department of Molecular Biology and Biochemistry, Faculty of Science, Simon Fraser University, Burnaby, BC, Canada.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Jin
authors.affiliation: British Columbia Centre for Excellence in HIV/AIDS, Vancouver, BC, Canada.
authors.forename: Guinevere Q
authors.initials: GQ
authors.lastname: Lee
authors.affiliation: British Columbia Centre for Excellence in HIV/AIDS, Vancouver, BC, Canada.
authors.forename: P Richard
authors.initials: PR
authors.lastname: Harrigan
authors.affiliation: Massachusetts General Hospital, Boston, MA, USA; Harvard Medical School, Cambridge, MA, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Bangsberg
authors.affiliation: University of California San Francisco, San Francisco, CA, USA.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Martin
authors.affiliation: Centre for Research in Infectious Diseases, National Institute of Respiratory Diseases, Mexico City, Mexico.
authors.forename: Santiago
authors.initials: S
authors.lastname: Avila-Rios
authors.affiliation: Centre for Research in Infectious Diseases, National Institute of Respiratory Diseases, Mexico City, Mexico.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Reyes-Teran
authors.affiliation: Faculty of Health Sciences, Simon Fraser University, Burnaby, BC, Canada; Department of Molecular Biology and Biochemistry, Faculty of Science, Simon Fraser University, Burnaby, BC, Canada; British Columbia Centre for Excellence in HIV/AIDS, Vancouver, BC, Canada.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Brockman
authors.affiliation: Faculty of Health Sciences, Simon Fraser University, Burnaby, BC, Canada; British Columbia Centre for Excellence in HIV/AIDS, Vancouver, BC, Canada. Electronic address: zbrumme@sfu.ca.
authors.forename: Zabrina L
authors.initials: ZL
authors.lastname: Brumme
mesh_terms: D015496:CD4-Positive T-Lymphocytes; D017931:DNA Primers; D015536:Down-Regulation; D014644:Genetic Variation; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics*; D054298:Human Immunodeficiency Virus Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020218:Response Elements; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics* / Q000378:metabolism; D000086082:Viroporin Proteins
chemical_list: D017931:DNA Primers; D054298:Human Immunodeficiency Virus Proteins; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins
keywords: BST2/tetherin; CD4; Downregulation; HIV-1 Vpu; Rev/RRE; Sequence diversity