title: A rev1-vpu polymorphism unique to HIV-1 subtype A and C strains impairs envelope glycoprotein expression from rev-vpu-env cassettes and reduces virion infectivity in pseudotyping assays.
abstract: Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev-vpu-env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1-vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1-vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01_A/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev-vpu-env cassettes since this increases Env expression irrespective of the presence of the polymorphism.
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Matthias H
authors.initials: MH
authors.lastname: Kraus
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: Parrish
authors.forename: Katharina S
authors.initials: KS
authors.lastname: Shaw
authors.forename: Julie M
authors.initials: JM
authors.lastname: Decker
authors.forename: Brandon F
authors.initials: BF
authors.lastname: Keele
authors.forename: Jesus F
authors.initials: JF
authors.lastname: Salazar-Gonzalez
authors.forename: Truman
authors.initials: T
authors.lastname: Grayson
authors.forename: David T
authors.initials: DT
authors.lastname: McPherson
authors.forename: Li-Hua
authors.initials: LH
authors.lastname: Ping
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Anderson
authors.forename: Ronald
authors.initials: R
authors.lastname: Swanstrom
authors.forename: Carolyn
authors.initials: C
authors.lastname: Williamson
authors.forename: George M
authors.initials: GM
authors.lastname: Shaw
authors.forename: Beatrice H
authors.initials: BH
authors.lastname: Hahn
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D015497:HIV-1 / Q000235:genetics* / Q000472:pathogenicity*; D054298:Human Immunodeficiency Virus Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics* / Q000378:metabolism; D054299:env Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis*; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000378:metabolism; D000086082:Viroporin Proteins
chemical_list: D054298:Human Immunodeficiency Virus Proteins; D011993:Recombinant Fusion Proteins; D054334:Viral Regulatory and Accessory Proteins; D054299:env Gene Products, Human Immunodeficiency Virus; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins